MECHANISMS OF CHOSEN ENZYME REACTIONS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 105

mechanism of the substrates oxidation catalyzed by six isozymes of hepatic P450 2B1.
Two principle finding were observed: 1) ratios of the products related to putative
“radical” and “non-radical” reaction pathways were found to be within a range 8-20
indicating only a small contribution of the former process, 2) the chemical nature of the
products indicates at least two active species are involved in the substrate oxidation
process. These experimental data were interpreted in the framework of the following
description: 1) because the rate of rearrangement of the 'clock” carbon-centred radicals
in a free state is very short (80-100 fs), formation of a majority of the products via
radicals produced in the active site is excluded, 2) two electrophylic oxidants are
produced in the natural course of P450 oxidation reaction, a peroxo-iron species and a
hydroperoxo species and 3) hydroxylation by both species occurs by a mechanism which
is similar to the Hamilton “oxenoid” mechanism, e.g. insertion of oxygen atoms across
the substrate C-H bond, 4) in the case of the hydroperoxo species the insertion runs as a
concerted process in which an oxygen atom attacks the substrate carbon with
simultaneous protonation of the atom and rupture of the species O-O bond. The first
product of the process is protonated alcohol. An analysis of products of epoxidation and
hydroxylation of olef

s by cytochrome P450 2B4 (Vaz et al., 1998) also supports the
concept that two species with different electrophilic properties hydroxo-iron and oxi-
iron, can affect epoxidation.
Apparently contradicting evidence for and against radical and non-radical
mechanisms of hydroxylation and epoxidation caused by the thermodynamic allowance
of different reaction pathways and the possible involvement of several active oxidizing
species, aroused special interest in the theoretical analysis of putative mechanisms of
these processes.
A theoretical model for the cytochrome P-450 hydroxylation of saturated cyclic
hydrocarbons (quadricyclane, cyclopropane) suggested by Bach et al., (1995) implicates
the formation of symmetrically bridged complex in the coordination sphere of the
heme ferric iron atom followed by its consequent transformation to an epoxide-like
positively charged complex. After the hetrolytic O-O bond cleavage, the complex
produces a cation which inserts across the substrate C-H bond by a barrierless
concerted mechanism. Two alternative mechanisms of hydroxylation catalyzed by
cytochrome P450, synchronous insertion of oxygen atom across C-H bond and a
synchronous two- step rebound process, were recently discussed (de Visser et al.,
2001a,b.c). Density function calculations and the conservation of orbital symmetry
analysis were performed to analyze energy and quantum mechanical factors affected by
the reaction of the ferryl structure [HS-Por-Fe=O] with ethane. The barrier for the
synchronous reaction was estimated to be at least 4 kcal/mole higher than one for the
asynchronous process. The estimation also indicated that the barrier for asynchronous
stepwise epoxidation of ethylene is about 11 cal/mole lower than for the synchronous
insertion. It was also stressed that the addition of O to

in

(or is a symmetry

forbidden process.
The first investigations of the cytochrome P450 by physicochemical methods
unequivocally indicated that the enzyme heme iron could exist in low- and high-spin
states, depending on reduction, binding of substrates, temperature, pH and chemical
modification (Peisach etal.1972; Coon et al., 1981; and references therein). According to