CYTOCHROME P450 : A MONOOXYGENASE 369
Conclusions that the reference 48 authors take from their calculations are
as follows: (1) Cpd I and Cpd 0 might coexist if the Cpd 0 to Cpd I energy
barrier is not too large compared to the barrier for substrate oxidation (highly
dependent, it appears, on the substrate itself); (2) energy levels for Cpd 0 and
Cpd I both depend on the protein environment (or, for model compounds, on
the porphyrin substituents); and (3) thr252 may not be needed as a proton
source but is important as a hydrogen - bond donor or acceptor [as an acceptor
it assists in the scission of ( 2 ) prot to yield Cpd I]. As evidence for conclusion
(3), it has been found experimentally that thrOCH 3 substituted for thrOH
forms a competent cytochrome P450 catalyst, indicating that the thr252 proton
is not crucial for intermediate protonation, but rather is needed for hydrogen -
bonding purposes. The reference 48 authors also speculate further about the
( 2 ) prot intermediate. This species might exist at low concentrations and have a
short lifetime but would be a strong oxidant. It may act as the oxidant when
Cpd I cannot form as experienced by Dawson and co - workers. 44a Further study
of the ( 2 ) prot intermediate should be forthcoming. Finally the authors state that
they have studied the direct protonation mechanism only in relation to the
PDB: 1DZ8 structure. Other mechanisms may exist and should be studied
further.
7.4.4 Analytical Methods: X - Ray Crystallography
In their Science magazine article of 2000,^35 the Schlichting group describes the
process involved in obtaining the X - ray crystallographic structures of interme-
diates in the CYP101 catalytic cycle using cryogenic temperatures and rapid
data - collection techniques (PDB: 1DZ4, 1DZ6, 1DZ8 and 1DZ9). Figure 7.14
identifi es the intermediates whose structures have been determined. The
researchers used the technique of freeze - trapping intermediates and deter-
mining their X - ray structures using monochromatic X rays. For instance, to
progress from the dioxygen intermediate (Figure 7.13 ) to the activated oxygen
species (Figure 7.17 ), the second electron in the CYP101 cycle was provided
using the X - ray beam itself.
First the ferrous enzyme [PDB: 1DZ6, Figure 7.14 ( 3 )] was obtained by
diffusion of dithionite reducing agent into the PDB: 1DZ4 crystal (Figure 7.14 ,
( 2 )). The dioxy intermediate [PDB: 1DZ8, Figure 7.14 ( 4 )] was then generated
by exposure to a high partial pressure of O 2. Following this structure determi-
nation, the PDB: 1DZ8 crystals were irradiated with long - wavelength X rays,
producing hydrated electrons within the crystal and presumably driving the
reaction toward the activated oxygen species. A third data set was collected
after the treated crystal was thawed and refrozen. This data set refi ned to show
a “ product complex ” PDB: 1DZ9 with a ferryl – oxo porphyrin system in posi-
tion to hydroxylate the C 5 position of the camphor molecule. [See Figure 7.14
( 6 ), active site shown in Figure 7.17 .] The reference 35 authors report that the
oxidation state of the iron [possibly Fe(IV)] and the electronic state of the