Kinetics of NO ligation with nitric-oxide synthase by flash photolysis andstopped-flow spectrophotometry

Nitric-oxide synthase (NOS) catalyzes conversion of L-arginine to nitric ox ide, which subsequently stimulates a host of physiological processes. Prior work suggests that NOS is inhibited by NO, providing opportunities for aut oregulation, This contribution reports that NO reacts rapidly (k(a) congrue nt to 2 x 10(7) M-1 s(-1)) with neuronal NOS in both its ferric and ferrous oxidation states. Association kinetics are almost unaffected by L-arginine or the cofactor tetrahydrobiopterin, There is no evidence for the distinct two phases previously reported for association kinetics of CO. Small amoun ts of geminate recombination of NO trapped in a protein pocket can be obser ved over nanoseconds, and a much larger amount is inferred to take place at picosecond time scales. Dissociation rates are also very fast from the fer ric form, in the neighborhood of 50 s(-1), when measured by extrapolating a ssociation rates to the zero NO concentration limit. Scavenging experiments give dissociation rate constants more than an order of magnitude slower: s till quite fast. For the ferrous species, extrapolation is not distinguisha ble from zero, while scavenging experiments give a dissociation rate consta nt near 10(-4) s(-1). Implications of these results for interactions near t he heme binding site are discussed.