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

被引:50
作者
Scheele, JS
Bruner, E
Kharitonov, VG
Martásek, P
Roman, LJ
Masters, BSS
Sharma, VS
Magde, D
机构
[1] Univ Calif San Diego, Dept Biochem & Chem, La Jolla, CA 92093 USA
[2] Univ Calif San Diego, Dept Med, La Jolla, CA 92093 USA
[3] Univ Texas, Hlth Sci Ctr, Dept Biochem, San Antonio, TX 78284 USA
关键词
D O I
10.1074/jbc.274.19.13105
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (k(a) congruent 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 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.
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页码:13105 / 13110
页数:6
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