CHAPTER Aerobic Respiration and the Mitochondrion rch a inccated by here woe ofthe hear an lungs. Onigen poor blood shown by 2 blue vessel) exporedto oxygen in the lang, where t becomes oxyge WHY WE NEED TO BREATHE Every breath you take, and every beat of your hear, out cells need oxygen. To satisfy cellular cd with hemoglobin. Much my and physiology is devoted to ensuring an ° oviding enough oxygens a of anesthesiologits, astronauts, and ‘everyone knows from experience. This oxygen is used to power cellular metabolism by providing energy through the Biochemical pathway of respiration, much of which takes place within mit happens because oxygen demas of human anat ur blood runs scuba divers, Exercis fe recall from (Chapter 1, aro: 30k up residence in th ‘early ancestors of eukaryotic cells. So the next time you find yourself gasping for breath after running up a flight of stars, lake @ moment and contemplate the fact that the oxygen you are so desperately trying to suck in is being consumed by bacterially derived endosymbionts living inside your coll In this chapter we wil learn how oxygen is coupled to energy hhondtia which, at from bacteria that Sout: Ralph Hutchings / Getty mages, ne. reduction, and what role the mitochondria play in the | CHAPTER OUTLINE 5.1. Mitochondtial Seucture and 5.4 Oxidative Phosphoryation in the 5.9. Using the Proton Gragien: Function Formation of ATP 5.10 Peroxisomes 5.2. Aerobie Metabolism inthe 5.5. Electron-Transport Complexes. «5.11 THE HUMAN PERSPECTIVE: Mitochoncrion 5.6 Establishment ofa roton-Motive Diseases that Result fom Abnormal 5.3 THE HUMAN PERSPECTIVE: Fores Mitochonal of Peroxisomal The Role of Anaerobic and 5.7 The Stuctue of ATP Synthase Function ‘Aerobie Metabolism 5.8 The Binding Change Mechanism 168 of ATP Formation5.1 Mitochondrial Structure and Function During the frst two bilion years or so that fe existed on Eat, the atmosphere consisted largely of reduced molecules, suchas molecu- lar hydrogen (H), ammonia (NH), and HO. The Earth of this period was populated by anaerobes organisms that captured and uulizedenergyby means ofoxygen-independeat (anaerobic) metab- olism, such as glycolysis and fermentation (Figures 3.24 and 3.2) Then, between 24 and 27 billion years ago, the cyanobacteria appesred—a new kind of organism that carried outa new type of photosynthetic proces, one in which water molecules were split apart and molecular oxygen (O,) was released. At some point fl- lowing the appearance of the cyanobacteria, the Earths atmosphere began to accumulate significant levels of oxygen, which set the stage for a dramatic change inthe types of organisms that would come to inhabit the plant. ‘As discussed on page M4, molecular oxygen can be a very toxic substance, taking on exira electrons and reacting witha variety of| biological molecules. The presence of oxygen in the atmosphere ‘must have been a powerful agent for natural selection. Over time, species evolved that were not only protected from the damaging eficts of molecular oxygen, bu posessed metablic pathways that tlized the molecule to great advantage. Without the ability fo use cxygen, organisms could only extract a limited amount of energy from thei foods, excreting energy-tich products such a lactic acid and ethanol, which they were unable to metabolize further. In contrast, organisms that incorporated 3; into their metabolism ould completely oxidize such compounds to CO, and 10 and, in the process, extract a much larger percentage oftheir energy com tent. These organisms that became dependent on oxygen were the arth frst aerobes, and they eventually gave rise to all of the onygen dependent prokaryotes and eukaryotes living today. In eukaryotes the utilization of oxygen asa means of energy extraction takes place ina specialized organelle, the mitochondrion. As discus sn Chapter 1, a massive hody of data indicates that mitochondria FIGURE 5.1 Mitocho Ihave evolved from an ancient aerobic bacterium that took up residence inside the cytoplasm of an anaerobic host cel. ‘Mitochondria are large enough to be seen in the light micro Scope (FIGURE 5.12), and thelr presence within cells hes been known for over a hundred years. Depending on the cell ype, mitochondria can have a very different overall structure. At one end of the spec- trum, mitochondria can appear as individual, bean-shaped organelles (Figure 5.16), ranging from 1 to 4ym in length. At the other end of the spectrum, mitochondria can appear as a highly branched, inter connected tubular network. Observations of fluorescently labeled mitochondria within living cells have shown them to be éynamic organelles capable of dramatic changes in shape. Most important, ‘mitochondria can fuse with one another,orsplitin twa (FIGURE 5.2). Ichas been knowa for many years that mitochondria and the endoplasmic reticulum (ER) engage in extensive interactions, but it ‘was a surprise to discover that mitochondrial fission is apparently induced by contact with thin tubules from the ER. As shown in Figure 5.26, BR tubules can encircle the mitochondrion lke a noose ‘These ER tubules appear to initiate constriction, which is then com- pleted through the action of soluble proteins thal are recruited to the ‘outer surface of the mitochondrion from the cytosol (Figute 5.2) “The alance between fasion and fission is likely a major determinant of mitochondrial number, length, and degree of interconnection. ‘When fusion becomes more frequent than fission, the mitochondria tend to become more elongated and interconnected, whereas 2 pre- dominance of fission leads tothe formation of moze numerous and distinct mitochondria, A number of inherited neueologic diseases are caused by mutations in genes that encode components of the ‘mitochondrial fusion machinery. ‘Mitochondria occupy 15 to 20 percent ofthe volume of an av age mammalian liver cell and contain more than a thousand diff ent proteins, These organelles are best known for their role in generating the ATP that is used to run most ofthe cells energy- requiring activities. To accomplish this function, mitochondria are often associated with fatty acid-containing oil droplets from which they derive raw materials to be oxidized. A particularly striking Mioenogaria fo a (ing fost viewed with a ghase-convast merseope. Mitochondia ae seen a elongated dark bodies. {0 Transmission elecvon micrograph of thin section through a mitocrenerion revealing the intemal structure ofthe organelle, particularly the membranous ‘ra ofthe nner membrane (]Lecalaton of mitechondain the midpigessrrouncing tre proximal porvon ofthe fagellum of» ba spor, Sean: a} Couey of Norman K. Weazl, Stanford Universi: (KR Portar/hoto Researcher, Ine) Don W. Faweort/Phote Researcher, In. 169, ans eupuoN2oN + 1S uns pus 8170 aoa +s uaLaves oupuoysoimy ox pve vonendeoy ® @ FIGURE 5.2 Mitochondrial fusion and fision. (a The dynamic nature of tees ‘cxgpnelie is capture inthe frames of hs movi, whien shows a parton ofa ‘mouse Sirablest whovemitochanda have been labeled with a fireacont rote. nthe Fst eve rames, two pairs of mitochandia whch have been Srieily colores) contact endco-ena and immediately fee Inthe fe thee {eames the ler ion product undergoes fusion and the daughter mitochon die move apart. (by Three-diensonal model of contacts between the ER (gener) and mtoctonciapurps) in 3 yous co Dazed on EM tomography {Section 18.5) ER tubules are seen to encrcle portions ofthe yeast mitochondrial network, making stes of ue fission, Simia’ events occur mammalian cl Bar equals 200 rm. (nthe model depicted hare, contact with ER tubules inates the proces of mitochond'al consti (step 1). Fission s subsequent sccomplahed by proteins (e.g, Dept in mammal thet esrb into helices around the outar surfs ofthe ritechoncon (step 2. Opt tm member af the dynamin amy 0! GTP-binding protens hat ae nvelved in severing membranous suuctutes see Figure 8.1}. GTP binding an hyerolyss causes @ conformational change inthe Drp1 helices thas fly canes te mitochondrion, siting it ino two smaller organeles step 3 ‘ounce: a) From David C. Chan, Cell 125:1242, 2006; © 2006, wit permission of Elsevier: [bl Image by M. West, rom Jonathan R. Friedman, etal, Science ERtbue © @ Fission ste 334359, 2011, sprinted with permission fom AAAS, arrangement of mitochondria occurs in sperm call, where they are ‘often locate inthe midpiece, jst behind the nucleus (Figure 5.10). ‘The movements of a sperm are powered by ATP produced in these ritochondria. Mitochondria are also prominent in many plat cells ‘where they ae the primary suppliers of ATP in nonphotosynthetic tissues, as wel as being a source of ATP in photosynthetic leaf cells during periods of dark ‘While energy metabolism has been the focus of interest inthe study of mitochondria, these organelles are also involved in other activities. Tor example, mitochondria are the sites of synthesis of ‘numerous substances, including certain amino acids and the heme groups shown in Figures 2.35 and 5.12. Mitochondria also play a vital role in the uptake and release of calcium ions, Calcium fons are cssenial triggers for cellular activities (Seetion 15.5), and mitochon- dria (along with the endoplasmic rticulur) play an important role in regulating the Ca®* concentration of the cytosol. For example, ‘when Ca" concentrations rise to abnormally high levels the cyto sol a Ca?" transporter in the inner mitochondrial membrane takes, up some of the excess Ca fons. The process of cll death, which playsan enormous role inthe life ofall multicellular animals, is also regulated o a large extent by events that occur within mitochondria (Seetion 158) Mitochondrial Membranes If you take a close look at the electron micrograph of Figure 5.1 sean be seen thatthe outer boundary of a milochondrion contains two membranes: the outer mitochondrial membrane and the snner ‘mitochondrial membrane. The outer mitochondrial membrane completely encloses the mitochondrion, serving ais outer boundary. The inner mitochondrial membrane is subdivided into two major domains that have different protein residents and carry out distinct functions, One of these domains, called the ner boundary mem- brane, lies just inside the outer mitochondrial membrane, forming & double-membrane outer envelope, The inner boundary membrane is particulary rich in the proteins responsible forthe import of mito- chondrial proteins (see Figure 8.47). The other domain ofthe inner ‘mitochondrial membrane is present within the interior of the orga- nelle as series of invaginated membranous sheets, called cristae, ‘The role of mitochondria as energy transducers i intimately tied to ‘the membranes ofthe cristae that are so prominent in electron micro graphs ofthese organelles, The cristae contain slagge amount of mem brane surface, which houses the machinery needed for aerobic respiration and ATP formation (see Figure 522). The organization of, the cristae is shown ina clearer profile in the scanning electron micro graph of FIGURE 5.20 and the three-dimensional reconstruction of Figure 3.3b. The inner boundary membrane and internal cristal mem branes are joined to one another by narrow tubular connections, of cristae junctions, as shown in the schematic lusrations of Figure 5.3. Exactly how such complex organization is created is aot yet fuly known, but an inner membrane associated protein complex called -MiXOS, (also known as MICOS or MINOS) is located atthe eristae Junctions and is required for normal organization of cristae. The membranes of the mitochondrion divide the organelle into ‘wo aqueous compartments, one within the interior ofthe mitochon rion, called the matrix, and 2 second between the outer and inner ‘membrane, called the intermembrane space. Tine mateix has 2 agellike consistency owing to the presence of a high concentrationStam) tm outer membrane Cristelménbrare Cistae Iner membrane | ATP synthase paricles o FIGURE 5.3 Th by oles ofthe nar memorane. (3) Th (fstaeunton tructure of mitochondrion (8 cerning elacon micrograph ofa macerated mitochondrion showing the intra mati enclosed ‘densioral constuction of mtacrondrion ested on 3 series of micragrhe taken wth 3 highswalage lection mictoscope of single thick section ef brown fat aus that hal been tked a various angles. Highwolage instuments accelerate elections st locos that alow them to penerat thickortssue sections (yp to 1.5 ym). This techrique suggests thatthe erste are presen as fattoned sheets (bret) tat communicate with the ince-membrane space by way of natram tubular openings ther than “wide-open” channel zi typiealy depicted Inti reconstruction, the inner mitechondial membrane i shawn in blue tthe peripheral regions ard n yellow when I penetrates inta the mat to form the erste (el Sehemate oiagrams showing the tree-dimensional intemal structure ep] and atin secon fettom) ofa mtechonction om bovine hear tise Sous (o) From K Tanaka and. Naguro nt. Rew Cyto 68:11, 1980; (5) Counesy of Guy A. Parkins ane Terence. Fay (ap to 500mg/ml) of water soluble proteins. The proteins of the intermembrane space are best known for their role in initiating cll suicide, a subject discussed in Section 15.17. ‘The outer and inner membranes have very different properties ‘The outer membrane is composed of approximately 50 percent lipid bby weight and contains a curious mixture of enzymes involved in such diverse activities asthe oxidation of epinephrine, the degradation of Uyptophan, and the elongation of faty acids, In contrast, the inner membrane contains more than 100 different polypeptides and ‘has a very high protein/lipid ratio (more than 3:1 by weight, which ung ve aunanvS LPUONDCUN «1S172, corresponds to about one protein molecule fr evry 15 phospholipids) ‘The inner membrane is virally devoid of cholesterol and rich in an unusual phospholipid, cardiolipin (diphosphatidylglycero; see Figure 46 for the structure), which are characteristics of bacterial plasma membranes, from which the inner mitochondrial membrane has presumably evolved, Cardiolipin plays an important role in facil- itating the activity of several ofthe large protein complexes involved fn electron transport and ATP synthesis, The outer mitochondrial membrane is thought to be homologous to an outer membrane present as part of the cell wall of certain bacterial cells, The outer ‘mitochondrial membrane and outer bacterial membrane bath contain ‘porns, integral proteins that havea relatively large internal channel (eg. 2-3nm) surrounded by a barrel off strands (FIGURE 5.4). The porins ofthe outer mitochondrial membrane are not static structures as was once thought but can undergo reversible closure in response to conditions within the cell. When the potin channels are wide ‘open, the outer membrane is freely permeable to molecules such as ATP, NAD, and coenzyme A, which have key roles to playin energy ‘metabolism within the mitochonérion. In contrast, the inner mito- chondrial membrane is highly impermeable; vitually all molecules and ions require special membrane transporters to gain entrance to the matrix ‘As will be discussed in following sections, the composition and ‘organization of the inner mitochondrial membrane are the keys to the bioenergetic activities of the organelle. The architecture of the inner membrane and the apparent fluidity of its bilayer facilitate the interactions of components that are requized for ATP formation, vevpuoyronn 942 ave voxeudsoy 290104 + S EaLEvHD) FIGURE 5.4 Porins. Gram-negative bacteria have a Tpid-contining| outer membrane outside oftheir plsma memiorane as part of their calls Thi outer membrane concave proveine alld porn, tat consist ofa bavel of sheet anc form an opening though which moderates2ed molecules can penetrate. This image shows the protein OmpW enipeded inthe outermembrane of E ol. The porn cantare mall hydrophobic compound within ts cenval channel varety of pois having diferent sed channels and selectvies ‘re ako founain she outer mitochondal membrane n eukaryotic call Sout: From Heedeok Hong, ea, Bil Chem 281, cover of #1, 2006: 12008, The American Society for Biochemistry and Molecule Bley Image courtesy of Bart van den Beg, The Mitochondrial Matrix Jn addition t an array of enzymes, the mitochondrial matrix also contains ribosomes (of considerably smaller size than those found in the cytosol) and several molecules of DNA, which is citcular in higher plants and animals (Figure 5.3). Thus, mitochondria posess their own genetic material and the machinery to manufactae their cows RNAs and proteins. This nonchromosomal DNA is important because it encodes a small number af mitochondrial polypeptides (13 in humans) that ae tightly integrated into the inner mitechon- drial membrane along with polypeptides encoded by genes residing ‘within the nucleus. Human mitochondeial DNA also encodes 2 bo- somal RNAs nd 22 (RNAs that ace used in protein synthesis within the organelle. Mitochondelal DNA (mtDNA) ia relic of ancient his- tory. is thought tobe the legacy fom a single aerobic bacterium that took up residence in the cytoplasm of a primitive cell that ult rately became an ancestor of ll eukaryotic cells (page 26). Most of| the genes of this ancient symbiont were either lost or transferred cover the cours of evolution to the ncleus of the host cell, leaving only a handfal of genes to encode some ofthe most hydrophobic proteins of the inner mitochondrial membrane Ii interesting to note that the RNA polymerase that synthesizes the various mito chondrial RNAS is not related tothe multisubunit enzyme found in prokaryotic and eukaryotic cells. Instead, the mitochondrial RNA polymerase isa single subunit enayme similar in many respects to certain bacterial viruses (bacteriophages) from which appears to have evolved. Fora numberof reasons, mtDNA is well suited for use in the study of human migration and evluton. A numberof com panies, for example, employ mtDNA sequences to trace the ancestry of clients who are searching for ther ethnic or geographic roots ‘We wil ern ter inthe chapter to discuss the molecular archi- tectue of mitochondeal membranes, but rst let vs consider the role ofthese organelesin te basic oxidative paways of eukaryotic cel, ‘hich is summarized in FIGURE 5.5. It might help to examine this overview figure and ead the accompanying iegend before moving on to the following detailed descriptions of these pathways eviews PI 1. Describe the metabolic changes in oxidative metabolism that must have accompanied the ‘evolution and success of the cyanobacteria, 2. Compare the properties and evolutionary history of the inner and outer mitochondrial membranes; the intermembrane space and the matrix 5.2 Aerobic Metabolism the Mitochondrion In Chapter 3 we described the initial stages in the oxidation of carbo- hydrates, Begining with glicose, the irs steps of the oxidation pro- cess are carried out by the enzymes of glycolysis, which are located in the cytosol (Figure 5.5). The 10 reactions that constitute the glycolytic pathway were illustrated in Figure 324; the major steps of the pathway are sammarized in FIGURE 5.6. Only a smal fraction of theFIGURE 5.5 An overview of carbohydrate metabolim in eukaryote calls. The reactions of gyeajss gonerate pyruvate and NADH in the eysol. nthe sence of Os, the pyruvate is educed by NADH to lactate (or anther product of fermentation such a ethanol in year 2 Figure 2.29 for deta). The NAD" formed into reactions eried inthe continuation of glycolysis In he prosoncs of, the rua mowes nt the matrix feitatod by » membrane waneporey, whore t's decarbaryated and linked to coenzyme A (CaN 2 reaction that generates NADH, The NADH produced during hcolys donates its high-energy electrons to a compound that costes the ine mitochon membrane as shown n Figure 5.9, The acyl CoA passes through the TCA cele (a shown n Figure 5.7), whim generates NADH and FADHs. The elecronsin chess various NADH and FADH2 molecuos ar passed slong the slecton-transport chai, which is made wp af eri that are embeded inthe inner mitochondial membrane, to molecular orygen(03\The energy tleasee during electon vansport used in he formato of ATP by a process cscused at length ata inthe chapter fal of the energy rom scion transpor ware robe vtlzed n AT? formation, approximately 36 ATPs euld be ganerateg fom a single molecule of uss. fire energy available in glucose is made available tothe cell during elycolysis—enough for the net synthesis of only two molecules of ‘ATP per molecule of glucose oxidized (Figure 5.6) Mos of the energy remains stored in pyruvate. Each molecule of NADH produced daring the oxidation of glyceraldehyde 3-phosphate (reaction 6, Figure 56) also carries a pair of high-energy electrons! The two products of glycolysis— pyruvate and NADH—can be metabolized in two very different ways, depending on the type of ell n which they are formed and the presence or absence of oxygen. Inthe presence of O, aerobic organisms are able to extract large amounts of additional energy from the pyruvate and NADH produced during glycolysis—enough to synthesize more than 30 Additional ATP molecules. This energy is extracted in mitochondria (Figure 5.5). We will begin with pyruvate and return to consider the fate of the NADH later in the discussion. Each pyruvate molecule produced by glycolysis transported across the inner mitochondrial ‘membrane and into the matrix, where itis decarboxylated to form a {wo-carbon acetyl group (—CH,COO"). The acetyl group is trans- ferred to coenzyme A (a complex organic compound derived from the vitamin pantothenic acid) to produce acetyl CoA. Pyruvate+HS~CoA+NAD! —> acetyl CoA +CO, +NADH +H" he term high-energy estrone an energy electrons arena away wel ceed by biochem bat ey convey a sll image Ar cessed om page 180, igh ergy lectone re ne ht rl with lowe ain ad ar ore edt fred from dane to an scepor than claw snegy elton, ‘The decarboxylation of pyruvate ad transfer ofthe acetyl group to CoA (Figures 5 and 57) along with the reduction of NAD" is catalyed by the giant multenzyme complex pyruvate dehydrogenase, whose structure was shown in Figure 241 The Tricarboxylic Acid (TCA) Cycle (Once formed, acetyl CoA is fed into a cyclic pathway called the tri- carboxylic acid (TCA) cycle, where the substrate is oxidized and its energy conserved. Other than succinate dehydrogenase, which is bound fo the inner membrane, all the enzymes of the TCA cycle reside inthe soluble phase ofthe matsx (Figure 5.5) The TCA cycle is also referred toasthe Krobs eyele alter the British biochemist Hans Krebs, who worked out the pathway inthe 1930, or the citric acid cycle, after a key molecule formed in the pathway. "The first step inthe TCA cycle is the condensation ofthe two- carbon acetyl group witha four-carbon oxaloacetate to form a six- ‘carbon citrate molecule (FIGURE 57, step 12). During the cycle, the ‘trate molecule is decreased in chain length, one carbon ata time, regenerating the four carbon oxaloacetate molecule, which can con dense with another acetyl CoA. Itisthe two carbons that are removed “during the TCA cycle (which are not the same ones that were brought in with the acetyl group) that are completely oxidized to carbon dioxide, During the TCA cycle, four reactions occur in which a pait of electrons are transferred from a substrate to an electeon-accepling 173, Uoupuowouy amp ui wsHoaeiay 2i9o.0y + 25174 vevpuoyronn 942 ave voxeudsoy 290104 + S EaLEvHD) “won WH 20. o Ie ot te o ie CHOPOT pot YI eo 4 eo 4 4 yeeraidehyse =O (ratecues) honor 2NVo* ° “NG NIDA + 2 ; rowotngte oR ¢ yeeate Hon trates) opor Ge 3Phosoho- ayceate (reales) ° Phosphoenot pyrwate (reales) ‘Glucose is phosphonates a the expanse of one ATP, rearranged stivctraly to fom fects phosphate, and then phosphonates again at the expense ofa second ATP. The two phosphate groups ve stated at ‘he to end (C1, C6) of fe rucose cha. ‘The sccarbon bisphosphate i spit ato two bree carbon manephesohates, ‘The treecaronaldemyce'soxiszed to an acid asthe electors removed ‘ram te sbstate are used to reduce the coerzyne NAD” to NAD Ia ‘auton he Cl acs phosphonate to form a ay phosphae, whch has a hn ohosohate groupiranstrpotental denotes byte yelow shading) ‘The phosphate group from 1 is transferred o ADP forming ATP by ‘Wot ste vel Shosshanfaton, Taw ATP are formed per ucase odes ‘hese reactons resutin the rearrangement and deitatio ofthe substrate fo form an enol phosphate atthe C2 poston that nas high Shosohate grouptvenster potent ‘he phosphate groups warfare to ADP forng ATP by substateve shosphorfaton, geteratig 2 atone at the G2 postion. Two ATPs are formed per gucose onde [Net reaction: [cucose+ 2000" + 290 + 2%, —e 2rynate + ZATP + 2MOH 2H = 2,0 FIGURE 5.6 An overview of glycolysis showing some ofthe key stops, These include the two reaction in whch phosphate groups ae transfered om AP tothe siczarbon sugar to produce fuctose 6b sphosphate (steps 7 3 she oxdation and phosphorylation of glyceraldehyde S-phosohate to prose 12-bsohorohoglyerate ana NADI tp 8 and the wansfr of shosphate groups rom thee-crbon phosphorlstes sortrates to ADP to Produce AIP by substrate ahospharylaton (ep 7 and 10). Keep in mind that two malecules of gleerldenyaed-shosphate ae fred fom ach rrolecule of glucose, so eacions& wough 10 shown here occur twice pe” glucase molecle oxidizedFIGURE 5.7 The tricarbonic acid (TCA) eee. Tho cycle begins withthe condanaation of oxaloacetate (OAA) and acetyl CoA reaction 12, The eaoons of those two compounds re marked with numoers oF Jeers. The two carbons lot during pessage through the cyl ate derved from oxaloacetate. The standard free energies fn eaial)andnames of enzymes a6 slip protded. Five pis of electrons ae removed fom substrate motecues by pyruvate dehydrogenase and the enzymes of the TCA eee, These highaneray tector are vanslerred ta NAD" orFAD and then pissed doar the electon-vansport chan for sein ATP prositon, The reacsons shown here aegin with umber 11 ects the pathy continves where th last reaction of lease umber 10 of Figue 58) leaves of coenzyme. Three of the reactions reduce NAD! to NADH, and one reaction reduces FAD to FADH; (Figure 5.7). The net equation forthe reactions ofthe TTCA cycle can be written as Acetyl CoA + 2H,0-+ FAD +3NAD® + GDP, >2CO,+FADH,+3NADH +31" + GTP+HS—Coa ‘The TCA cycle is a critically important meta bolic pathway. Ifthe position ofthe TCA cycle in the overall metabolism of the cell is considered (FIGURE 5.8; see also Figure 3.22), the metabolites of this cycle are found to be the same compounds generated by most of the cells catabolic pathways, Acetyl CoA, for example, is an important end product of a number of catabolic paths, including the disassembly of fatty acids, which are degraded ‘within the matrix of the mitochondrion into two carbon units (Figure 5.8). These two-carbon compounds enter the TCA cycle as acetyl CoA. The catabolism of amino acids also generates meta bolites of the TCA cycle (Figure 5.86), which enter the matrix by means of special transport systems in the inner mitochondrial membrane. It is apparent that all of a cells energy-providing macromolecules (polysaccharides, fats, and proteins) are broken down into metabolites of the TCA eyele. Thus, the titochondrion becomes the focus for the final ee oe by, boo Succinate energy- conserving steps in metabolism regardless ofthe nature of the starting material The Importance of Reduced Coenzymes in the Formation of ATP Wisevident from the net equation of the TCA cycle thatthe primary products of the pathway are the reduced coenzymes FADH, and [ADIL which contain the high-energy electrons removed irom various substrates as they are oxidized. NADH was also one of the products of glycolysis (along with pyruvate). Mitochondria are not able to i ho 0 sh oH, Prwsate Hs-con Nap 2 5 Ree Cok poo Eto wp meen ho Stcoo- booty, e he, ‘orate Xoo SUDE? E owosceate at=75——otae L9 : _ Spa Moe electrons (rom coo atoms Seer) a to be used in “Coo = SAT production soctrate MO sate sees = *oo0r * PF HO" Han ie G Gee eG % sms 82 stare, ae ‘SuccinyCoA “aKetoglitarate Import the NADH formed in the cytosol during glycolysis. Instead, the electrons of cytosolic NADH ate used to reduce alow molecular ‘weight metabolite that can either (1) enter the mitochondrion (by @ pathway called the malate-aspartate shutle) and reduce NAD* to "NADH, or (2) transfer its electrons to FAD (by a pathway called the elycerol phosphate shutle, which is shown in FIGURE 5.9) to produce FADH,, Both mechanismsallow the electrons from cytosolic NADH to be fed into the mitochondrial electron-transport chain and used for ATP formation. "Now that we have accounted for the formation of NADH and PADI by both glycolysis and the TCA cycle, we can turn to the 175, Uoupuowouy amp ui wsHoaeiay 2i9o.0y + 25176 vevpuoyronn 942 ave voxeudsoy 290104 + S EaLEvHD) Hof FATTY ACID CYCLE i cH CH= CHE -S-c0h Ronco scot FFIGURE 5.8 Catabolic pathways gonerato compounds that are fod into the TOA ey.) Orcationo nt acide The ft step in he ‘oxidation of a fatty acid ists acvation by Inkage tothe thio (SH) ‘group of coenzyme. whch oceure ar te ety ay Oru | ‘araported seus the inner nitachondl membrane in asocation with 8 caer protein not shown). the mizochondion, the faty acy Caf molecule undergoes stopuio cirassomalyn hin an acy CoA (shown nue) emoved om the faty ae chain with each ound oF theccle. I addon tothe molacule of acetyl CoA thats fed ito the TCA cyl, each ound of hefty acid eel produces one NADH and lone ADK tx apaarent fom examination of his seis of reactions ‘thy fats are auch arch storehouse of chemical energy. (Input of mine 98 nte she TCA cel. FIGURE 5.9 The glycerol phosphate shuttle Inthe glycerol phosphate shut, electrons are ansiered rom NADH to dnydronjacetone ‘Phosahate(DHAP) term aleeral-phosahste, whieh ste ter into the mitochondrion, These elections then reduce FAD atthe ner mito chondhal membrane, fring ADH, whien can transfer he elecwons toa carter ofthe elacronsrarspor chain sce Peyidoe Seite et Gycine Leucine i ‘Serne Lysine oar-b-s-con Toots an few con | j scan Pnte Aeace cok NF oe fea Coh <2 he Tosa | = a ioe sence re da ote Qi ate a ‘TCA CYCLE | ae Stemate sends Loa hin scenes ie ee ‘ine vine ® steps that utilize these reduced coenzymes to produce ATP. The ‘overall process can be divided into two steps, which can be summatized as follows Step 1 (FIGURE 5.10). High-energy electrons are passed from FADE, or NADH to the first ofa series of electron carriers that make up the electon-ransport chain, which is located inthe inner ‘mitochondrial membrane. Flectrons pass along the electron-teansport chain in energy-releasing reactions, These reactions ate coupled to ‘energy-requiring conformational changes in electron carriers that ‘move protons outward across the inner mitochondrial membrane. ‘Asa result the energy released during electron transport is stored in the form of an electrochemical gradient of protons across the mem- brane. Eventually, the low-energy electrone are transferred to the terminal electron acceptor, namely, molecular oxygen (Q,), sehich becomes reduced to water. Step 2 (Figure 5.10). The controlled movement of protons back ‘across the membrane through an ATP-syathesizing enzyme pro- Vides the energy required to phosphorylate ADP to ATE. ‘The importance of proton movements during electron trans- port and the formation of ATP was fist proposed in 1961 by Peter “Mitchell ofthe University of Edinburgh, who named it the chemios- ‘motic mechanism, Further analysis of the two steps summarized above will occupy us for much ofthe remainder ofthe chapter.0320 a ection energy +o82v FIGURE 5.10 A summary ofthe process of oxidative phosphorylation Inthe Brat step ofthe proce, subctraies such ea ocirata and succinate se oxdzed gute 57) and the elecvons are arslrred to the coonaynes NAD" or FAD to form NADH or ADH These high-anergy elecrone are thon transfered through 2 ores ofeiectan carer ofthe secon transport chan. The energy eased is ued to anslocate rotons fom the matrict he intermembrane space, establishing a proton elacvochomical ‘grdient wero the inner mitochondal membrane in step 2, the protons move down the elactachemical gradient, tough an ATPsynthesiing complex. The energy stored inthe gradients used 1 syrthasize ATP. These two essential step of exdatve phosphorylation frm the basi of the chemiosmotie mechanism proposed by Pete Michell s 1961 Each pair of electrons transferred from NADH to oxygen by 177 meant of the electron-transport chain releases sullicient energy (0 drive the formation of approximately three molecules of ATP. Each pair donated by FADH, releases suficient energy forthe formation of approximately two molecules of ATP. If one adds up all of the ATs formed from one molecule of glicase completely catabolized ‘by means of glycolysis and the TCA cycle, the net gain is about 36 ATPs (which includes the GTP formed by each round of the TCA cycle, step 16, Figure 5.7). The actual number of ATPs formed per molecule of glucose oxidized depends on the particular activities in Wwiich the cells engaged. The relative importance of glycolysis ver- sus the TCA cycle, that is, of anaerobic versus aerobic oxidative metabolism, in human skeletal muscle function is discussed in the accompanying Human Perspective Review PIA 1. How are the two praducts of glycolysis connected to the reactions of the TCA cycle? 2. Why is the TCA cycle considered to be the central pathway in he energy metabolism of a cell? 3, Describe the mechanism by which NADH produced in the cytosol by glycolysis is able to feed electrons into the TCA cycle. 5.3 THE HUMAN PERSPECTIVE The Role of Anaerobic and Aerobic Metabolism in Exercise Muscle contraction expends large amounts of energy in sliding Actin- ana myosin-containing filaments over one another, ae dis cussed in Chapter 9. The energy that drives muscle contraction '5 derived irom ATR The rate of ATP hydrolysis increases move ‘than 100-oll ina skeletal muscle undergaing maximal contrac- tion compared to the same muscle at rast itis estimated that the average human skeletal muscle has enough ATP available to fuel 8 2-to Second burst of vigorous contraction. Even as ATP 's hydrolyzed, itis important that addtional ATP be produces: ‘otherwise the ATP/AD® ratio would fall and witht the free energy available to fuel contraction. To solve this problem, muscle cals contain a store of creatine phosphate (CrP), one of the com- pounds with a phosphate transfor potential greater than that of [ATP (sae Figure 3.28) and thus can be utiized to generate ATP in the following reaction CrP HAD? > Cr+ ATP ‘Skeletal muscles ypicallyhave enough stored creatine phosphate 0 maintain elevated ATF lovels for approximately 15 seconds, This consumption of creatine by muscle is the reason that body cells have a limited supply of both AvP and crestine phosphate, 'tfollove that intense ot sustainad muscular activty raqurres the formation of adsitional quantities of AT, which must be obtained by oxidative metabolism Human skelatal muscles consist of elongated, contractile calls known az floors. Muscle foere aro of two general types (FIGURE 1: fasttwitch fers, which can contract very rapidly e.g 1540 mseq), and slow-twitch foers, which contract more slowly (c.g. 40-100 msec) Fast-twitch fibers aro se0n under the electron microscope to be neat devoid of mitochondria, indiating that ‘hese cell are unable to produce much ATP by aerobic respira ‘ion, Slow-twtch fiers, on the other hand, contin large numbers fof mtochonatia, These two types of skeletal muscle bers ae utes for different pes of acties, For example, iting waichts ff running sprints depends primanly on fstetwiten fiber, which are able to generate more force than ther slow-twitch counters pars. Fastwitch fers produce nearly all of their AIP anaer- obically as a result of glycolysis. Even though glycolysis only produces about 5 percent as much ATP per molecule of glucose Dordized compared with serobic respiration, the reactions of continued sna2ecning veun oul» eS178 soy 2iolay « S¥aLdvHD oupuoypouny ays pve usenet 8 FIGURE 1. Skeletal muscles conten ic of fast-titch or typeI) ‘bes (darkly stained and slowewtch (or yoe U foes Nightly stared, Souec: Couey of Dunean MacDovgal alyeolysis occur much mare rapidly than those of the TCA cycle and electron transpon; thus, the rate of anaerobic ATP produc: tion i actually higher