CYTOCHROME P450 : A MONOOXYGENASE 367
that explains most mechanistic questions by invoking low - spin and high - spin
states for the compound I intermediate. One recent analysis by the Shaik
group of Compound I reactivity, with styrene as substrate, shows that the reac-
tion features multi - state reactivity (MSR) with different spin states (doublet
or quartet), different electromeric situations involving radicals, cations, and
Fe III and Fe IV oxidation states.^41 The researchers concluded that low - spin path-
ways lead to styrene epoxide products via a concerted mechanism that may
or may not result in stereochemical scrambling. High - spin pathways have bar-
riers that may prevent ring closure to epoxide and may include intermediates
with suffi cient lifetimes to allow stereochemical rearrangement and side prod-
ucts such as phenylacetaldehyde or 2 - hydroxostyrene.
One interesting discussion in the literature involves alkene epoxidations
catalyzed by hydroperoxoferric P450, intermediate ( 5b ) in Figure 7.14. First,
it was found that removing thr252, a highly conserved threonine on P450 ’ s
distal side near the site of dioxygen binding, impaired the ability of P450 CAM
to hydroxylate camphor.^42 Next, epoxidation of alkenes was investigated in
mammalian threonine – alanine mutants, where it was found that substrate
alkenes were both epoxidized and hydroxylated but greater amounts of epoxi-
dized product were formed in mutants compared to wild - type enzyme.^43
Further study of the T252A (thr252ala) mutant in the Dawson and Sligar labo-
ratories showed that epoxidized but little or no hydroxylated products were
formed and that the peroxide shunt from ( 5b ) back to ( 2 ) in Figure 7.14 was
operating.^44 All these results were interpreted to mean that Compound I was
not being formed (or being formed in very small concentrations) in these
mutants. Rather a second electrophilic oxidant, hydroperoxoferric P450 ( 5b )
was responsible for the epoxidation products. This postulation carried the
“ two - oxidant ” name. Dawson and co - workers were able to show that the
hydroperoxoferric P450 ( 5b ) has suffi cient lifetime to be detected using low -
temperature EPR/ENDOR methods.^45 Shaik and co - workers, as stated above,
postulated the “ two states ” theory — namely that high - and low - spin states of
the compound I intermediate were responsible for differences in cytochrome
P450 reactivity.^46 Their calculations also showed that the hydroperoxoferric
intermediate should be a poor oxidant with a large energy barrier to over-
come.^47 However, their calculations did not explain why the T252A P450 CAM
mutant produced little or no hydroxylated camphor.
More recently, Shaik and co - workers used density functional theory as well
as QM(DFT)/MM calculations to show a pathway from the peroxoferric inter-
mediate ( 5a ) via the hydroperoxoferric ( 5b ) intermediate to compound I ( 6 )
(see Figure 7.14 ).^48 In Figure 7.16 , using Shaik ’ s terminology, the peroxoferric
intermediate is labeled ( 1 ), the hydroperoxoferric intermediate is labeled ( 2 ),
Cpd 0, and the ferryloxo intermediate is labeled ( 3 ), Cpd I.
The reference 48 authors used an UB3LYP unrestricted DFT method,
Jaguar 5.5 and Gaussian 03 software, beginning calculations with an
LACVP (Fe)6 - 31G (rest) basis set. See Section 4.4.3. The researchers assumed
a direct protonation mechanism involving a framework of amino acid residues