Cytochromes catalyze electron migration

C1
Cytochromes
• Enzymes that catalyze redox reactions or proteins
that are redox active (electron or oxygen carriers)
• Enzymes: oxidoreductases (many are
oxygenases)
• Transfer e- from reductant to oxidant using a
heme at the active site
• Carry out crucial oxygenation steps in the
production of many 2o products
• Numerous cytochromes in cells; cyto C, cyto bc,
cytochrome c oxidase, cyto P450 (but rarely
covered in a biochem course)
• In particular, cyto P450 important to 2o
metabolism - Carries out key hydroxylation
reactions
 C2

• Cytochromes generally do not have the “Xase”

enzyme designation, so it is easy to forget that
they are enzymes (i.e., Oxidoreductases)
• We will cover:
– General Cytochrome review
– Cyto P450 in more detail
• Two types of major cytochrome processes:
– e- transport in bioenergetics (i.e., in mitochondria and
chloroplasts)
– Hydroxylation reactions in biosynthetic pathways
(especially 2o metabolism)
• But all are redox reactions
 C3
Examples of Cytochrome catalyzed reactions
Cytochrome P450 Reactions (each done by a different cyto
P450): CH2OH NADPH CH2OH
H+ NADP+
C O O2 H2O C O
OH HO OH

O O

11-deoxycortisol Secondary Metabolism cortisol

NADPH
H+ NADP+
O2 H2O

Oxygenation of O
Benzo(a)pyrene contaminants Benzo(a)pyrene-epoxide
Promutagen Mutagen
 NADPH OH C4
H+ NADP+
O2 H2O Other generic
term for these
CH2 CH2 P450s is MFO
(mixed function
NH3+ NH3+ COO-
COO- oxygenases)
Phe Primary Metabolism Tyr

Electron Transport:
Cytochrome C
4 CytoCreduced + O2 oxidase 4 CytoCoxidized + 2 H2O
Mitochondria (e.g., reduction of O2. Note: cannot really oxidize ½ an O2)
OH O
Cytochrome B6/f
CH3 CH3
(CH2-CH=C-CH2)9H (CH2-CH=C-CH2)9H
OH PQH2 + 2 PCox O PQ + 2 PCred

Chloroplasts (electron transport from PSII to PSI uses a cytochrome)

 C5
Hemes
Common theme The heme catalyzes
in the above e- transport from one
examples is a substrate to another.
Very
redox active hydrophobic
heme at the tail. Firmly in
There are only three
active sites of membrane. types of hemes. All
each are porphyrins
cytochrome. (tetrapyrroles + Fe).
So heme part is fairly
Composition of constant.
all cytochromes • cyto b6/f in
chlorplasts.
is a protein plus • Cyto P450.
The protein scaffold
a redox active • The heme in varies greatly,
hemoglobin. depending on the
heme.
reaction being
catalyzed. So the
b and c hemes: No protein dictates the
isoprene tail. Not Cyto c: soluble reaction.
as hydrophobic. In e- carrier.
Bound to protein by sulfide
soluble hemes.
bond across C=C. (add rxn??)
 C6
Redox activity of the heme
• The Fe in the heme is redox active
• The ½ rxn: Fe3+ + e- Fe2+
• In the reduced heme, the electron on the Fe is
delocalized in the heme ring
• Thus: Heme good electron carriers for 2 reasons:
– Fe is a redox active metal
– Conjugated -structure of heme allows e- to be
delocalized into heme ring
• All hemes essentially the same. It is the protein
that dictates the reaction specificity.
• Even Hemoglobin, the reaction is essentially the
same:
– Iron in the hemoglobin resting state is: Fe2+
– Reaction is: Fe2+ + O2 (Fe3+ ● O2-)
 C7

Function of cytochromes
• Cytochromes in respiration
• Cytochromes in photosynthesis
• Cytochromes in biosynthesis

• Respiratory e- transport chain in

mitochondria and bacteria
– Transfer of e-’s from NADH to O2
– Requires e- transport through several
cytochrome hemes
Mitochondrial electron transport
 C8
Mitochondrial Electron Transport
Succinate dehydrogenase
Succinate Fumarate
Each complex is made
up of several protein
2e- 2H+
sub-units + e- carriers