than that achieves by aerobic resp ration The probiems with producing ATP by glycoiyss are the rapic use of the fibers available glucoss (stored as glycogen) and the pro duction ofan undesirable ond product, lactic aid. Let's cons der this latter aspect further, Recall snat the consinuation of glycolysis raquires the agen eration of NAD*, which occurs by fermentation eage 108). Muscle cals regenerate NAD" by reducing pyruvate—the end product af fgycelysie—to lactic acid. Most of the lactic acid diuses aut of the active muscle cel's into the bloed, where is carried to the Iver and converted back o glucose, Glucose produced nthe lver is relansed into the blood, wnere can be retumad tothe active muscles to continue to fuel the high eves of glycolysis. However, the formation of latie acids associates with a drop in pH within the muscle Ussue (from about pH 7.00 to 6.35), which may pro- duce the pain and cramps that accompany vigorous exercise. Increased acidity, along with accumulation of phosphate and conformational changes in myosin protein, contrbutes to loss of ‘contrac strangth in muscles curing pralonged exertion I, instead of tying to use your muscles to lift weights or run sprints, you were to engage in an aorobie exercise, such as bicycling oF fost walking, you would be able to continue to per form the activity for much longer periods of tme without fesling muscle pan oF fatigue, Aerobic exercises, as ther rame impos, 218 designed to allow your musclas to continue to perform acr obically, that's, to contique to produce the necessary ATP by clacton transfer and oxidatve phosphorylation. Asrobic axer ses depend largely on the contraction of the slow-twitch fibers (ofthe sialetal muscles. ARhough thace Soars generate less force, they ean continue to function for long periods of time due to the continuing aerobic production af ATP without production of late aid Acrabic exercise is initally uoled by the glucose molecules stored as glycogen in the muscles themselves, but after @ few minutes, the muscles depend mare and more on fre faty acids released into the blood from adipose (fat) tissue. The longer the period of exercise, the greater the dependency on fatty acids, [After 20 minutas of vigorous aerobic exercise, tis estimated that about $0 percent of he calories being consumed by the muscles ‘are derive from fat Aerobic exercise, such as jogging, fast walking, Swimming, or bicycling, is one of the best ways of reducing the bodys ft contort “The ratio 0! fast-witeh to slowetwitch fibers wares from one particular muscle to another. For example, postural muscies in the back that alow 2 person to stand consist f a higher proportion of slowstwiten bors than arm muscles used to throw or ft an ‘object. The precise ratio af fast-twitch to slovatwiten bare in @ particular muscle is genetically determined and quite variable from person to person, allowing a particular indvidual to excel in corain types ef physical actvitiae. For example, world-class sprinters and weight liters tend to have a higher procortion of fGstenwiteh fivers in thet muscles than long-distance runners. In addition, training for sport such as weight iting leads to 9 le proportionate enlargement of the fat-twtch fib “The muscle tissue of the heart must aso increase its lovel of activity during vigorous exercise, but unike skeletal muscle tissue, the heart can only produce ATP by aerobic metabolism. In fact, aporoximately 40 percent of the cytoplasmic space of @ human heart muse cals occupied by mitochondria, 5.4 Oxidative Phosphorylation the Formation of ATP Mitochondria aze often described as miniature power plants. Like power plants, mitochondria extract energy from organic materials and store it, temporarily, inthe form of electrical energy. More spe cifically, the energy extracted from substrate i utilized to generate an ionic gradient across the inner mitochondrial membrane. An ionic gradient across a membrane represents a form of energy that can be tapped to perform work We saw in Chapter 4 how intestinal cells ulize anionic gradient across their plasma membrane to tans- port sugars and amino acids out ofthe intestinal lumen, just as nerve cells use a similar gradient to conduct neural impulses. The use of ionic gradients asa form of energy currency requires several compo- nents, incuding a system to generate the gradient, a membrane capable of maintaining the gradient, and the machinery to utlize the gradient to perform work. Mitochondria utilize anionic gradient across their inner mem- ane to drive numerous energy-requiring activites, most notably the synthesis of ATP When ATP formation is driven by energy that 4s released from electrons removed during subsrate oxidation, the process is called oxidative phosphorylation and is summarized in Figure 5.10, Oxidative phosphorylation can be contrasted with substrate level phosphorylation, as discussed on page 105, in which [ATP is formed directly by transfer ofa phosphate group from a sub- strate molecule to ADP, According to one estimate, oxidative phos- phorylation accounts for the production of more than 2x10" molecules (> 60 kg) of ATP in our bodies per day. Unraveling the basic mechanism of oxidative phosphorylation has been one ofthe crowning achievements inthe field of cell and molecular biology: fling in the remaining. gaps continues to be an active area of research. To understand the mechanism of oxidative phosphoryl tion, itis frst necessary to consider how substrate oxidation is able to release fee energy.Oxidation-Reduction Potentials Tone compares a variety of oxidizing agents, they can be ranked in 2 series according to their affinity for electrons the greater the aft, the stronger the oxidizing agent. Reducing agents can also be ranked according to their affinity for electrons: the lower the affinity (the more easly electrons ae released), the stronger the reducing agent. To put Uisinlo quantifiable terms, reducing agents are ranked according to clectron-transfer potentiak thore substances having «igh electon- transfer potential, such as NADH, ae strong reducing agents, whereas ‘hose with a low electron-transfer potential, such as HO, are weak reducing agents. Oxiizing and reducing agents occur as couples, such as NAD" and NADH, which dif in their number of electrons. Strang reducing agents are coupled to weak oxidizing agents, and vice versa. Tor example, NAD’ (ofthe NAD'-NADH couple) isa weak oxidizing agent, whereas O; (f the O,-H0 couple) ia strong oxidizing agent. Because the movement of electrons generates a separation of charg, the affinity of substances for electrons can be measured by Instruments that detect voltage (FIGURE 5.11). What is measured for a given couple is an oxidation-reduction potential (or redox potential) relative to the potential for some standard couple. The Standard couple has arbitrarily been chosen as hydrogen (H'—H), ‘swith free-energy changes, where the standard fre-energy change, |AG?, was used, a similar assignment is employed for redox couples. ‘The standard redox potential, for a given couple is designated as the volage produced by half cell (with only members of one cot ple present) in which each member of the couple is present under standard conditions (es in Figure 5.11). Standard conditions are LOM for solutes and ions and latm pressure for gases (eg, Hat 25°C. “The standard redox potential forthe oxidation-reduction reaction involving hydrogen (2 H’ +2 electons ->H,) is 0,00V. Table 5.1 gives the redox potentials of some biologically important couples. “The value forthe hydrogen couple inthe table is not 0.0, but -0.42V. ‘This number represents the value when the concentration of H is 10” M(pHi of 7.0) rather than 1OM(pH of 0.0), which would be of, Volueter @ [ KO: bidge l litle physiologic use. When calculated at pH 7, the standard redox potential is indicated by the symbol Ej rather than Ey, The assign ‘ment of sign (positive or negaive) to couples is arbitrary and varies among different disciplines, We will consider the assignment in the following way. Those couples whose reducing agents are better donors of electrons are assigned more negative redox potentials, For example, the standard redox potential for the NAD"-NADIL couple is-0.32V (Table 5.1). Acetaldehyde isa stronger reducing agent than NADH, and the acetate acetaldehyde couple has a standard redox potential of 0.58 V. Those couples whose oxidizing agents are better electron acceptors than NAD", that is, have greater affinity for elec- ‘ons than NAD', have more postive redox potentials. Just as any other spontaneous reaction is accompanied by a loss in free energy s0 too are oxidation-reduction reactions. The stand= aed free-energy change during a reaction of the type Aion * Biot) = Avet* Bron can be calculated from the standard redox potentials ofthe two cou ples involved in the reaction according tothe equation, AG" =-nF AF, ‘where ris the numberof electrons transfered inthe reaction, Fis the Faraday constant (23.063 kcal/V*mol), and Bsis the diference in volts between the standard redox potentials of the two couples. The greater the difference in standard redox potential between two cou ples, the farther the reaction proceeds under standard conditions to ‘he formation of products before an equilibrium state is reached. Lets consider the reaction in which NADH, a strong reducing agent, is oxidized by molecular oxygen, a strong oxidizing agent. NADH+10,+H* >H,0+NAD* ‘The standard redox potentials ofthe two couples can be written as 40; +2H' 426 91,0 B,=+0.82V NAD" +2H" +2e7->NADH4H* av ‘TABLE 5.1 Standard Redox Potentials of Selected Halt Reactions Reference halt-cell_ Sample hatcell FIGURE 5.11 Measuring the standard oxidation-reducton (redox) potenti, The sample hall contains the oxiied and reduced embers ofthe couple, bath present at 1M. The reterence halal contains 21M solion of” that sin equilorum with Mycogen gas at atm pressure. fn slctie exes formed by connecting the hall-cals rough 2 ‘omer and a al bridge. electrons fon prferentally fom the sample halal tothe vterence hace, then the tandare redox porenal(f) oF the somple couple a neta: the electron fis the oppo ‘recton, the tancard redox potential ofthe sample couple postive, The sa Orga, wien conse of atrateg KC solton, provides 3 path for counterions to move between thehalels and maintain elec neutral inthe to compartments, Electrode equation Succinate +00, 421426 = s hstgiarate 1,0 Actaie +H 2 ctl 2H 2¢ wal; teKeogtarse +00, 421427 = wocteae Cyne 12H" 2 22 yaeine NAD'2H'426¢°SONADHH EE NADP" 42H +26 <= NADPH Aedaldehyle +210 426 =ehanal Pyruvate 2H +26 slate Onloaeate+ 2142 malate FAD 2172.6 = FADH, in avoprotains) Ubiguloone+2 H+ 2 = biguoo! 