I

Chapter

EI\ERGY

E,I\ERGY
Living organisms are not in equilibrium. Rather they require a continuous influx of free
energy to maintain order in their internal structures or minimalizing disorder,
Metabolism is the overall process though which Iiving organisms acquire and utilize
the free energy they needed to carry out their various functions. They do so by coupling the
exothermic reactions of nutrient oxidation to the endothermic processes required to maintain the
living state as:
. perforrnance of mechanicalwork;
. the active transport of molecules against concentration gradients;
. biosynthesis of complex molecules.
Phototrophs (plant and some bacteria) acquire free energy from the sun through
photosynthesis (light energy powers the reaction of CO, and HrO to form carbohydrates).
Chemothrops obtain their energy by oxidizing organic compounds (carbohydrates, lipids,
proteins)obtained from other organisms, ultimately phototrophs. Additionally,the nutrients are
broken down in a series of metabolic reactions to common intermediates that are used as
precursors in the synthesis of other biological molecules.
A remarkable property of living organisms is that they maintain a steady state. For
example, over 40-year time span, a human adult (70 kG average weight)consumes literally tons of
nutrients and drinks over 20 000 L of water. He does so without significant weight change.

Generation of energy in the cell

,\t

\.

of

tn the living cells are carried out the steady reactions of degradation and formation

compounds sonnected with consuming and generation of free energy. These processes are called
as metabolism. The metabolism is often divides into two categories: catabolism and anabolism. The
reactants of mentioned processes, their intermediates, and products are referred to as metabolites.
Catabolism is involved in degradation of complex metabolites of into simpler products.
Summary free energy, AG, of this process is negative - catabolism is exothermic and it is free
energy source for anabolism. In humans the final products of catabolism are: C02, H2O, urea, uric
acid and creatine, which are secreted mainly by the lungs and kidneys.
Anabolism. lt is a biosynthesis of complex molecules from simpler reactants using
energy generated in the course of catabolism. Summary free energy, AG, of the anabolism is
positive. This is endoenergetic process.
Simplifying, it can be assumed that the source of energy is the burning of hydrogen in
oxygen. The donors of hydrogen mainly are: glucose, and other simple sugars, fatty acids, ketone
bodies and hydrocarbon skeleton of amino acids.
The burning of H, in 0, in vitro is explosive and strongly exothermic and its energy is
fully converted into heat.
The burning of hydrogen invivo (metabolic process) is slow, multistage process in which
c.a. 40o/o of energy released is stored in the chemical form. lts main carrier of this free energy is
ATP (adenosine triphosphate).

ATP
The endothermic processes that maintain the life of cell are driven by exothermic reactions of the
nutrient oxidation. ln these reactions are synthesized a few types of ,,high-energy" intermediates
whose consumption drives endoenergetic processes. The centra! role in energy metabolism
plays ADEN0SINE TRTPHOSPHATE, ATR which consists of one adenosine moiety to which three

phosphoryl groups are sequentially linked via phosphoester bond followed by two
phosphoanhydride bonds. ADP and AMP are similarly constituted but wrth only two or ono
phosphorylgroup.
phosphoester

The structure of ATP indicates its

relationship to ADP and AMR and
adenosine" The phosphoryl grouPS,
stafting with that on AMR are referred
to as a, p, and y phosphates.
Note the difference between
phosphoester and phosphoan hyd ride

:]rir-irhtiii*ir.r.,'iir-iril
l.rr it-itis-.,
,-

)--*--. o-*l o-A

p-O +
O-p;.()-p.--.rl-*
v
I

i? +li ,l+

'i itl'i
ol
"l
ir

::;HOOH:
I
I
I
|

!
rrl
rlt-I

bonds

'iffil
it"

+,

LATP

rr\_r

"tr_J

Adenosine

Transport of chemical energy by ATP in different

metabolic processes

_;

NDOENERGETI
PROCESSES

-fpyruvate

(neurotransmition

EXOEi{ER.GtrC

L-/

PR.OCESSES

ATP- energy carrier

- it plays important role in catabolic

and anabolic processes,

Adenine nucleosides with high (ATB ADP) and low (AMP) bond energy of
phosphoryl groups
NH.