Some subunits hold e- FAD Succinate-CoQ

carriers, some just reductase
structural FeS complex

NADH

FeS (2 cyt b’s) Cyt c1

2e- FMN FeS CoQ Cyt c
NAD+ + H+
NADH-CoQ CoQH2-Cyt c reductase
H+in reductase H+out H+in Complex- 280 kD H+out
complex
200 kD

Cu Cyt a ( Cu
Cyt a3 ) 2e-
2H+
+ 1/2 O2

Cyt c oxidase complex H2O

H+in 200 kD H+out
 Electrons in Mito e- chain flow downhill C9

• Always in the direction of - G, i.e., exothermic reactions

• The e- flow from more negative to more positive redox
potentials (o)
• e- wants to flow away from the more negative o
Redox potentials of some respiration e- carriers
Reaction o____ Note: Each cyto
NADH/NAD+ - 0.32 v has a different
Cyto bred/Cyto box +0.04 v redox potential.
Cyto cred/Cyto cox +0.26 v This is due mostly
Cyto ared/Cyto aox +0.29 v to diff protein
environments
O2/H2O +1.00 v
• i.e., if o(ox) - o(red) is positive, based on Nernst equation,
G = -n F o, G will be negative.
• e.g., for Cyto cred + Cyto aox Cyto cox+ Cyto ared
• o(ox) - o(red) = 0.29 – 0.26 v = 0.03 v
• G = -(1)(23)(0.03) = - 0.69 kCal/mol
Mitochondrial Membrane – Site of Electron C10
Transport

NADH-CoQ CoQH2-Cyt c Succinate-CoQ

oxidoreductase reductase oxidoreductase
Through a series of cytochrome and other e- carriers, NADH is oxidized and O2 is
reduced. A pH gradient is generated which is used to make ATP.
NADH/CoQ oxidoreductase C11

• Reaction: NADH + CoQ + H+ NAD+ + CoQH2

• Each is a substrate and a 2 e- carrier:
NAD+ + 2 e- + H+ NADH (Substrate from TCA Cycle)
O OH

+ 2 e- + 2 H+

O n OH n

CoQ CoQH2
CoQ (Coenzyme Q) is a membrane soluble
substrate
• FMN (flavin mononucleotide) is also a 2 e- carrier
• FeS (iron sulfur center) is a 1 e- carrier, but there
are two FeS’s
NADH-CoQ • 4 H+ are pumped through the membrane during
oxidoreductase the reaction
• CoQH2 carries the 2 e- to the next complex:
CoQH2/Cyto C reductase
CoQ/Cyto C reductase C12

• Reaction: ½ CoQH2 + CytoCox ½ CoQ + CytoCred

• 1 e- is carried on a linear path to CytoC
• Problem is that CoQH2 has 2 e- and CytoC can only
hold 1 e-
• CoQH2 CoQ + 2 e- + 2 H+
CytoCox + e- CytoCred
• To accommodate this stoichiometric problem an extra
pathway is used – the Q cycle (later)
• H+ are pumped (also later)
• CytoCred carries the e- to CytoC oxidase

CoQ/Cyto
reductase
Cyto C oxidase C13

• Rxn: 4 CytoCred + O2 + 4 H+ 4 CytoCox + 2 H2O

• Another stiochiometric problem
• We will look at this mechanism also
 The Q-cycle on CoQ/Cyto C reductase C14

Cyt C

NADH 2 H+
NAD+

Point of the CoQ is a 2 e- carrier

pathway is Cyt C is a 1 e- carrier
to transport Stoichiometric problem dealt with by dividing the
H+ across pathway in two
the memb 1 e- linear to Cyt C, 1 e- cycled back to CoQ
 The Q-cycle on CoQ/Cyto C reductase C15
Remember CoQ (Q) is a substrate present in excess

Cyt C

NADH 2 H+
NAD+
1CytC
Step 1 ox
1CytC Step 2 2CytC
ox Step 3 3CytC
ox
1QH 2QH 2CytC 3CytC
2 1e- red
2 1e- red 1e- red
c1 c1 c1
Note: extra
1Q 2Q 3Q H+s pumped
b 3Q- b 1e- b
3Q2- 3Q2-
1e- 1e- 3QH
2
b b 4Q b
3Q 2H+
 C16
The Q-cycle
evolutionarily conserved Q-cycle

