Mammalian NO synthases catalyze the monooxygenation of L-arginine (L-Arg) to N-hydroxyarginine (NOHA) and the subsequent monooxygenation of this to NO and citrulline. Both steps proceed via formation of an oxyferrous heme complex and may ultimately lead to a ferrous NO complex, from which NO must be released. Electrochemical reduction of NO-bound neuronal nitric-oxide synthase ( nNOS) oxygenase domain was used to form the ferrous heme NO complex, which was found to be stable only in the presence of low NO concentrations, due to catalytic degradation of NO at the nNOS heme site. The reduction potential for the heme-NO complex was approximately - 140 mV, which shifted to 0 mV in the presence of either L-Arg or NOHA. This indicates that the complex is stabilized by 14 kJ mol(-1) in the presence of substrate, consistent with a strong H-bonding interaction between NO and the guanidino group. Neither substrate influenced the reduction potential of the ferrous heme CO complex, however. Both L-Arg and NOHA appear to interact with bound NO in a similar way, indicating that both bind as guanidinium ions. The dissociation constant for NO bound to ferrous heme in the presence of L-Arg was determined electrochemically to be 0.17 nM, and the rate of dissociation was estimated to be 10(-4) s(-1), which is much slower than the rate of catalysis. Stopped-flow kinetic analysis of oxyferrous formation and decay showed that both L-Arg and NOHA also stabilize the ferrous heme dioxy complex, resulting in a 100-fold decrease in its rate of decay. Electron transfer from the active-site cofactor tetrahydrobiopterin (H4B) has been proposed to trigger the monoxygenation process. Consistent with this, substitution by the analogue/ inhibitor 4-amino-H4B stabilized the oxyferrous complex by a further two orders of magnitude. H4B is required, therefore, to break down both the oxyferrous and ferrous nitrosyl complexes of nNOS during catalysis. The energetics of these processes necessitates an electron donor/acceptor operating within a specific reduction potential range, defining the role of H4B.
- SINGLE-TURNOVER CONDITIONS
- REDUCTASE DOMAIN