MECHANISMS OF CHOSEN ENZYME REACTIONS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 114

heme, binding and its activation by the second electron and proton transfer with the
formation of oxoferryl structure [S-Fe(IV)=O] similar to those of peroxidase Compound
I but with sulfur ligand instead of histidine (Fig. 3.14). According to the NOS
crystallographic model (Crane et al., 1998; Raman, 1998; Fishmann; 1999), the
guanidinium group of Arg is adjacent to the heme ring and can be directly attacted by the
[S-Fe(IV)=O] center. In contrast, the cofactor is located at the opposite site of the
heme ring. Kinetics of argenine hydroxylation catalyzed by NOS is shown in Fig. 3.14
(Wei et al., 2001)
Recent investigations have shed light on peculiarities of the NOS action mechanism:
the role of the cofactor and CaM, and cooperativity in kinetic and thermodynamic
properties of different components of the nitric oxide synthesis system. Stop flow
experiments with eNOS (Abu-Soud et al., 2000) showed that calmodulin binding caused
an increase in NADH-dependent flavin reduction from 0.13 to at 10 °C. Under
such conditions, in the presence of Arg, heme is reduced very slowly Heme
complex formation requires a relatively high concentration of NO (>50 nM) and inhibits
the entire process: NADH oxidation and citrulline synthesis decreases 3-fold and
increases 3-fold. NOS reactions were monitored at subzero temperatures in the presence
of 50% ethylene glycol as an anti-freeze solvent (Bec et al., 1998).

At 30°C in the absence of Arg, the ferrous-oxi complex transforms very slowly to the
ferric state. In the presence of substrate and a new species with the 12-nm shifted
Sorey band is detected. A decay of this species is accompanied by the formation of
Because the presence of is necessary for these reactions, the
main function of this compound is to be a reducing agent. This suggestion is supported
by experiments on the stabilizing effect of ascorbic acid on the chemical stabilization of
tetrahydropterin in the endothelial nitric oxide synthesis (Heller et al., 2001). At the
same time, a significant increase in the half lifetime of in solution is demonstrated.
As is shown (Wei et al., 2001), a ferrous-dioxy intermediate in iNOS forms for
and then is transformed to the [S-Fe(IV)=O] state. The rate of the [S-Fe(IV)=O] decay is
equal to the rate of radical formation and the rate of Arg hydroxylation. In contrast,