l"

ilT\
=niT
I

_O= $Hr-O-

NZ

oH oH

ooo
ri , ,
P-O-P-O-P-O

S.A

oo
Ja 8-6-

cH,-o-$-o-$-o-

OH OH

ATP

ADP

il

cHz-o-P-o$tuvP

Phosphoanhydride bond (red) Hight energy bond

Phosphoester bond (black) - Low energy bond

Other "high energy" phosphate compounds

'!'

oo'!'

o
lt

q ,o-

P-O-

8-

9",

lo
lil

n\ T-t',
N:Q 0

I
I

H-C-OH O
Err-o-#-oI

c-o-P-oilil
CHz

6.
l

13 -

b is- p h o s p h o glyc

r ate

ri

O-

phosphoenolopyruvate

t-$-o-

ti

8-

phosphocreatine

The values of their AGo are given above. They do not provided free energy indirectly,
but they are involved in the synthesis of ATP via substral-level phosphorylation. lt is a second
metabolic path (beside of oxidative phosphorylation) of synthesis of ATR very impoftant for cells
realizing the anaerobic metabolism (without oxygen). For example, phosphocreatine is ,,stor+
house" of energy needed for muscle cells to contract. lndirectly this energy is released byATR
which is quickly regenerated by transfer of phosphoryl group from phosphocreatine to ADP
catalped by enzyme creatine kinase.

Dhnanha'-r.aa*ina
l rr\ru]rrr\rvrgGrLrrls &r

n nD
nrJr

(-

ATD
rr
I

nl

lar.aalina
\rt Erllt rg

Other compounds containing ,,rich" energy bonds

H3c-ci3_"oo
acetyl-S-CoA
'

HsC
NHz
Ao-

'-'ttO
C:_-

S-CoA
palrnitoyl-S-CoA

C-S

bond is

,,rich" gngrgy
bond

o---'o-i:o
I

ocarbarnoyl phospate

C-S bond (thioester bond) between atom of S of coenryme A, CoA, and carbonyl
group, >C0, of residue of carboxylic acid is also "rich" in energy. Fatty acids in the active form
as acyl-CoA (where "-" represent a rich energy bond)can participate in catabolism (Boxidation) and anabolism (synthesis esters of glycerol or esters of cholesterol).

Phosphate compounds with low energy bond

.tr,[-o.

t/al

N+"
loH

glucose-6-ph osphate

H
I

H-C_OH

HO_C_H
I

n-8-o-$-oll_

Hse,e T
?
HIC-N-C-C-O-P-OH,C ri
d

i,

HO-

phosphocholine

glycerol-6-phospate

The vatues AGo of their hydrolysis reaction are below 17 kJmol. To these

compounds belong the phosphate esters of glycerol, inositol, aminoalcohols, and all
monophosphate n ucleotides.
ATP is placed in middle position between phosphate compounds possessing
bonds with very high and low energy. ln metabolism ATP plays the role of energy
transmifter. In cell there are not other mechanisms, which allow the transfer of phosphate
groups from high energy donors to low energy acceptors without the participation of ATP.

Mitochondrion
The mitochondrion is the site of eukaryotic
oxidative metabolism. lt contains the enzymes
that mediate this process, and enzymes and
redox proteins involved in electron transfer and

oxidative phosphorylation.
It is calied as the cell's "powei' plant".
The mitochondrion is bounded by outer
membrane and contains an extensively
invaginated inner membrane, called cristae.
The inner mitochondrionon compartment, named matrix (gel-like substance), which contains the
enzymes of oxidative metabolism (e.9. Krebs cycle enzymes), as well as substrates, nucleotide
cofactorc, and inorganic ions. The matrix also contains mitochondria genetic machinery - DNA,

RNAand ribosomes.
The first acceptor of H atoms spitted from substrate is mainly NAD*, next FMN followed
by coenzyme Q. Allelements of respiratory chain are proteins (except coenzyme Q). Some of
these are enzymes and other are nonenzymatic proteins, which contain the iron.sulfur clusters
known as prosthetic group of iron-sulfur proteins (nonheme iron proteins). These Fe:S clusters
participate in etectron-transport process. The most common types, designated as [2Fe'2S] and
protein Cys
[aFe- S] clusters consist of equal number of Fe and S ions, are coordinated to four
sulfhydryl groups. (See the next slide).
10

lron-sulfur cluster [4Fe4S] coordinated with NADH-dehydrogenase

NADH dehydrogenase protein
fg2+r3+

(i

.S-Cys

:
cy,s

-S \

.;H

FE

v\n

M Gp

transfer the electrons between FMN and

coenzyme Q, and between cytochrome
and cytochrorne c,.
The oxidized and reduced states of Fe:S
clusters differ by one formal charge. This
is because the Fe atoms in each cluster
form a conjugated system and thus can
have oxidation stages between tlte +2
and +3 values possible for individual Fe
atoms. E.g. the Fe:S cluster can contain
two Fe(ll) and two Fe(ll[.

