Kinetics of NO ligation with nitric-oxide synthase by flash photolysis and stopped-flow spectrophotometry.
Nitric-oxide synthase (NOS) catalyzes conversion of L-arginine to nitric oxide, which subsequently stimulates a host of physiological processes. Prior work suggests that NOS is inhibited by NO, providing opportunities for autoregulation. This contribution reports that NO reacts rapidly (ka congruent with 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 amounts of geminate recombination of NO trapped in a protein pocket can be observed over nanoseconds, and a much larger amount is inferred to take place at picosecond time scales. Dissociation rates are also very fast from the ferric form, in the neighborhood of 50 s-1, when measured by extrapolating association rates to the zero NO concentration limit. Scavenging experiments give dissociation rate constants more than an order of magnitude slower: still quite fast. For the ferrous species, extrapolation is not distinguishable from zero, while scavenging experiments give a dissociation rate constant near 10(-4) s-1. Implications of these results for interactions near the heme binding site are discussed.
Scheele
J S
JS
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093-0358, USA.
Bruner
E
E
Kharitonov
V G
VG
Martásek
P
P
Roman
L J
LJ
Masters
B S
BS
Sharma
V S
VS
Magde
D
D
D000818:Animals; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010782:Photolysis; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013057:Spectrum Analysis
D011994:Recombinant Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat