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

Jürgen S. Scheele, Eric Bruner, Vladimir G. Kharitonov, Pavel Martásek, Linda J. Roman, Bettie Sue Siler Masters, Vijay S. Sharma, Douglas Magde

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

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) ≃ 2 x 107 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.

Original languageEnglish (US)
Pages (from-to)13105-13110
Number of pages6
JournalJournal of Biological Chemistry
Volume274
Issue number19
DOIs
StatePublished - May 7 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Scheele, J. S., Bruner, E., Kharitonov, V. G., Martásek, P., Roman, L. J., Masters, B. S. S., Sharma, V. S., & Magde, D. (1999). Kinetics of NO ligation with nitric-oxide synthase by flash photolysis and stopped-flow spectrophotometry. Journal of Biological Chemistry, 274(19), 13105-13110. https://doi.org/10.1074/jbc.274.19.13105