CYTOCHROME P450 : A MONOOXYGENASE 363
7.4.2 Cytochrome P450: Structure and Function,
In this discussion, we will concentrate on the Pseudomonas putida P450 CAM
(CYP101) enzyme structure and function, especially as concerns the iron –
heme center, the enzyme ’ s mechanism of activity as a complex series of indi-
vidual steps involving protein redox partners and cofactors such as NAD(P)H,
and how the interactions of heme iron and dioxygen are controlled by the
protein environment. The cytochrome P450 iron heme b prorphyrin ligand,
protoporphyrin IX, shown in Figure 7.1 , is the same as that found in hemoglo-
bin and myoglobin. The heme group is held in the catalytic pocket through a
covalent bond between the iron and a cysteine amino acid side chain from the
protein (cys357 – S – Fe(III) in PDB: 1DZ8 = 2.2 Å ) and other hydrogen - bonding
interactions with amino acid side chains and specifi c solvent (water) molecules.
There are three important N – H · · · S hydrogen bonds to leu358, gly359, and
gln360 that help retain cysteine ’ s sulfur atom coordination to iron as the metal
ion passes through the various ferric, ferrous, and ferryl oxidation states of the
catalytic cycle. (See Figures 7.13 and 7.14 .)
Several CYP101 X - ray crystallographic structures were reported by the
Schlichting group inScience magazine in 2000^35 (PDB: 1DZ4, 1DZ6, 1DZ8,
1DZ9). References 34 and 35 form the basis for the catalytic cycle found in
Figure 7.14.
The resting state of the enzyme, also called met - P450, consists of a six -
coordinate, low - spin (l.s.) iron(III) center ( 1 — bold numbers in parentheses
refer to structures in Figure 7.14 ). In addition to the four planar porphyrin
nitrogen ligands, the Fe(III) is axially coordinated to the sulfur of a conserved
cysteine (cys357 in CYP101) and a water molecule. Upon addition of substrate,
camphor in this case, the iron(III) center loses its water ligand becoming fi ve -
coordinate ( 2 ). Electron paramagnetic resonance (EPR) spectroscopic results
identify the iron(III) center as high - spin (h.s.). X - ray crystallographic struc-
tures of this moiety include PDB: 1DZ4, 2CPP,^36 and 1UYU.^37 Next, an elec-
tron is added to produce an Fe(II) metal center ( 3 ). The X - ray crystallographic
structure of this intermediate has been published by the Schlichting group as
PDB: 1DZ6.^35 This step is followed by the addition of molecular dioxygen ( 4 ).
Questions remain as to the oxidation state of iron in this intermediate, and
evidence exists for both the ferrous – O 2 state (Fe 2+ – O – O) and the ferric – super-
oxide state ( Fe^3 +−−O 2 ). The PDB: 1DZ8 X - ray crystallographic structure rep-
resents this intermediate — the active site is modeled in Figure 7.13. This
structure is the last relatively stable intermediate in the cycle, although it is
known to decompose via the autoxidation shunt producing the physiologically
dangerous superoxide radical anion. Addition of an electron to ( 4 ) generates
the ferric – peroxo state, Fe 3+ – OO 2 − ( 5a ), and then addition of a proton gener-
ates the ferric – hydroperoxo moiety, Fe 3+ – OOH − , ( 5b ). These non - equilibrating
intermediates, found in many heme - containing oxygenase and peroxidase
enzymes, have been studied using cryogenic radiolysis.^38 The technique involves
radiolysis of the ferric protein in frozen aqueous – organic solutions at 77 K to