MECHANISMS OF CHOSEN ENZYME REACTIONS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 113

1. Formation of a protein radical, for example Cys 151, RS, which promotes the
synchronous insertion of oxygen atoms across the substrate C-H bond (Waller and
Limscomb, 1996, Shilov, 1997). The absence of rearranged products of the radical clock
substrates for MMOH isolated from M. capsulatus raises the possibility in principle, of
such a mechanism.
2. Drawing a parallel of the compound Q to the cytochrome P450 Compound I
(Newcomb et al., 2000), a nonsynchronous concerted mechanism in MMO was
suggested . According to this mechanism, the difference in the bond vibration of C-H
and Fe-O bonds causes the insertion of oxygen atoms across the C-H bond via a
transition state in which the substrate possesses a radical character. Such an elementary
process is possible in the approach of the substrate to the ferryl oxygen.
3. A concerted mechanism assuming the elecrophylic attack of one oxygen atom of
the diiron ferryl to the C-H bond carbon with nucleophylic assistance of the second
oxygen atom with the formation of a pentavalent carbon intermediate (Shteinman, 1996).
4. Using the analogy of model reactions of alkane oxidation in mixtures of Fe(II) and
dioxygen in solvents, a mechanism invoking the formation of intermediate with an iron-
carbon bond followed by interaction with soxygen was proposed (Waller and Limscomb,
1996; Shilov, 1997).
The mechanism of C-H bond activation was examined in recent theoretical work
with the use of ab initio density functional methods (Dunietz et al., 2000; Gherman et
al., 2001).
3.4. Nitric Oxide Synthase
Nitric oxide (NO) is a tiny molecule with enormous biological impact. NO mediates a
large number of physiologic and pathophysiologic processes including vascular
relaxation, inhibition of platelet aggregation, regulation of endothelial cell adhesivity,
preservation of the normal vessel wall structure, etc. (Stuehr 1999; Stuehr and Ghosh,
2000); and references therein). NO is generated in an enzymatic process of oxidation of
L-arginine (Arg) by dioxygen catalyzed nitric oxide synthase (NOS) in the presence of
NADPH. The process involves stepwise oxidation of Arg to N-hydroxyl-L-arg, which is
converted to cirulline and nitric oxide. Both reactions occur within the hydroxylase
domain of NOS containing heme, the cofactor tetrahydrobiopterin and the Arg
binding site. The second, the reductase domain, containing FMN, FAD and NADPH,
provides electrons for the active site reduction. Two and 1.5 NADPH are consumed
for each NO. The third component of the enzyme system is calmodulin (CaM), which
lies between these two domains and promotes electron transfer from NADH to heme.
CaM binds only at certain concentrations of Three isomers of the enzyme are
intensively investigated: neuronal (nNOS), endothelial (eNOS), and cytokine-inducable
(iNOS).
A set of structural and kinetic investigations indicates that the heme active structure
of NOS and the mechanism of Arg hydroxylation are similar to those for cytochrome
P450 (Bec et al., 1998; Stuehr, 1999; Adak et al., 2001a,b; Abu-Saud et al., 2000; Wei et
al., 2001; Wolthers, 2002; Lange et al., 2001). The mechanism involves the reduction of