Cytochrome P450:

Mechanism

.
+ I
V

Petsko wt. al., Science, 2000, 287,1615

 The “Rebound”
C
H C
e H
O O H

FeIV FeIV

H C
O

FeIII
 HAT Reactions
• The E0 of aliphatic C-H bonds exceeds 3 volts
• Oxidation by simple ET would be very energy intensive and
also oxidize the protein
• These oxidations are carried out at ~ 1 V
• The polarity between C and H is also low
• The Proton and Electron are simultaneously transferred which
reduces the energy barrier
• It is a QM process- involves tunnelling
• In this case depends on the redox potential of the oxoiron
intermediate and the basicity of the oxygen bound to the iron
 Proton Couple Electron Transfer
(PCET) Reactions
• Proton-coupled electron transfer is a fundamental mechanism in biology
Enzymes often rely on the coupling of electrons and protons to affect
primary metabolic steps involving charge transport and catalysis.
• Without the coupling of the proton and electron, many processes in biology
would not be possible
• For instance, consider the oxidation of tyrosine the electron and proton must
transfer in a concerted fashion if high energy intermediates are to be avoided
• If the electron were to transfer in the absence of proton transfer, then the
very strong acid TyrOH•+ (pKa = −log Ka = −2, where Ka is the acidity
constant) is produced. Also a very oxidizing potential is needed (E = 1.46 V
vs NHE)
• The high-energy pathways are avoided if the electron and proton transfer
together. In this case, the TyrO• radical is produced, and the high-energy
process of first removing an electron (or, vice versa, first removing a proton)
is circumvented.
 Horseradish Peroxidase
Horseradish peroxidase catalyzes the oxidation of organic
substrates with H202 as the ultimate electron acceptor
AH2 + H202  A + 2H20
The first step of the peroxidase reaction path involves the
addition of H202 to the FeIII resting state to form a high-valent
iron-oxo derivative known as compound I, which is formally
two oxidation equivalents above the FeIII state (see next slide).
Compound I is then reduced back to the FeIII state in two steps
through compound II.
 CytP450 vs HRP

Cyt P450
-
OOC O COO- HRP
X= S-Cysteine,
N IV N
X= N-Histidine,
Efficient C-H activation Fe
N N

X= cysteine, tyrosine or histidine

25
CytP450 vs HRP

Enzymes pKa Eo (V)

HRP <3.5 0.97

CytP450 >12 1.0

Groves et al. Nature Chemistry, 2014, 6, 89

Green et al. Science, 2004, 304, 1653 26
 Function dependent on axial ligand and
cavity
Cytochrome b5 Cytochrome P450
HRP
• The enzyme ''Cytochrome c oxidase'' is a large transmembrane protein complex
found in bacteria and the mitochondrion
• It is the last protein in the electron transport chain

• It receives an electron from

each of four cytochrome c
molecules, and transfers them
to one oxygen molecule,
converting molecular
oxygen to two molecules of
water
• In the process, it translocates
four protons, helping to
establish a chemiosmotic
potential that the ATP
synthase then uses to
synthesize Adenosine
triphosphate (ATP).

Chem Rev. 2018 Mar 14; 118(5): 2469–2490

• The complex is a large lipoprotein composed of several metal prosthetic
sites and 13 protein subunits in mammals.
• The complex contains two hemes, the ''a'' and ''a3'' hemes, and two
copper centers, the CuA and CuB centers.

Subunit 1
Metal Cofactors in Subunit 1 Heme-Cu Active site in Subunit 1
 CuB

a3-CuB
Subunit 2: CuA

CuA
 Thermodynamics: Aerobic Metabolism
Oxidation of food generates electrons
- 0.32 V        +0.82 V
NAD+ + H+ + 2e-  NADH
O2 + 4H+ + 4e-  2H2O
Cytochrome c Oxidase

Adenosine diphosphate (ADP)

+ PO43- Adenosine 5'-triphosphate (ATP)

ATP Synthases
• The O2/H2O acceptor redox couple has a midpoint redox potential
at pH = 7 (Em,7) of 815 mV

• Cytochrome c is the donor of the four electrons, and mammalian

cytochrome c and many bacterial c-type cytochromes have
an Em,7 of ca. 250 mV

• Hence, under standard conditions the free energy change (per

electron) for reaction 1 is 565 meV ( 13 kcal/mol).