MECHANISMS OF CHOSEN ENZYME REACTIONS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 112

formation of the hydroxylated product (rebound mechanism). The formation of radical
and carbocation intermediates is assumed on the basis of the formation of 1-
methylcyclobutanol from the substrate (Ruzicka et al., 1990).
The rebound mechanism is not unequivocally supported by the radical clock
investigation of MMO from M. capsulatus (Liu et al., 1993). The intramolecular kinetic
isotope effect of indicates the involvement of the substrate C-H bond in an
elementary act of hydroxylation. Nevertheless, products, expected in the case of radical
intermediate rearrangement with have not been detected. In a recent
investigation (Jin and Lipscomb, 2000), the rearrangement products were observed
during oxidation of 1,1,2,2-tetramethylcyclopropene with the rate constant of the carbon
centered radical rearrangement at 30° but not for cis- or trans-1.2-
dimethyl cyclopropene or trans-2-phenylmethylcyclopropane
The authors concluded that the bulky radical clocks have sterical obstacles
to recombination and, therefore, can be rearranged before recombination. In contrast,
more elegant substrates produce radicals able to reach the reduced diiron ferryl cluster.
Carbon-centered radical intermediates were proved by the spin-trapping technique in
reactions of MMO from M. capsulatus (Bath) (Deighton et al., 1991). Results of elegant
experiments with chiral substrates (R)-and in reaction of the enzyme
from M. trichosporium and M. capsulatus were reported (Priestly et al., 1992).
According to data, both (R)-and ethane underwent about 35%
inversion of configuration. This means that a radical intermediate, ethyl radical, can
rearrange its configuration in the active site before the formation of ethanol.
Strong evidence in favor of the rebound mechanism was obtained in experiments on
the kinetic isotope effect (KIe). Upon hydroxylation of methane and ethane catalyzed by
MMO in the steady-state kinetics condition, relatively low KIE was
observed (Belova et al., 1976)). A very high KIE (50-100) in the decay of compound Q
in the presence of and was reported (Waller and Limscomb, 1996). The use of
and showed a linear decrease of the decay rate constant. These
results were interpreted as support for the rebound mechanism. The observed KIE is
significantly high than the KIE detected in other hydrogen and proton transfer reactions
(Section 1.2.1). Such high values of KIE can be explained in the framework of a “quasi-
reversible” mechanism of the reaction in the active site (Waller and Lipscomb, 1996).
According to this mechanism, the hydrogen abstraction reaction can be generated in an
altered form of compound Q (Q'). This form is equilibrium in the compound Q-substrate
complex and with compounds

and which make possible secondary

processes.
The mixed-valent [Fe(II)Fe(III)] state of MMOH from has the ability to
accommodate simultaneously several molecules (methanol, water and DMSO) as
recently demonsrated by ENDOR spectroscopy (Willems et al., 1998). The structure of
the binuclear iron-methanol complex and the detailed mechanism of the complex
dissociation were investigated with the use of density function methods (Bash et al.,
2001a.b).
Among other discussed concepts concerning the MMO substrate hydroxylation in the
compound Q active site, the following suggested mechanisms should be mentioned.