4 H+ per e- pair CoQH2 thru Complex III

1. CoQH2 binds to Qo site outer site

2a. releases 2H+ into perimito space
2b. 1e- passed to Cyto-C via an FeS
3. 1e- passes to cytoBL-->BH
& reduces CoQ at matrix Qi site
4. oxidized CoQH2 dissociates at Qo site
5. 2nd CoQH2 binds to open Qo
6a. repeat of step 2a.
6b. repeat of step 2b.
7. repeat of step3.
8. 2H+ of matrix reduce CoQ --> CoQH2
9. CoQH2 dissociates from Qi site
10. open Qi site binds new CoQ
Q-cycle accomplishes two things:
1. Turns a 2 e- pathway into a 1 e- pathway
2. Pumps extra H+ across the membrane
 Cytochrome C Oxidase C17
CytoC is a 1 e- Carrier
O2 requires 4 e- to be fully reduced
Stoichiometric problem dealt with by using 4 1-e- carriers on Cyto C oxidase
Orchestrates the rxn: 4 CytoCred + O2 + 4 H+ 4 CytoCox + 2 H2O
4H+
4 Cyt C Intermembrane space
e-
Cua
4 e- carriers provide a
e-
reservoir (buffer) of e- -
Cyt a e
capacity so O2 can be Cyt a3
e - Oxygen binding site
fully reduced in a
concerted set of Cub
steps

Matrix

4H+ 4H+
2 H2 O
O2
 Mechanism of O2 reduction on Cyto C oxidase C18
2H+
e- OH-
6 Fe3+ Cu2+ 7 2 H2O
H -HO
Fe4+=O2- O Cu2+
H Fe3+ Cu2+
Cyto a3 Cub

e-
5
e- + 2H+ 1

- -
Fe3+ -O-O- Cu2+
Fe3+ Cu1+

4
2 e-

Fe2+ O=O Cu1+

3 Fe2+ Cu1+
O2
Cytochromes in Photosynthesis
C19
 C20
Photosynthetic Electron transport
 C21

The Thylakoid Membrane

Cytochrome P450 - Hydoxylations
 C22
Cytochrome P450 Occurance
• Used in 1o and 2o biosynthetic pathways.
• Large family of enzymes that mostly carry out
hydroxylation reactions. (RH + O2 NADPH ROH + H2O)
• All are a b-type heme + a protein scaffold.
• The 450 from the peak absorbance of the heme. Absorb
at 450 nm. Some slightly different (e.g., 448 nm).
• Name originally from being a cyto of unknown function
that absorbed at 450 nm.
• Also referred to as mixed function oxygenases (MFOs),
aryl hydrocarbon hydroxylase, etc., relating to their
function
• Like other cytochromes, catalyze e- transfer from a donor
to acceptor (oxidoreductases)
• In this case one of the donors is the biomolecule or
toxicant to be hydroxylated. The other donor is NADPH.
The acceptor is O2.
 C23
Cytochrome P450 Occurrence
• Found in all eukaryotes. In some prokaryotes.
• In animals, highest concentrations in liver cells. Used
there to detoxify contaminants.
• Highest sub-cellular concentration: smooth endoplasmic
reticulum (SER).
• Also found in mitochondria, but hard to distinguish from
other cytochromes.
• There are multiple forms of cyto P450. Each is an
isozyme.
• Each isozyme has a different reaction specificity.
• e.g., Mammals have > 60 different cyto P450 isozymes
(each coded by its own gene)
• With so many isozymes, cyto P450s can carry out a large
variety of reactions, each varying in RH substrate. Thus,
MFO activity before variety of isozymes understood.
 C24
Cytochrome P450 & Drug resistance
• Specific substrates induce expression of specific
cyto P450s.
• e.g., Phenobarbital, toxicants, ethanol and pro-
carcinogens increase activity of different cyto
P450s. Control is at the gene expression level.
• Part of a phenomenon known as multi-drug
resistance.
• Creates a problem.
• For example: EtOH increases the expression of
cyto P450s that degrade drugs, including
antibiotics.
• Thus, in alcoholics, antibiotics are often less
effective.
 Cyto P450 Reaction and energetics C25

RH + O2 + NADPH + H+ ROH + NADP+ + H2O

RH and NADPH are oxidized, O2 is reduced

Highly exothermic. Even just for hydroxylation of RH.

o
R+ + H+ + 2 e- RH - 0.2 v
½ O2 + 2 e- O2- +1.0 v

RH + ½ O2 ROH +1.2 v

G = -n F o = - 2(23)1.2 = - 55.2 kCal/mol

But this does not account for the NADPH, which will also be
oxidized
 Cyto P450 Reaction and energetics C26

RH + O2 + NADPH + H+ ROH + NADP+ + H2O

RH and NADPH are oxidized, O2 is reduced.

