Abstract
Nitric-oxide synthases (NOS) catalyze the conversion of l-arginine to NO, which 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 motif (CX(4)C) that participates in a ZnS(4) center, which stabilizes the dimer interface and/or the flavoprotein-heme domain interface. Previously, the Cys(331) --> Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding of l-Arg and tetrahydrobiopterin (BH(4)). Because binding l-Arg and BH(4) to wild type nNOS profoundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has behavior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding l-Arg and BH(4) has little effect immediately but substantial effect after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS. Such incubation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is related to a structural rather than a catalytic role in maintaining active nNOS.
Highlights
Nitric-oxide synthases (NOS)1 constitute a family of hemethiolate-liganded proteins that catalyze the conversion of L-Arg to NO and L-citrulline, requiring NADPH and molecular O2 [1]
Because conformational defects on either the proximal or distal side of the heme can have dramatic effects on ligand binding, it seemed useful to us to characterize the kinetics of heme ligation for CO and NO
The kinetics of CO binding for both the C331A mutant and WT Neuronal nitric-oxide synthase (nNOS) display two phases for concentration-dependent bimolecular combination that differ in rate by a factor of 100
Summary
Nitric-oxide synthases (NOS) constitute a family of hemethiolate-liganded proteins that catalyze the conversion of L-Arg to NO and L-citrulline, requiring NADPH and molecular O2 [1]. A recent study [9] prepared nNOS (rat brain) protein with the point mutation C331A expressed in Escherichia coli. Because L-Arg substrate and BH4 bind near the heme, the findings [9] suggested that “the primary dysfunction . As to the reason for that dysfunction, the suggestion was made that Cys331 is required for binding zinc at a tetrathiolate site [10] (discovered just as the work with the mutant was being completed) and that loss of zinc perturbs the structure, especially near the heme site. Because conformational defects (as might be introduced by mutations) on either the proximal or distal side of the heme can have dramatic effects on ligand binding, it seemed useful to us to characterize the kinetics of heme ligation for CO and NO. That would test the conclusion that the pocket is initially distorted and would provide a very sensitive test of the ability of incubation with L-Arg to restore normal functionality
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