2eytochome by + 22 = coca By 2eyrociome jog +2 = 2eyrocheome ea 2eytochome tag *2€ wo 2 gochey Jo,sans2r= #0 179, {Ly Jo wonesiog 98 u voneRiousrous ONaEPO + ¥S180 ‘The voltage change forthe total reaction is equal to the difference between the two F values (AF,) a which is a measure ofthe free energy released when NADH is oxi- dized by molecular oxygen under standard conditions. Substitting this value into the above equation, (-2)(23.068 keal/V-mol) 14 V) 52.6 kcal/mol of NADH oxidized 20.82 V-(-0.32V)=1.UV ac the standard fee-energy difference (AG*) is 52.6 kealimol. As with other reactions, the actual AG values depend on the relative concentration of reactants and products (oxidized and reduced ver sions of the compounds) present in the cell at a given instant. Regardless, it would sppear thatthe drop in fee energy of pair of clectrons as they past from NADH to molecular oxygen (AG" =~52.6keal/mal) shold be sufficient o drive the formation of several molecules of ATP (AG"=+7.Skeal/mol) even under conditions in the cel, where ATP/ADP ratios are mich higher than those at standard conditions. The transfer ofthis energy from NADH \o.ATP within the mitochondrion occurs in a series of all, energy releasing steps, which willbe the primary topic of discussion in the remainder ofthe chapter. lecuons are tasfered to NAD" (or FAD) within the ito chondtion from several substrates ofthe TCA cyte, namely isocivate, avketohstarate, malate and succinate (reaction Mi, 15-16 18, and 17 of Figure 57, respectively). The fr three ofthese intermediates have redox potentials of relauvely high negative values (Table 5.1)— sulicietly high to transfer electrons to NAD" under conditions that ‘preva nthe el? Ta contrast, the oxidation of succinate to fn ‘which as a more positive redox potential, proceeds bythe reduction Of FAD, a coenzyme of greater elecion affinity than NAD oupuoypainy ox: pve uaneudtoy na0H0y » 5 Baldy Electron Transport ive ofthe nine reactions illustrated in Figure 5.7 are catalyzed by dehydrogenases, enzymes that transfer pairs of electrons from sub strates to coenzymes. Four of these reactions generate NADH and ‘one produces FADH,, The NADH molecules, which are formed in ‘the mitochondrial matrix, dissociate from thei respective dehydro- {genases and bind to NADH dehydrogenase, an integral protein of the inner mitochondrial membrane (see Figure 5.17) Unlike the other enzymes of the TCA cycle, succinate dehydrogenase, the enzyme ‘that catalyzes the formation of FADH, (Figure 57, reaction 17), isa component of the inner mitochondrial membrane, In either case the high-energy elecizone associated with NADH or FADU, are tcanslerred through a series of speciic electron carriers hal constitute the electronstransport chain (or respiratory chain) of the inner ‘mitochondrial membrane, as indicted in Tale 5.1, the standard rede potential (2) ofthe malonate nla couple more postive than hat ofthe NAD"-NADI couple Ths, th ond op of malate to grlacette has positive AG” rene 1, Figure 7) and cn proceed’ the oration ef naloaetate oly when the ato produto reacts Flap below tht of tanda onde The AG othe econ skp negative by lone of very low onleuceateconceneatons, which i pose Because the ew reaction nthe cele reaction 12 Fgere 57) highyexergoniand ete ofthe major sate-coatling enya fhe TCA ye Types of Electron Carriers ‘The electyon-transport chain contains five types of membrane bound electron carriers: flavoproteins, cytochromes, copper atoms, ubiquinone, and iron-sulfur proteins. With the exception of wbiqui none, all ofthe redox centers within the respiratory chain that accept and donate electrons are prosthetic groups, that is, non-arino acid ‘components that are tightly associated with proteins + Flavoproteins consist ofa polypeptide bound tightly to one of, ‘wo related prosthetic groups either flavin adenine dinucleotide (EAD) or flavin mononucleotide (FMN) (FiGURE 5.122). The prosthetic groups of favoproteins are derived from riboflavin {vitamin 8), and each is capable of accepting and donating two protons and two electrons. The major flavoproteins ofthe silochondsia are NADH debydrogenaie ofthe electron-ransport chain and succinate dehydrogenase of the TCA cyee {© Cytochromes ae proteins that contain heme prosthetic groups (Such as that described for nyoglbin on page 86). The ron ator of a heme undergoes reversible transition between the Fe” and Fe® oxidation sates asa result ofthe acceptance and los of single electron (Figure 5.128). There are three distinct cytochrome ‘ypesmab, and e—present inthe eleeton-transport chain, which Afr fom one another by substistons within the heme group (indicated by the blue shaded portions of Figure 5.128) «© Three copper atoms, all located within a single protein complex ofthe inner mitochondrial membrane (see Figure 5.20), accept and donate a single electron as they alternate between the Gu!" and Cu states. {© Ubiquinone (Ug, or coenzyme Q) is lipid: soluble molecule containing along hydrophobic chatn composed af ve-cazbon isoprenoid units (Figure 5.12), Like the Mavoprotens, each ‘ubiquinone i ble to accep and donate two electrons and two protons, The paraly reduced molecule isthe fre radical Ubisemiguinone, and the fell reduced molecule is ubiquinol (UQH,). UQUQH, remains within the lipid bilayer of the ‘membrane, where itis capable of rapid lateral diffusion, « Iron-sulfur proteins aze iron-containing proteins in which the iron atoms ae not located within a heme group but instead are linked to inorganic sulfide fons as part of an iron-sulfur center. ‘The most common centers contain either two or four atoms of son and sulfur—designated [24-28] and [48e-4S]—linked to the protein at cysteine residues (FIGURE 6.13). Even though a single center may have several iron atoms, the entire complex is capable of accepting and donating only a single electron. The zedax potential of an iron-sulfur center depends on the hyde: phobicity and charge ofthe amino acid residues that make up is local environment. As a grovp, iron-sulfur proteins have potentials ranging from about ~700mV to about +300 mV, corresponding lo # major portion of the span over which cleetzon transport occurs, More than a dozen distinct iron-sulfur centers have been identified within mitochondria The carriers of the eleciron-transport chain are arranged in ‘order of increasingly positive redox potential (FIGURE 5.14). carrier is reduced by the gain of electrons from the preceding carrier in the chain and is subsequently oxidized by the loss of electrons to the carrier following it, Thus, electrons are passed from one carrier to the next, losing energy as they move “downbill” along the chain “The final acceplor of this electron “bucket brigade” is O,, whichot Mee ney ly 0 Vb toe baorot xc ‘arene apg eM ya i cit A bay io FIGURE 5.12 suuctrs of the emai ec Colne and reduced forms of hee Sines at {ypes of electron carries. [a MN of of ewee NADH dehysrogenae, theme goupototedramec,andi@ubqusene 7 Courgireah Theme geupsotte gy cat tae hs vatouseecromer ofthe lcton- ey 1 trarepor chain erin the sbattions ORES By m0 en the porpnyen ngs ndcte by he cy tue shading! ana typeof kage f the ve proten Cyocromescan seep oniyone aes matt Slecuon, wheres FAN and quinones can Pvt a Sccepttwo electrons ard we protein fezenive reactors, as shown FAD diene from FMN innoving an adenosine group bonded the pospete -o ors oo ors FIGURE 5.13 lron-sulfur canters Souci of (2-25) (a) and | [4Fe5} (8) oresulur center Both types ef rer-slur centers are joined to the poten by linkage toa sulfur atom {shown in orange ofa cysteine resi. Inorgan'e nln (5) 378 shown in yellow Both ype of iromsillur centers accept anya single election, whose carge is ditouted {among he various ion atoms. = 181 3 ota i <= pore “by Jo wonewiog 94 cs tte he 4s » 04 a 2 o2ey BE ea 20 ao, I ozzy fp.02 oFhcaye vot oss 408 came 408 0 FIGURE 5.14 The arrangement of several carirsin the electron- ‘anspor chan. The diagrom i usttater te approximate redox potenti! of the caters end the decline in fee energy as election pass move along the respiratory nan t0 molecular oxygen. The merous ten-sur enters 8 notincested in hie Faure forthe sake of simply. Ae dacvsed in the folowing section, each ofthe thee electron wansers hat re denoted by red arows yield sficent energy to move protons across he inner rmtechendal membrane, whieh in tom provides the energy required 0 generate A from ADP. Source: Based ona drawing frm A. L Lbninger, Biochem, 22,1975,182 accepts the energy depleted clctrons and is reduced to water. The specific sequence of carriers that constitute the electron-transport cain was worked out by Brition Chance and co-workers at the University of Pennsylvania using a variety of inhibitors that blocked electron transport at specific sites along the route, The concept of. these experiments is depicted by analogy in FIGURE 5.15. In each case, an inhibitor was added to cells, and the oxidation state of the various electron carriers in the inhibited cells was determined. This determination can be made using a spectrophotometer that meas ures the absorption of light ofa sample at various wavelengths, This ‘measurement reveals whether a particular carrer isin the oxidized or reduced state In the case depicted in Figure 5.15, addition of rote- none blocked electron transport ata sit that left NADH and FMNH, in the reduced state, and left cytochromes®, c,and a in the oxidized sate. This result indicates that NAD and FMN ate situated “upstream” ofthe block. In contrast, an inhibitor (eg, antimycin A) that actsbetween cytochromes b and c would eave NADH, FMNH., (QU; and cytochrome b in the reduced state. Thus, by identifying reduced and oxidized components in the presence of differen inhib ‘tors, the sequences ofthe carriers could be determined, “Te tendency for electrons to be transferred from one carrier to the next depends on the potential difference between the two redox centers, but the rate of transfer depends on the catalytic activities of, the proteins involved. This distinction between thermodynamics and kinetic is similar to that discussed on page 90 regerding the activity of enzymes, Studies indicate that electrons may travel con siderable distances (10-20A) between adjacent redox centers and that electrons probably flow through special “tunneling pathway consisting ofa series of covalent and hydrogen bonds that stretch oupuoypainy ox: pve uaneudtoy na0H0y » 5 Baldy Poi of ihibton Percent each ectron carers reduced FIGURE 5.15 Experimental use of inhibitors to determine the squance of carers inthe electron-transport chain, n this hyo analogy, eaten’ of mitacrondta with the ihibitor rotenone aves thera {aes on the upsteam (NADH sid of the pont of inhibition nthe uly recuced mate and thors carries onthe downtraa (0; 49 ofnhoiton nthe ily oxidiced state, Comparizon ofthe elect of overal inhibitors revealed the order of the carers within the chai, Sounce Based on a drawing rom AL Lehninger, Biochemisty, 26, 1975. FIGURE 5.16 Electron-tunnsling cytochrome «peroxidase complex. The heme group of blu, and tet of etochromec peroxidase (whichis nota rlochondaa electro-transport chan but provides an analogous election Scceptorfor which ngheselston crystal scares are aval is eo Several desined patiays yell) exit for he movement of elevors rom ecrons th ups. ftean be ‘of slacron trarsler have boon proposed. ‘several amino acd resus stuted between the heme noted that other moe Sou: By Jey J, Regan an N. Onuche, om David Baatan ot Science 2581741, © 1992. Reprinted wth permission om AAAS, across parts of several amino acid residues, An example of such @ pathway involving cytochrome cis shown in FIGURE 5.16 Review PI 1. Describe the steps by which the transport of electrons down the res ratory chain leads to the formation of a proton gradient. 2. Of the five types of electron carriers, which has the smallest molecular mass? Which has the greatest ratio of ron atoms to electrons carried? Which has a component that is located outside the lipid bilayer? Which are capable of accepting protons and ele and which only electrons? 3. Describe the relationship between a compound’ affinity for electrons and its ability to act as a reducing agent What is the relationship between the electror transfer potential of a reducing agent and the ability the other member ofits couple to act as an oxidizing agent? 