Accounting for the whole reaction
o
R+ + H+ + 2 e- RH - 0.2 v
NADP+ + H+ + 2 e- NADPH - 0.32 v
1 O2 + 4 e- 2 O2- +1.0 v

RH + O2 + NADPH + H+ ROH + NADP+ + H2O +1.52 v

G = -n F o = - 4(23)1.52 = - 140 kCal/mol

Why is the NADPH required?

 Cyto P450 Mechanism C27

NADPH P-450 Reductase (ox) P-450 (red) RH + O2

NADP+ P-450 Reductase (red) P-450 (ox) ROH + H2O

1. Activation: NADPH reduces the P450 Heme

NADPH + CytoP450ox NADP+ + CytoP450red H2O2

Enzyme: Cyto P450 reductase. 2 e-, P450 heme is reduced 2 separate times
A flavo protein with a Flavin Adenine Dinucleotide (FAD) and
a Flavin MonoNucleotide (FMN)

2. Substrate oxidation: Reduced P450 Heme reduces O2 to H2O and drives

oxygenation of the substrate.

CytoP450red + O2 + RH + 2 H+ ROH + H2O + CytoP450ox

Enzyme is CytoP450. 1 heme at active site. 2 e- from NADPH via the heme
2 e- come from RH.
 Cyto P450 Mechanism C28

• Cyto P450 e- transport chain

• Composed of two enzymes
• Cyto P450 reductase with FAD and FMN co-factors or
e- carriers
• Cyto P450 with the Heme as the e- carrier
Cyto P450 reductase Cyto P450 RH + O2 + 2H+
NADPH 2e-
2e- 2e- 2e-
FAD FMN Heme

NADP+ + H+ 1 e- at a time ROH + H2O

• RH and NADPH are both e- donors

• O2 is the only e- acceptor
• The O in the H2O takes the 2 e- from NADPH
The Cytochrome ROH
C30
RH NADPH
P-450 Cycle
Fe3+
9 1
FAD
(ROH)Fe3+ (RH)Fe3+
e-
8
2 #1 FMN
(R●)(Fe ●OH)3+
(RH)Fe2+
7 O2
3
(RH)(Fe-O)3+
H2O (RH)Fe2+(O2)
6
4
2H+ 5
(RH)Fe3+(O2-2)
(RH)Fe3+(O2•)

e-
#2
 C31

(+) (-) (+)

• Net: RC:H
(-)
+ O 2 + 2H + + 2e- H2O + ROH

• W/O Substrate: O2 + 2H+ + 2e- HOOH

 C32

• Three-dimensional concept of the Cyto P450

system in the endoplasmic reticulum
• P450:Reductase ratio is ~ 10:1
 C33
Cyto P450 reaction in the SER
RH + O2
NADP+ ROH + H2O
NADPH

2e-
2e- FAD Heme
2e- FMN 2e-

Heme in region that

Cyto P450 wraps around the
SER reductase reductase
Membrane

All Cyto P450s use this structure and mechanism. All bind same reductase.
 Types of Cyto P450 reactions C34

