Kinetics of CO and NO ligation with the Cys(331) -> Ala mutant of neuronalnitric-oxide synthase

Nitric-oxide syntheses (NOS) catalyze the conversion of L-arginine to NO, w hich then stimulates many physiological processes. In the active form, each NOS is a dimer; each strand has both a heme-binding oxygenase domain and a reductase domain. In neuronal NOS (nNOS), there is a conserved cysteine mo tif (CX4C) that participates in a ZnS4 center, which stabilizes the dimer i nterface and/or the flavoprotein-heme domain interface. Previously, the Cys (331) --> Ala mutant was produced, and it proved to be inactive in catalysi s and to have structural defects that disrupt the binding of L-Arg and tetr ahydrobiopterin (BH4). Because binding L-Arg and BH4 to wild type nNOS prof oundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has beh avior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding L-Arg and BH4 has little effect immediately but subst antial effect after extended incubation. 3) Incubation for 12 h restores be havior similar but not quite identical to that of wild type nNOS. Such incu bation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is rel ated to a structural rather than a catalytic role in maintaining active nNO S.