,lo

ions placed in Fe:S clusters

S--Cys
.nnnn^

:
l1

The change of adsorption spectrum of nicotinic acid in course of oxidation

Reduction of NAD* to NADH leads
to change of structure of
nicotinic acid ring (part of NAD* ),
which can be detected by UV
spectroscopy. Pair of electrons
(2e') is transferred to next
elements of respiratory chain.

340

wavelength (nm)

zH* + 2e'

(
.L

The change of UV absorption spectrum

derived from change of structure of
nicotinic acld ring. A new maximum at

-{\NHz

$r
I

NAD*

IQ^NHZ

{
zH* +

ze-

NADH

+ H*

340 nm is formed.

\2

Conversion of structure of dimethylisoalloxasine ring as a result of reduction of

FMN into FMNH2 or FAD into FADH,
FMN lub

F.AD

Di methylisoalloxasine is

FVINH, lub FADH,

a pafi of FMN and FAD

as well as FMNH, and

Abs

FADH2.

?o
Hsc
rr
H,cMT^TAo

zH* + ze'

H,cnz*\Ao*

HscM,'nA*-\o

zH* + ze'

dimethy lisoalloxasi rre

dimethyisoalloxasine

(oxidized)

(reduced)

The changes oi absorption in UV specirum. The resuit of ring reriuciion ihe

rnaxirnum at 450 nm is shrinking.

Koenzyme

0 (ubichinon)
n :6 to 9

H3c
H:

13

o-A-cH:

t lt

-oT(CFI2
o

-cH

{ ?H,
-CHz) nH

cxidized fonn

zH'+

\
fr"

-r2H*+

2e

\
OH

Hsc

-o./

reduced form

HgC-O\

FH,
CH:C--CHzhH

T4

Heme C in cyt*chroffie c bonded to protein

CHs
I

,r"
H,cr
-ooc-crrz

,&-s-cy'to

"7{*rLT.*-o,

ln" L ^1

ll

-*,X"-1t -=H;ll - S-cy,r

l_-l
r' cHr

CII3

ffi;
Cytochrome c is soluble in water. Fe atoms in cytochrome are easy oxidized and
Fe3*), and it is reversible process. Thus, the cytochromes can be donors
reduced (Fez+
and acceptors of electronS in respiratory chain. ln cytochrome C heme in covalently bonded
with enrymatic protein by two S atoms from residues of cysteine contained in amino acids

chain.

15

Cytochrome C

The Cytochrome c is a small heme protein found

loosely associated with the inner membrane of the
mitochondrion, lt is a highly soluble protein,
unlike other cytochromes, lt is an essential
component of the electron transport chain, where

it carries one electron. lt is capable of undergoing

oxidation and reductionn but does not bind
oxygen.

Three.dimensional structure of cytochrome c

(green) with a heme molecule coordinating a
central lron atom (orange).

f'T

Heme Afound

in cytochrome a + as
Cytochrome a + os
is a synonim for
cytochrame oxidase
associated with the

*ir"
QH

pumping of protons and

the resultant
phosphorylation of ADP
to ATP. The reaction is
the terminal event in the
electro transport scheme
by which oxygen is usecl
for fuel combustion. lt is
a part of the respiratory

CH-CHz

o).
H

-OOC-CHz-CHz

f",
CHz

chain.

A*Metso

Cytochrome a + a, is the last unit in electron transport chain. lt transfer electrons to molecular
oxygen. ln this place electrons, molecular orygen and free protons bond together and molecule
of water is formed. Heme A is not covalently bonded with enzymatic protein, but it forms only
coordinative bonds with histidine(18) residue and methionine(80) residue.
lt
LI

Standard red uction potentials

18

Eo' tVI

- 0.74

- a.67

Acetaldehl,de

- 0,60

o,

or-

- 0.15

zIJ+

H,

- 4,42

Pyrurrate* CO, +H+

malate

- 0,33

+ H+
NADP+ + H+

I{ADH

- 0^32

I\ADPI{

- 432

FMI{ (bonded w'ith enzyme + 2H.)

