Kinetics of CO ligation with nitric-oxide synthase by flash photolysis and stopped-flow spectrophotometry

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

Research output: Contribution to journalArticle

41 Scopus citations

Abstract

Interaction of CO with hemeproteins has physiological importance. This is especially true for nitric-oxide synthases (NOS), heme/flavoenzymes that produce ·NO and citrulline from L-arginine (Arg) and are inhibited by CO in vitro. The kinetics of CO ligation with both neuronal NOS and its heme domain module were determined in the presence and absence of tetrahydrobiopterin and Arg to allow comparison with other hemeproteins. Geminate recombination in the nanosecond time domain is followed by bimolecular association in the millisecond time domain. Complex association kinetics imply considerable heterogeneity but can be approximated with two forms, one fast (2-3 x 106 M-1 s-1) and another slow (2-4 x 104 M-1 s1-). The relative proportions of the two forms vary with conditions. For the heme domain, fast forms dominate except in the presence of both tetrahydrobiopterin and Arg. In the holoenzyme, slow forms dominate except when both reagents are absent. Geminate recombination is substantial, ~50%, only when fast forms predominate. Stopped-flow mixing found dissociation constants near 0.3 s- 1. These data imply an equilibrium constant such that very little CO should bind at physiological conditions unless large CO concentrations are present locally.

Original languageEnglish (US)
Pages (from-to)12523-12528
Number of pages6
JournalJournal of Biological Chemistry
Volume272
Issue number19
DOIs
StatePublished - May 9 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Scheele, J. S., Kharitonov, V. G., Martásek, P., Roman, L. J., Sharma, V. S., Masters, B. S. S., & Magde, D. (1997). Kinetics of CO ligation with nitric-oxide synthase by flash photolysis and stopped-flow spectrophotometry. Journal of Biological Chemistry, 272(19), 12523-12528. https://doi.org/10.1074/jbc.272.19.12523