CYTOCHROME P450 : A MONOOXYGENASE 371
hydrogen bond with the hydroxyl group of thr101. Reduction by dithionite to
the ferrous P450 – camphor complex with freeze - trapping to prevent re -
oxidation produces the PDB: 1DZ6 structure — one that is very similar to the
ferric analog (PDB: 1DZ4). Next, diffusion of molecular oxygen into the
reduced enzyme crystals produced the P450 – camphor – O 2 complex (PDB:
1DZ8). This unstable ternary complex ’ s structure was determined with short -
wavelength X rays to prevent radiolysis. As can be seen in Figure 7.13 , the
diatomic oxygen molecule is bound end - on ( η^1 ) to the heme iron [Fe(II) or
Fe(III) – O distance equals 1.8 Å , O – O bonding distance equals 1.25 Å ]. The O 2
distal oxygen (furthest from the iron ion) is within 3.5 Å of thr252 ’ s hydroxyl
group. The camphor molecule has moved slightly away from its previous posi-
tion but is still within van der Waals contact with the dioxygen (or superoxide)
ligand. The porphyrin ring has become fl atter and the iron ion is slightly above
the porphyrin plane. Obvious changes from PDB: 1DZ4 or 1DZ6 structures
upon dioxygen binding include the presence of an ordered water molecule,
H 2 O 901 , close to both the dioxygen molecule and the hydroxyl group of thr252.
New conformations of conserved residues asp251 and thr252 are seen as the
carbonyl oxygen of asp251 has fl ipped by 90 ° toward asn255 and the amide
nitrogen of thr252 has rotated toward the heme pocket. H 2 O 901 sits in a groove
in the distal pocket and interacts with H 2 O 902 that in turn interacts with other
water molecules (H 2 O 687 , H 2 O 566 , H 2 O 523 ) in a chain reaching toward the highly
conserved glu366. (See Figure 7.13 .)
A second electron is now added by irradiation of the P450 – camphor – O 2
complex (PDB: 1DZ8) with long - wavelength X - rays that results in changes in
electron density around the active site. After prolonged exposure to the long -
wavelength X rays, thawing and refreezing of these crystals, data collection,
and refi nement led to a difference map indicating that O – O bond cleavage
had taken place (PDB: 1DZ9). The electron density was most consistent with
a ferryl – oxo iron ( Compound I ), Fe IV = O (P + • ), complex that had been previ-
ously observed in cytochrome c peroxidase and catalase structures.^51 In this
structure, shown in Figure 7.17 , the Fe(III) or Fe(IV) – O bond distance has
shortened to 1.67 Å and the to - be - hydroxylated camphor C 5 atom is within
3 Å of the oxo oxygen atom. Water molecules H 2 O 901 and H 2 O 902 cannot be
detected, but electron density for H 2 O 903 appears to establish a similar
hydrogen - bonding network reaching toward the highly conserved glu366. The
structure is similar to that found by the Poulos research group when P450 CAM
was co - crystallized with 5 - exo - hydroxycamphor.^52 In the PDB: 1DZ9 structure,
the asp251 carbonyl has fl ipped back to its original position in ferric P450 CAM
(PDB: 1DZ4).
How are the conserved residues asp251 and thr252 believed to take part
in the catalytic mechanism? It is known that D251N (asp251asn) mutants
exhibit catalytic rates decreased by two orders of magnitude. Replacement of
thr252 leads to a large proportion of uncoupling (moving into the peroxide or
oxidase shunts), leading to the conclusion that thr252 ’ s role is to provide an
important hydrogen bond. The reference 35 authors believe that the P450 CAM