Different isozyme for each reaction type and substrate

 C35
Oxidative de-alkylation is a hydroxylation

H O
NADPH
RNH CH3 RNH CH2
O2

RNH2 + H2C=O
 C36
Number of isozymes and reactions
• For each of the above reaction types, there are multiple
Cyto P450 isozymes
• In total, ~ 60 different isozymes in mammals
• Each for a different “hydroxylation” reaction on a different
substrate
• Each isozyme has a different substrate binding site. But
similar Heme, O2 and reductase binding sites
• However, each isozyme can handle 5 to 10 related
substrates (flexible enzyme)
• Thus, assuming 60 isozymes and 5 to 10 substrates per
isozymes, we can assume 300 to 600 possible reactions
from the P450 system
• The 60 genes are placed in various gene families
• We will look at two examples
– Cyto P450 III and Cyto P450 XI – Steroid metabolism
– Cyto P450 I – Contaminant oxidation
C37
C38
 C39
Different Cyto P450 isozyme for each reaction type
• Homologous regions of all P450s
•Reductase binding region (e.g. membrane region)
•Heme binding region
•O2 binding region
• Heterologous region of all P450s
•Substrate binding site

Proposed model of the membrane topology of a P-450 isozyme

C40
 IN SMOOTH ENDOPLASMIC RETICULUM C41
O CH 3 O CH 3 O
2 OH 2

HO HO HO
PREGNENOLONE 17α-HYDROXYPREGNENOLONE DEHYDROEPIANDROSTERONE

O CH 3
O CH 3 O
2 OH 2

O= O=
PROGESTERONE O=
17α-HYDROXYPROGESTERONE ANDROSTENEDIONE
CH2OH CH2OH
3 C=O
3
C=O
OH 1 P450 scc

O=
2 P450c17α
O=
11-DEOXYCORTICOSTERONE 11-DEOXYCORTISOL 3 P450c21

4 P450c11β
 O CH 3 CH2OH CH2OH
C=O
C42
C=O
OH

HO O= O=
PREGNENOLONE 11-DEOXYCORTICOSTERONE 11-DEOXYCORTISOL
CH3 CH2OH
CH2OH
C=O 4 C=O
4 C=O
HO OH
HO

HO O= O=
PREGNENOLONE CH3 CORTICOSTERONE CH2OH CORTISOL
HC
4 HO C=O
1 HO

18-HYDROXYCORTICOSTERONE
HO O= O=
CHOLESTEROL O CH2OH 1 P450 scc
4 HC C=O

HO
2 P450c17α
ALDOSTERONE
3 P450c21

Mitochondrion O=
4 P450c11β
 Cyto P450 IA1 12 1 C43
Bay region 11 2
PAH, PCB and dioxin 10 Benzo(a)pyrene
3
hydroxylation 9
4
8
7 6 5 K
region

7,8-Oxide P-450 Mono-oxygenase

Simple epoxide 4,5-Oxide

O
O
Epoxide hydratase
7,8- Dihydrodiol
4,5-Dihydrodiol
Dihydrodiols Nonreactive
HO OH

OH OH
P-450 Mono-oxygenase

O 7,8-Diol,9,10-oxide
Diol-epoxide
HO Ultimate carcinogen: highly reactive
electrophile
OH
 C44
Cyto P450IA1
• Oxidoreductase is induced by PCBs, dioxins and PAHs
• Initiates metabolism (and detoxification) of these
contaminants

Assay of Cyto P450IA1

EROD: Ethoxy-Resorufin-O-Dethylase - measures
activity of Cyto P450IA1
• Reaction: CH3CH2O H
N
O O
HO H
N
O O
P450IA1
NADPH
N N
+ CH3CHO
7-ethoxy-resorufin 7-hydroxy-resorufin
weakly Fluorescent Strongly Fluorescent
Abs: 450 nm Em: 550 nm

• Time frame for appearance of P450IA1 after exposure of

an organism to a PAH or a PCB is on the order of days
 C45

• Oxidation of Benzo(a)Pyrene by Cytochrome P450

• Epoxide + Diol Formation

+
P-450
NADPH + O2 + +H NADP+ + H+

+
O
 C46

·(FeO)3
+
H
·O· H Radical
Abstraction
recombination
of e- to FeO

·(FeO)2
·+ +
H
OH
CH3 H2O
H
Transfer
O·-
H
· HO
+ Fe3+ HO H
CH3 CH3
O H
·
 C47

Hydroxylation

Starting with the radical para to the oxygen

H O
OH

Note: The phenol has the same

oxidation state as the epoxide & diol.
 C48

Or by the hydroxylation mechanism in the P-450 cycle

Removal · (FeO·)3+
+ FeO3+ of one π e-

H
H· transfer

· (FeOH)3+

Radical
recombination

+ Fe3+

OH