4, Lock at Figure 5.12 and describe how the serniquinene slates of ubiquinone and FMN are similar 5 Electron-Transport Complexes When the inner mitochondrial membrane is disrupted by detergent, the vatious electron caries can be isolated as part of four distinct, asymmetric, membrane-spanning complexes, identified as complexes 1, I IL, and IV (FIGURE 5.17). Each ofthese four complexes can be assigned a distinct function in the overall oxidation pathway. ‘Two components ofthe electzon-transport chain, cytochrome c andMatrix Comtex Complex it Non ‘ytectrome by erydrogenase Subunits (Wanna) [ONA encodes 7 1 DNA ercoced| 38 10 ‘ora % is oecilar mass (Da) 980,000 240,000 FIGURE 5.17 The elecvonvtransport chain ofthe inner mitochondrial memb tnd to other cartes labiquinane and cytochrome c that ar independent Leo jai it Complexil Comex Succrate Cytochrome ¢ ehyerogenese oxidase ° 3 4 10 a a 5 200,000 ‘The reepirtory chain consis of fur complenes of electron carers Electrons enter the chain fom ether NADH va complex) or FADH2 spas {apa of complx il Flectons are partes fom eer complex orto ubiquinone (UO), which extra 9 pool within the lpia bilnyr Cacrone ae subsequent pessed fom reduced ubiquinone ublaune! o compiex land Etecvons ar sarsfered rom cyachvame cto complex IW (orechrome oxida then to the peripheral protein epochremee, which thought tebe mabe 6 and then 1.0; fa frm H,0. The ses of proton tansocaion fam the matic to the ororel ae ineieted, The pects number of proton anegcate at each ste remains conrovril the number inated i ganar cantons Keepin mind that the proton rumbar shown are the goneated by each pa of eacron transporte, which suficent 0 reduce only ane-hl of a5 O> rolecule (The tanslocation of protons by eompex Il occurs by way ofa Qc ele, which ia seres of sequential nterconversions of uiquinone and ubiquino ‘coupled to movement of protons across the memerane, The O eco an be vided ino sW0 steps, e367 of which load to the eleaso of to proton int he tesol) ubiquinone, are not part of any ofthe four complexes, Ubiquinone ‘occurs as a pool of molecules dissolved in che lipid bilayer, and cytochrome c is @ soluble protein in the intermembrane space. Ubiquinone and cytochrome ¢ are thought to move within or along the membrane, shuttling electrons between the large, relatively immo- bile protein complexes. Once within one of the large, muliprotein complexes electrons travel along defined pathways (ofthe type ilus- trated in Figure 5.16) between adjacent redox centers whose relative Positions are fixed in place. ‘When NADHs the electron donor, electrons ener the respiratory, chain by means of complex I, which transfers electrons to ubiquinone, generating ubiguinol (Figures 5.12 and 5.17). Unlike NADH, which can diffuse away from its soluble dehydrogenases, FADH, remains covalently bound to succinate dehydrogenase, a component of complex Il. When FADH is the donor, electrons are passed directly to ubiquinone, bypassing the upstream end of the chain, which has oo negative a redax potential to accept the less energetic electrons of the flavin nucleotide (Figure 5.14) If one examines the redox potentials of successive carriers in igure 5.14, itis apparent that there are three places in which the transfer of electrons is accompanied by a major release of free energy. Each ofthese coupling ites, as they ae called, occurs between carri= cers that ae part of ane ofthe three complexes, I, IT, and IV. The free energy, which i released as electrons are passed across these three site, is conserved by translocation of protons from the matrix across the inner membrane into the intermembrane space, These three protein complexes are often described as proton pumps. Unlike other ions (eg, Na" oF CI’) that have to diffuse by themselves across the entire distance being traversed, H" ions can "hop" througls a channel by exchanging themselves with other protons present along the pathway (as illustrated in Figure 5.196). Such proton-conduction pathways (or “proton wires") can be identified because they consist of strings of acidic residues, hydrogen-bonded residues, and/or ‘rapped vater molecules. The translocation of protons by these three clectzon-iransporting complexes establishes the proton gradient that drives ATP synthesis. The ability of these remarkable molecular machines to aet as independent proton-trandlacating units can be demonstrated by purifying each of them and incorporating them individually into artificial lipid vesicles. When given an appropriate tlectzon donor, these protein-containing vesicles are capable of accepting electrons and using the released energy to pump protons across the vesicle membrane (see FIGURE 5.18). Great strides have beer made in the past few yeats in elucidat ing the molecular architecture ofall the protein complexes of the inner membrane. Researchers are no longer asking what these pro- ‘eins look like but are using the wealth of new structural information tounderstand how they work. We will briefly examine the mamma- lian versions of each ofthe four electron transport complexes, which together contain about 70 different polypeptides. The bacterial ver- sions are considerably simpler than their mammalian counterparts 183 ‘sovojsuo) uodsue-vonsen3 + 65184 vevpuoyronn 942 ave voxeudsoy 290104 « SEaLEvHD. pH eletode Oytetrome exitase ‘tectrome c Liposome membrane Inside aaueous phase FIGURE 5.18 Experimental demonstration that cytochrome oxida i ' proton pump. When purfied cytochrome oxidee is incorporated it ‘he atic blayer fa ipasome, the medium becomes sede fellowing she aaditon of reduces cytochrome c. This inciate that a electrons 38 transfered fom cytochrome cto cytochrome oxisse, and Op seeing reduced to water protons ae being tansicated from te compartment within the vere tothe extemal medium, This experiment was orginally Caried out inthe late 1960s by Marten Wkstrom and colleagues at the Unvarsty of Hlsne in Finland Source: Raprinted with permission from Macmillan Publier td: M. Verthovaly et, Nate 400:487, 1999, comin 199, and contain far fewer subunits, However, the additional subunits of plex rather than electron transport. In other words, the basic process ff electron transport during respiration has remained virtually ‘unchanged since its evolution billion of years ago in our prokary- otic ancestors Complex | (NADH Dehydrogenase) Complex Tis the gateway tothe ceetron-transport chai, eatalyaing the transfer of a pir of electrons ftom NADH to ubiquinone (UQ) to form ubiquinol (UQH,). The mammalian version of complex lis a huge L-shaped conglomerate, containing 45 diferent subunit that onstitste « molecular mas mass of nearly one million daltons. As {indicated in Figure 5.194, roughly half ofthe complex consist of & hydrophilic domain that projects into the matrix, with the remsi- ing hydrophobic portion of the complex embedded in the mem brane, Seven ofthe subunits—all hydrophobic, membrane-spenning polypeptides—are encoded by mitochondrial genes and are homo fogues of bacterial polypeptides. Together, the hydrophilic and sembrane-embeddled portions ofthe complex carry out the two di- ferent activities that are required of the electon transport chain: electron transfer and proton transocation. All of the components required for electron transfer reside ‘within the hydrophilic portion ofthe complex (FIGURE 5.198). The ‘the mammalian complexes do not contain redox centers and are ont ‘thought to function in either the regulation or assembly ofthe com ¥ pe w FIGURE 5.19 Structure anda proposed mechanism of action of complex ofthe respiratory chain (s)Cystalsvuctre ofa bactefl frm of complex The peripheral hyaropniie domain ana the namely, FAN (magenta sphere) the seven Fe-S centers (ad saheres), ad the quinone binding st (0}—re nailed, Heli HL of the membrane domain, “hich i proposed to act a connecting ro or piston, ie seen running roughly parallel to the alane of the membrane. (bl Proposed mechanism by which ‘loctan transfer inthe peripheral nysophilic arm is eouped by conformational ehange® to proton tansocation aero the membrane domain. Reduction of Ubsgunone at the Ot leads ta 2 rightward movement of hal HL nceated by the upper blac arr inthe reduced version), which eases conforma ‘ional changes inthe vansmemirane heices, leading tothe movement o protons acoss the membrane. Crucial charged residues in subunits af the rmemorane domain are indieted by ccos Red cies represent unprotonated gitar ac recs, anole crcoerapesentproansted lysine eaves The protonated and unprotonsted forms af these resus se soen as empty cls, reepectvely. The movement of protons between these residues, which is dive by eonformatonal changes inte wansmembrane helices, lorms te basis fr proton vanslocaton, Source: From RouslanG, Eftemow and Laon A, Sazanoy, Nature 476:414, 2011; eprinted by permission trom» Macmillan Publishers Lidchain of electron ers begins with an PMN. containing Davopro tein that oxidizes NADH atthe tip ofthe giant complex. Electrons are subsequently transferred through seven distinct iron-sulfur centers within the body of the hydrophilic domain, ending withthe wansfer of electrons toa protein-bound ubiquinone molecule situated near the membrane boundary (An eighth Fe-S cluster and possibly ‘second quinone molecule are also present but not apparently inthe direct path of electron transfer) The passage ofa pair of electrons from NADH to ubiquinone is thought to be accompanied by the "movement of four protons from the matrix into the intermembrane space. The determination of the structure of complex 1 (Figure 5.192) has led to a proposal that explains how electron transport through the peripheral arm ofthe complex can drive proton translo- cation through the distant membrane-embedded portion, It can be seen in Figure 5.19b that the membrane embedded domain of the complex contains very long a helix, denoted HI. (shown in purple), that is oriented roughly parallel to the membranes surface. This helix, which rans nearly the entire length of the membrane domain, {sin direct contact with three discontinuous transmembrane helices (show in purple, blue, and olive green). Each ofthe discontinsous helices is part of a half chansel. Each pair of half channels is con nected by a network of polar amino acid side chains to provide & potential whole channel for the translocation of protons across the ‘membrane. fourth proton-conducting channel ie probably located near the interface with the soluble domain as shown in Figure 5.198. sposed thatthe movement of electrons to ubiquinone induces 1 conformational change in the complex that causes the lateral ‘movement of helix HL (toward the right in Figure 5.198). This in turn leads to the tilting movement of the transmembrane helices, which changes the ionic environment of certain protoa. transferring residues, leading to the movement of protons across the membrane (black downward arrows). The authors of this proposal compare the ‘mechanism of action of complex Ito that of a steam engine, with helix HI acting as the piston that drives the movement of the down- stream elements of the machinery 1k ean be noted that complex I dysfunction has been linked to certain neurodegenerative disorders, as discussed in the Human Perspective on page 195, Complex Il (succinate dehydrogenase) (Complex Il consists of four polypeptides: two hydrophobic subunits that anchor the protein in the membrane and two hydrophilic subu nits that comprise the TCA cycle enzyme, succinate dehydrogenase. Complex 11 provides a pathway for feeding lower-energy electrons (ones dose to mV} from succinate to EAD to ubiquinone (Figures 5.14 and 5.17). The path from FADH, at the enzyme catalytic site to ubiquinone takes the electrons over a distance of 40 A through three tron sulfur clusters. Complex If also contains a heme group, whichis thought to attract eseaped electrons, thereby preventing the forma tion of destructive superoxide radicals (page 34), Flectron transfer through complex I is not accompanied by proton translocation Complex Ill (cytochrome be;) Complex III catalyzes the transfer of electrons from ubiquinel to cytochrome c, Experimental determinations suggest that four pro- tons are translocated across the membrane for every pair of electrons lransferred through complex IIL. The protons are released into the intermembrane space in two separate steps powered by the energy released as a pair of electrons are separated from one another and ‘passed along diferent pathways through the complex. Two protons are derived rom the molecule of ubiquino! that entered the complex. Two additional protons are removed from the matrix and translo- cated across the membrane as part of a second molecule of ubiqui- nol. Three of the subunits of complex Ill contain redox groups cytochrome b contains two heme b molecules with diferent redox potentials, cytochrome cand an con sulfur protein. Cytochrome bis the only polypeptide ofthe complex encoded by a mitochondrial gene Complex IV (cytochrome ¢ oxidase) The fina step of electron transport in a mitochondrion isthe succes- sive transfer of electrons from reduced cytochrome ¢ to oxygen according o the reaction Doe 42H 420, 92 e7t O* +0 To reduce a whole molecule of 0, A eyt HAH +0, 94 eyt 2 +2 HO The reduction of O, is catalyzed by complex IV, a huge assembly of polypeptides referred to as cytochrome oxidase, Cytochrome oxidase was the first component of the electron-transport chain shown to act asa edox-diven proton pump, This was demonstrated in the experiment depicted in Figure 5.18 in which the purified enzyme vas incorporated into vesicles containing an artificial lipid bilayer (liposomes). Addition of reduced cytochrome cto the medium was accompanied by the expulsion of H ions from the vesicles, which was measured as a drop in the surrounding pH. Whether inside a liposome or the inner mitochondrial membrane, translocation of protons is coupled to conformational changes generated by the release of energy that accompanies the transfer of electrons. For every molecule of O; reduced by cytochrome oxidase, eight protons are thought to be taken up from the matrix. Four ofthese protons are consumed in the formation of two molecules of water as indicated above, and the other four protons are translocated across the mem brane and released into the intermembrane space (Figure 5.17) Consequently, the overall reaction can be written: Ate” 8 Higa $0; 94 G78” 42044 Hoy Humans produce about 300 ml of “metabolic water” from this reac- tion per day. Tt ean be noted that a number of potent respiratory poisons, including carbon monoxide (CO), azide (N7), and cyanide (CN), have their toxic effect by binding to the heme ay site of cytochrome oxidase. (Carbon monoxide also binds to the heme group of hemoglobin.) Cytochrome oxidase consists of 13 subunits, three of which are encoded by the mitochondrial genome and contain al four of the proteins redox centers. A major challenge for investigators is to explain how carriers that are only able to transfer single electrons can reduce a molecule of Oto two molecules of H,O, a process that requites four electrons (together with four protons). Most impor- lanlly, the process must occur very efficiently because the cell is 135, ‘ox9}8uu09 sodsuei-vaa2ay186 dealing with very dangerous substances; the “accidental” release of| partially reduced oxygen species has the potential to damage virta ally every macromolecule in the cell (page 34) “The movement of electronsbetween edaxcentersofcytochrome ‘oxidase is shown in FIGURE 5.20, Electrons are transferred one at 2 time from cytochrome c through a bimetallic copper center (C4) of, subunit Il toa heme (heme a) of subunit 1. From there, electrons are passed to a redox center located in subunit I that contains a second hheme (heme as) and another copper atom (Cu) situated less than 5A apart. Acceptance of the first two electrons reduces the as-Cus, binuclear center according to the reaction Fe scape te" ‘Once the binuclear center acepts its second electron, an cil binds tothe enter. The OO double bond is then broken as the O atoms accepta para lectons from the reduced a-Cug bin clear center, likly forming a reactive peroxy anion OF- FeO, * ‘ocu'* oupuoypainy ox: pve uaneudtoy na0H0y » 5 Baldy In subsequent steps the binuclear center accepts two more electrons from cytochrome c and four protons from the matrix, which leads tothe formation of two molecules of wetr, or each proton removed Intermerane soace Membrane ant au Translocttion Substrate wari FIGURE 5.20 A summary of cytochrome axidateactiviy. A model showing te fow of electrons trough the lout eax centers of etocive ‘nies, on atoms are shown re ephare, copper atoms as yellow spheres. Electron are though to be passed ane ate fom etectrome © to the dimeric caper center Cu), then on tothe fon athe heme grup ne onto the binuclear redox center consisting ofa wheres gro of eftochrone a) und copper ton (igh redex centers ar neta Biophysics ACTA he sttures and suggested orientations Source: Reprinted From M. Wikatrom et al, Siac 1459'515, 2000, with permission fom Elsvir) from the matrix, an excess negative charge (in the form of an OH) fs left behind, thus contributing directly tothe electrochemical gra- dient across the inner mitochondrial membrane “Asnoted above, protons taken up from the matrix are utlized in two very different ways. For every molecule of O, reduced to 2 H,0 by cytochrome oxidase (1) four H" ions are consumed in this chem- ical reaction and (2) four additional Ht ions are translocated across the inner mitochondrial membrane, The first four protons can be referred to as “substrate” protons and the latter four as “pumped protons. Movement of both groups of protons contributes to the electrochemical gradient across the membrane ‘With the publication ofthe three-dimensional crystal structare of cytochrome oxidase, it became possible to search for pathways through which substrate and pumped protons might move through the huge protein Researchers have identified potential proton con enzyme—bound ATP +#,0 can occur spontaneously without the input of energy. This does rot mean that ATP can be formed from ADP without energy expenditure, Instead, the energy is required for the release of the Lightly bound product from the catalytic site, rather than the phosphorylation event ise . Each active site progresses successively through three distinct conformations that have different affinities for substrates and product. Recall thatthe F, complex has three catalytic sites, one ‘on each ofthe three subunits. When investigators examined the properties of the three catalytic sites in a static enzyme (one that ‘was not engaged in enzymatic turnover), the diferent ites exhib sted different chemical properties. Boyer proposed that, at any fone time, the three catalytic sites are present in different conformations, which causes them to have different affinity for nucleotides. At any given instant, one site is inthe “loose” or {conformation in which ADP and Pare loosely bound: a second site i in the “tight” or T conformation in which nucleotides (ADP+P, substrates, or ATP product) are tightly bound; and the third site isin the “open” or O conformation, which, because i has a very low affinity for nucleotides, allows felease of ATP ‘Although there were differences among the three catalytic sites in a static enzyme, Boyer obtained evidence that all of the [ATP molecules produced by an active enzyme were synthesized by the same catalytic mechanism. It seemed, in other words, that all thee of the catalytic sites of the enzyme were operating identi- cally. To explain the apparent contradiction between the asym- retry ofthe enzyme’ structure and the uniformity ofits catalytic mechanism, Boyer proposed that each ofthe three catalytic sites ‘passed sequentially through the same L, T, and O conformations (see Figure 5.27) | ATP is synthesized by rotational catalysis in which one part of| the ATP synthase rotates relative to another part. To explain ‘the sequential changesin the conformation ofeach ofthe catalytic sites, Boyer proposed that the o and 8 subunits, which form a hexagonal ring of subunits within the P, head (Figure 5.24), rotate relative tothe central stalk. In this model, which i referred to as rotational catalysis, rotation is driven by the movement of protons through the membrane via the channel in the F, base. Thus, according to this model, electrical energy stored in the proton {gradient is transduced into mechanical energy ofa rotating stall, hich i transduced into chemical energy stored in ATP, Evidence to Support the Binding Change Mechanism and Rotary Catalysi ‘The publication in 1994 of detailed atomic model of the F, head by John Walker and his colleagues of the Medical Research Council in England provided a remarkable body of structural evidence to sup port Hoyers binding change mechanism. Firs, it revesled the struc- ture of each ofthe catalytic sites in a static enzyme, confirming that they differ in their conformation and their affinity for nucleotides. Structures corresponding to the L, T, and O conformations were ‘identified in the catalytic sites of the three & subunits. Second, it revealed that the subunit ofthe enzyme is perfectly positioned within the ATP synthase to transmit conformational changes from the F, ‘membrane sector tothe F, catalytic sites. They subunit could be seen to extend as a shaft from the F, sector through the stalk and into a central cavity within the F, sphere (FIGURE 5.26), where it contacts each ofthe three subunits differently (Figure 5.268), Te apical end of the subunit is highly asymmetric, and at any given instant, different faces of the subunit interact with the difer- ent subunits causing them to adopt different (LT, and O) confor- ‘mations. As it rolates in steps of 120°, each binding site on the 7 subunit interacts successively with the three subunits of F, During single catalytic cele, the subunit rotates ful 360°, causing each catalytic site to pass sequentially through the LT, and O conforma- tions, This mechanism is illustrated schematically im FIGURE 5.27 and discussed in detail inthe accompanying legend. As indicated Figure 5.27, condensation of ADP and P, to form ATP occurs wile each subunit is in the T conformation, As indicated in Figure 5.244, the ¢ subunit is associated withthe “lower” portion ofthe y subunit, and the two subunits rotate together as @ unit. Direct evidence for the rotation of the subunit relative to the ‘af subunits has been obtained ina variety of experiments. "seeing {is believing” then Uhe most convincing demonstration came in 1997 from the work of Masasuke Yoshida and colleagues at the Tokyo Institute of Technology and Keio University in Japan, These esearch cers devised an ingenious system to watch individual enzyme mole- cles catalyze the reverse reaction from that normally operating in the cell. First, they prepared a genetically engineered version of the F portion of the ATP synthase, consisting of three a subunits, three B subunits, and ay subunit (asP7) (FIGURE 5.280). This polypeptide complex was then fixed toa glass coverslip by its head, and a short, fluorescently labeled actin fllament was attached to the end of the ‘y subunit that projected into the medium (Figure 528a). When the preparation was incubated with ATP and observed under the micro- scope, the fluorescently labeled actin filament was seen to rotate like microscopic propeller (Figure 5.288), powered by the energy released as ATP molecules were bound and hydrolyzed by the catalytic sites of the P subunits. ‘More recenly, similar type of F complex was “forced” to per- form the more challenging activity that t normally carries out within the cell-the synthesis of ATP. In this study, a microscope magnetic bead was atached to the subunit of a single F molecule and then ‘caused to rotate ina clockwise direction by subjectingthe preparationisi a w oneuioa aay jo wsivey FIGURE 5.26 The structural base of catalytic ite conformation. (34 section trough theF head shows the spatial organaton of hoe af ts subunits he subunit is constructed of two exlnded and intertwined a helices This helical tak projects into the central envy of the and between thew and 2 subunits on each side. The conformaton ofthe eatalic ste o the f subunit shown onthe lel is determined by is contact with hey subunit (Atop view ofthe; head showing the arangoment of the sia and f subunit ehown in red an yo) sound tne asymm y subunit shown in Be). The Y subunit tn postion to rotate relive tothe srrauncing subunte. kt ileo evident that hey subunit makes contac: na erent way wih eae ofthe tee subunits, ining each of them to adapta diferent conformation. cmesponds to te O confarmation, Pr tothe L conformation, an Foy tthe T conformation Souecr From J.P Abrahams, et al, Nature 370424, 627, 1994, opined by Pe sian from Macmillan Pubshers Lid. Courety of Jan E. Wilk. {oa revolving magnetic field. When one of these F, molecules was from the model, three molecules of ATP were synthesized with each placed within a tiny transparent microchamber in the presence of 360° turn, When the magnetic field was switched off, the subunit ADP and P, the forced rotation of they subunitled tothe synthesis of rotated spontaneously inthe reverse direction, driven bythe hydrolysis ATP, which built up to UM concentrations. As would be expected of the recently synthesized ATP. Taken together, these innovative seh, e ° ° oP ow, a0 + a> — 4 ssonareoss v “ Atom, P+ Fi- Sportaneous. a weve» ARiomaton 4 Ae + fi edcored © saya oe € ae — 4 1 a AS. v fe nN ad FIGURE 5.27 The binding change mechanism for ATP synthesis (Schematic craving showing changes na singe catalytic ste during acl of catalyes. At the beginning of the cele, the #6 inthe pen (0) conformation, and substrate ADP ane? ae entering the sto In sop, the movernent ‘of protons through the membrane induces a shift tothe lose () confermaton in which the substrates are laoely bound: In step 2, the movernent of 2ditonal protons induces a shift to the tight (7) canformason in which he aft for substates increases, causing them tobe tghty bound 0 the Catalytic te. In step 3, the igh bound ADP ana, spontaneously cancense to form 3 ightly bound AIP no changs in conformation i required for 1c movement of addiienal proionsnauees 3 sil lathe apes (0) canfrmalion which the ater ATP greatly deveatd, eis he sep nse slowing the product o be released fom the site. Once the ATP has dasocate, the catayic ste i valabe for svosvate binding, andthe repeate.