文摘
Nitric oxide synthases (NOSs) are flavo-heme enzymes that require (6R)-tetrahydrobiopterin(H4B) for activity. Our single-catalytic turnover study with the inducible NOS oxygenase domain showedthat a conserved Trp that interacts with H4B (Trp457 in mouse inducible NOS) regulates the kinetics ofelectron transfer between H4B and an enzyme heme-dioxy intermediate, and this in turn alters the kineticsand extent of Arg hydroxylation [Wang, Z.-Q., et al. (2001) Biochemistry 40, 12819-12825]. To investigatethe impact of these effects on NADPH-driven NO synthesis by NOS, we generated and characterized theW457A mutant of inducible NOS and the corresponding W678A and W678F mutants of neuronal NOS.Mutant defects in protein solubility and dimerization were overcome by purifying them in the presenceof sufficient Arg and H4B, enabling us to study their physical and catalytic profiles. Optical spectra of theferric, ferrous, heme-dioxy, ferrous-NO, ferric-NO, and ferrous-CO forms of each mutant were similarto that of the wild type. However, the mutants had higher apparent Km values for H4B and in one mutantfor Arg (W457A). They all had lower NO synthesis activities, uncoupled NADPH consumption, and aslower and less prominent buildup of enzyme heme-NO complex during steady-state catalysis. Furtheranalyses showed the mutants had normal or near-normal heme midpoint potential and heme-NO complexreactivity with O2, but had somewhat slower ferric heme reduction rates and markedly slower reactivitiesof their heme-dioxy intermediate. We conclude that the conserved Trp (1) has similar roles in two differentNOS isozymes and (2) regulates delivery of both electrons required for O2 activation (i.e., kinetics offerric heme reduction by the NOS flavoprotein domain and reduction of the heme-dioxy intermediate byH4B). However, its regulation of H4B electron transfer is most important because this ensures efficientcoupling of NADPH oxidation and NO synthesis by NOS.