FMl\Hr(bonded n'ith

-0'3 0

-0.29

a.29

Reductant

Oxidtzer
Acetate

+ CO, +2I{+

Succinate* CO, +2F{Acetic acid

ZIj{+

IYAD+

Pyruvate +FI?O

u-ketoglutarate

* HrO

/a

enryme)
Dehydnoliponate + Z}I"
1

r3-&is-phosphoglycerate + 7I{+

dihS'droliponate
3-phosphoglycerol
alcteh3,de + Pi

Glutatione (oxidizecl) +7}J+

2 glutatione reducecl

-4,23

EAD +

Fi\BH,

-0.22

2Ftr+

Standard reduction potentials (cont 1.)

Reductant

Oxidant

E6'

tvl

Acetaldehyde + 2H*

ethanol

-0,24

Pyruvate + 2[J*

Iactate

-0,19

Oxaloacetate + 2H+

malate

-0,17

cr-ketoglutarate + NHo*+ 2H*

glutaminian + HrO

-al,4

Fumarate* 2H*

siccin ate

-0,03

CoQ+ZIJ-

CoQII2

4,0+

Cytochrome b (F*'*)

cytochrom e b (Fe2*)

0,47

Dehydroascorbinate

askorbininate

0,08

Cytochrome cr (Fe3*)

cytochrome ct (Fe3*)

0,23

Cytochrome c (Fe'*)

cytochrorn e c (Fe3*)

0,25

Cytochrome a (Fe'*)

cytochrom e fi (F*3*)

4,29

%or+ Hro

HrO,

0,3 0

Cytochrome 0, (Fe3*)

cytochrom frs (Fe'*)

0^5 5
19

Standard reduction potentlals (cont

Es' tv]

Fe2-

4,77

HrO

0,92

Reductant

Oxidant
Fe3*

2.1

20

Mitochondrial respiratory chain complexes

The proteins embedded into inner mitochondrial membrane are organized into 5 respiratory
complexes of electron-transfer chain. Each complex consists of several proteins components
that are associated with the redox-active prosthetic groups with successively increasing
reduction potentials.
6H* (or 4H*)

oo

n (]ffifl

()()

(XXX)

()()

oooo

succinate

umarate

3ADP

3Pi

zPi

or

2ADP

oo

^. .*
6H

or
4H*
21

Respiratory complexes

Components of respiratory chain called as complexes are designated by Latin

numbers from I to V. The complexes Ito lV are composed with proteins,
allosteric groups, coenzymes and they are the parts of mitochondria
respiratory chain. Complex V last in respiratory chain, is the enzyme called
ATP synthase.

22

Complex
Complex I composed with oryreductase NADH: (dehydrogenase NADFI) and coenzyme Q,
CoQ (ubiquinone). lt contains one molecule of molecule of FMN (flavin mononucleotide), which
is a redox-active prosthetic group, and 6 to 7 iron-sulfur clusters (Fe:S) . FMN accepts 2 hydrogen
atoms (2H* + 2e) forming FMNHT. Complex ltransfers hydrogen atoms to ubquinone.
Cornplex I is coupled with phosphorylation process.

-^:::

Complex

X::-X:: X::CoQ- coenryme Q (CoQ or ubiquinone (oxidized or quinone form).

CoQH, - coenzyme GoH, or ubiquinol (reduced or hydroquinone form).
Fe2+S (Fe s+) - Fe : S (iron- sulfur clusters)
23

Cornplex ll
Complex llcomposed with enzymatic protein: succinate-coenzyme Q reductase and FAD , and
also Fe:S clusters and cytochrome b* . This complex participates in oxidation of succinate to
fumarate . lt converts ubiquinone into ubiquinol (hydroquinone form).