() Schematic crowing showing changes aa three extavie soso the enrye simultaneously, The maverant of protons trough te Fs par of the enzyme causes the rotation ofthe aymmeti y subunt, wien euplys thee diferent faces tothe eataltic subunts Ae the y subunit oat, induces tof he subunits, causing each catalytic ste ro pass sucessvly rough he T,O, and L conformations changes inthe conformation ofthe cta192 oupuoypainy ox: pve uaneudtoy na0H0y » 5 Baldy ‘Covers coated wits NTA FIGURE 5.28 Direct observation of rotational catalysis (To ceny out tho experiment, amosied version of portion ofthe ATP synthase consisting ofa fy was prepared. Each subunit wat modified canain 1Ohist ine (Hs) esdues at ts Necermins, a ste feated onthe outer (rata fae of ref head. Tho de enn of histidine have a hin afnty fora substance (NNTAY, hich was used to coat he coverslip The ¥ subunt as mode by replacing one ofthe seine residues nea the fond of the stale witha cyte res duo, hich provided a means o attach ‘he fusesceny labled sein flament inthe presence of ATP the ain flamer was observed to rotate counterocewiee (when viewed fom the membrane se, At low ATP concentrations the actin flaments could be instep af 120 [e)Azequenes of fur ames rom a wideo of arotating acti fiament Source: From H. Nal et a, Nature 386: 299, 1997, epinted by permission ‘rom Naemilan Puolsvors ic Courtesy of Masato Yorn bioengineering experiments demonstrate unequivocally that ATP synthase operates asa rotary motor Rotary machines are common in our industrialized society; we suse rotary turbines, rotary drills, rotary wheels, and propellers, to name just afew. But rotary devices are extremely rare in living organ isms, There are, for example, no knawn rotary organelles in eukary oti cell, rotary joints in animals, or rotary feeding structures inthe biological world. Infact, only two types of biological structures are in rotating parts: ATP synthases (and related pro- teins that act as ionie pumps) and bacterial flagella (which are pic- ‘ured in Figure 1.14, inset), both of which can be described as rotary “anomachines” because their size is measured in nanome ters, Engineers have begun to invent nanoscale devices made of known inorganic materials that may one day carry out various types of sub ‘microscopic mechanical activities. The construction of nanosized ‘motors poses a particular challenge, and attempts have already been ‘made to use the ATP synthase to power simple, inorganic devices Someday humans may be using ATP instead of electricity to power some oftheir most delicate instruments RevieW Po 1. Describe the steps in ATP synthesis according tothe binding change mechanism 2. Describe soma of the evidence that sup binding change mechanism. rts the 5.9 Using the Proton Gradient By 1997, a detailed understanding of the working machinery of the F complex was in hand, but major questions about the structure and Junction ofthe membrane-bound , portion ofthe enzyme remained tobe answered. Most important among them were: What i the path taken by protons as they move through the F, complex, and how does this movement lesd to the synthesis of ATP? It had been postulated that 4. The ¢ subunits of the F, base were assembled into a ring that, resides within the lipid bilayer (asin Figure 5.244). 2. ‘thee ring i physically bound tothe Y subunit ofthe stalk, ‘The “downhill” movement of protons through the membrane drives the rotation ofthe ring of subunits 4, The rotation of thee ring ofF, provides the twisting force (torque) that drives the rotation ofthe attached subunit, leading to the synthesis and release of AIP by catalytic subunits ofthe Fring Allof these presumptions have been confirmed. We willlookat each ofthese aspects in more detail The Role of the F, Portion of ATP Synthase in ATP Synthesis ‘A body of evidence, including X-ray crystallography and atomic force microscopy, has shown thatthe e subunits are indeed org zed intoa circle to form aring_shaped complex (Figure 5.25). Hig resolution electron micrographs indicate that the wo B subunits and the single a subunit of the F, complex reside outside the ring of « subunits, as showen in Figure 5.24, The b subunits are thought to be primarily structural components ofthe ATP synthase. The two clon- gated b subunits form a peripheral stalk that connects the E, and portions of the enzyme (Figure 5.248) and, along with the d subunit of are thought to hold the a8, subunits in a fixed position wile the Y subunit rotates within the center ofthe complex. The rotation ofthe ¢ ring ofF, during ATP synthesis has been demonstrated in numerous experiments, confirming that both the 6 ring and Y subunit act as rotors during enzyme activity, How are these two “moving parts” connected? Each ¢ subunit is built like a halrpin: it contains two transmembrane helices connected by a hydrophilic loop that projects toward the F, head, The hydrophilic loops situated onthe tops of thee subunits are thought to form abind= ing ste for the bases of the and € subunits, which together act like a “foot” that i firmly altached to the ring (Figures $24 and 5.28). As a result of this attachment, rotation of the ¢ ring drives rotation of the attached + subunit, wich ean occur at rates of more than 100 revolutions per second, Themechanism by which H” movements drive the rotation ofthe «ring is more complex and less well understood, FIGURE 5.29 pro- vides'a model as to how H ions might flow through the F, complex. Keep in mind in the following discussion of this model (1) thatthe subunits of thee ring move successively pasta stationary a subunit and (2) that protons are picked up from the intermembrane space fone ata time by each subunit and carried completely around a circle before they ae released into the matrix. In this model, eacha subunit bas two half-channels that are physically separated (ole) from one another. One half channel leads from the intermembrane (cytosolic)FIGURE 5.29 A model in which proton difsion i couple rotation ofthe ering ofthe F complex. As discussed nth tox, the number af subunits n thec rng arable. For the sake af imply, hie ring cons ef 12 subunits Is proposed in ths med that each proton ‘rom the intermembrane space enters a hal-channel thin ye 9 suoun' nd then bine to an aide resi (pin € ca hate accesible on fore ofthe c subunits Proton binding induces a conformational change thet Causes the rng to move by approximately 20% The bound proton i eared inal eile By he rtation ofthe ering and the releazed nto 3 second hal-channal that opens into the mate A succession of protons that engage inhi actty euses the cring to rotate in 9 counterclockwise ‘Srecton 9 shown space into the middle ofthe a subunit, nd the other lads from the riddle ofthe a subunit nto the matrix. Its proposed tha cach pro- {on moves from the intermembrane space through the hall-chaasel and binds to a negatively charged acidic residue situated atthe sut~ face of the adjoining c subunit. This acidic residue is apparently an aspartic acid in E cali Figure 5.29), bt ia platamie acid reside in animal mitochondria. Binding ofthe proton tothe carboxy] group of| that residue causes that subunit to rotate approximately 308 in a counterclockwise direction. The moverent of the recently proto- rated c subunit brings the agjoning subunit in the ring (ich was protonated at an ealier step) into alignment withthe second hal Channel of thea subunit, Once there, the acidic residue releases its associated proton, which diffuses into the matrix. Following disto- ciation ofthe proton, the csubuait returns to ts original conforma- tion and is ready to accept another proton from the intermembrane space and repeat the cle ‘According to this model, the proton-binding residue of each subunit actlike s revolving proton carrie. proton hops onto the carrie a a selected pick-up sil and is then carried around a circle before being released ata selected drop-off ate. Movement ofthe ring isdrivenby the conformational changes associated wih the sequential protonation and deprotonation of the acidic residue of each ¢ sub- 193 uit. Consequently, the numberof protons translocated foreach rota tion of thee ring is generally considered tobe equal tothe number of subunits that make up the ring, although this is not always experi ‘mentally validated. Thec ring has been shown to contain between 8 and 15 subunits, depending on the source. For the sake of simplicity, we will describe ac ring composed of 12 subunits as illustrated in Figure 5.29. In this case, the association/dissociation of four protons in the manner described would move the ring 120°. Movement of ‘thee ring 120° would drive a corresponding rotation ofthe attached ¥ subunit 120%, which would lead othe release of one newly synthe- sized molecule of ATP by the F, complex. According to this stoic cometzy; the translocation of 12 protons would lead tothe full 360° rotation of thee ring and subunit, and the synthesis and release of three molecules of ATP. the ring were o contain greater or fewer ‘han 12 subunits the H'/ATP ratio would change, but this can easly ‘be accommodated within the basic model of proton-driven rotation shown in Figure 5.29, rewornoisg + OF Other Roles for the Proton-Motive Force in Addition to ATP Synthesi Although the production of ATP may be the most important activity of mitochondria, these organelles are engaged in numerous other processes that require the input of energy. Unlike most organelles ‘hat rely primarily on ATP hydrolysis to power their activities, mito- chondria rely on an alternate source of energy—the proton-mative force. For example, the proton-motive force drives the uptake of ADP and P into the mitochondria in exchange for ATP and H, respectively: Exchange of ATP for ADP across the membrane is accomplished by the adenine nucleotide transiocase (ANT), which makes the ATP produced within the mitochondria available to meet the energy needs ofthe remainder ofthe cell.'These and ther actvi- ‘es that take place during aerobic respiration are summarized in FIGURE 5.20. In other examples, the proton-molive force may be used asthe source of energy to “pull” caleium ions into the mito chondrion; to drive the events of mitochoncril fusion; and to cause specifically targeted polypeptides to enter the mitochondrion from ‘he matrix (Figure 847) Review PIAA 1. Describe a proposed mechanism whereby proton diffusion from the intermembrane space into the matrix drives the phosphorylation of ADP. 5.10 Peroxisomes Peroxisomes are simple, membrane-bound vesicles (Figure 5.312) with a diameter of .1 to 10 um that may contain a dense, crystalline core of oxidative enzymes, Peroxisomes are multifunctional orga nelles, containing mote than 50 enzymes involved in such diverse activities as the oxidation of very-long-chain fatty acids (VLCFAs, ones whose chain typically contains 24 to 26 carbons) and the syn- ‘hess of plasmalogens, which are an unusual class of phospholi