Cornplex

-!SlJt-LrlllClLE

-a-

fu rnarate

X::X::X::24

Complex lll
Complex lll is composed with enzymatic protein: cytochrome c-coenzyme Q reductase.
Complex lll contains also Fe:S clusters and two cytochromes b and one cytochrome c . This
complex participate in oxidation of succinate to fumarate . lt converts ubiquinone into ubiquinol
(hydroquinone form). tt is capable to one-electron reductions. Therefore, it provides an electron
conduit between the two-electrons donor NADH and the one-electron acceptors, the
cytochromes.
Complex lll is coupled with phosphorylation process.

Complex

CoQHz

cYt bo*.

III

=e2*s

cytcTox.

Cyt c

red.

cYt cr

cYt c

o*.

)f
CoQ

Fe3*S

-^--

rea.

cyt - cytochrome, ox. - oxidizcd, red- - reduced

25

Complex lV
Complex lV is composed with enzymatic protein: Oxidoreductase and reduced cytochrome
and molecular oxygen.
Complex lV contains cytochromes a, and
cytochrome c to molekule of orygen.

a, and transfer the electrons from reduced

Comptex lV is coupled with phosphorylation reaction.Sometime it is called cytochrome

oxidase.

Complex
cYtao*

cYtcreo

\f
cytco*.

-\

clta3red

1zcytsreo"

a2

)a
clts3ox */

z,zo

cyt - cytochrome, oX. - oxidized, red. - reduced

26

c:

Gomplex V
Synthase ATP. Each carrier takes electrons from donor and transfers it to acceptor (next in
respiratory chain). Orygen atom is the final acceptor of electron pair. ln this end step 9z-ion is
formed, to which 2 protons (H+) are added and the molecule of water (HrO) is formed. ln this
process the organism uses the most oxygen taken from atmosphere .

Complex

V (ATP synthase)

ADP + Pi

-ATP
phosphate
Pi = inorganic

27

Scheme of electrons transport from substrate to oxygen molecule

amytal retenone

antimycin

XHz

2"'

NAD

2e'

'"- I

ATP

3-

2cyt

b 1.*

ATP

2cyt c

1,.'
I

2cyt a +

a3

CN.-LI\
I ,""?.
aztae

ArP

ttzOz

The blue narrows show the inhibition sites of electrons transport.

lnhibitors: amytat retenone, antimycin, CN- ion, caron oxide (CO)and azide.

XH, - reduced substrate

28

Oxidation

of NADH by FMN

General scheme of reaction

ox idative-red

NADH

uctive

+ H*

pa

irs

FNIVI

+2e' +

zH*

/
\r

Eo--0,32V

/\
/ \
NAD* + 2e- + 2H*

Componets of ned-ox reaction wEth

trr-'o

FMNH,

Eo =

-0,30

oxidatlve-reductlve pairs

29

Elimination of HrO, (and (ROS) by coupled action of glutathione reductase and

gl utath ione peroxidase

MDPH + H*

G-S.S.G

2HzO

(oxidixed)

Some substrates are oxidized or

qfut\ thione

Loil tase

*/

N1qpp*

G-S-H
(reduced)

G-SH- reduced glutathione

G-S-S.G

HzOz

reduced without participation of

respiratory chain. ln these
processes the ATP is not released.
Glutathione red uctase catalyzes
the NDPH-dependent reduction of
glutathione disulfide (GSSG) to
glutathione (GSH).

- oxidized glutathione

The major function of GSH is to eliminate HrO, and other organic peroxides, the
toxic products of
various oxidative processes damaging the cell's structures. Peroxides are etiminated
ihrough the
action of glutathione peroxidase yielding glutathione GSH. The coupled action of these
two
enzymes defense cell structures from attacks of reactive oxygen species (RoS)
30

Formation of peroxide anion radical

OH

o'

OH

OH

+o2- + H*
peroxide
anion radical

ubilrydo+rincx'te

semiquinone
radical

o'

+o1- +

H*

peroxide
anion radical

OH

semiquinone
radical

Oxidation of ubiquinone leads to formation of peroxide anion radical (ROS).

For simplifying,in the scheme a ring of ubiquinone is presented only - ring with hydroxyl groups(rino-side substituents),

31

Formation and elimination of oxygen reactive species (ROS)

Oz

!. - o)
pero<ide anlon

2e'

2e'
I

Hzoz

radicd

Sffi

> 2'OH '*__- HzO +ltzOz.

hydroxyl radical

'

:)2oH

ttzOz

--'-

Hjo

G-SS-G

2 G-SH

glutathione oxidized

glutathione reduced

32