Rapid calmodulin-dependent interdomain electron transfer in neuronal nitric-oxide synthase measured by pulse radiolysis

K Kobayashi, S Tagawa, S Daff, I Sagami, T Shimizu

Research output: Contribution to journalArticlepeer-review

Abstract

Electron transfer within rat neuronal nitric-oxide synthase (nNOS) was investigated by pulse radiolysis. Radiolytically generated 1-methyl-3-carbamoyl pyridinium (MCP) radical was found to react predominantly with the heme of the enzyme with a second-order rate constant for heme reduction of 3 x 10(8) m(-1) s(-1). In the calmodulin (CaM)-bound enzyme a subsequent first-order phase was observed which had a rate constant of 1.2 x 10(3) s(-1). In the absence of CaM, this phase was absent. Kinetic difference spectra for nNOS reduction indicated that the second phase consisted of heme reoxidation accompanied by formation of a neutral flavin semiquinone, suggesting that it is heme to flavin electron transfer. Experiments with the heme proximal surface mutant, K423E, had no second phase, confirming that the mutation blocks interdomain electron transfer. With the autoinhibitory loop deletion mutant, Delta 40, the slow phase was observed even in the absence of CaM consistent with the role of the loop in impeding interdomain electron transfer. The rate of heme to FMN electron transfer observed in the wild-type enzyme is similar to 1000 times faster than the FMN to heme electron transfer rate predicted during catalysis from kinetic modeling, suggesting that the catalytic process is slowed by kinetic gating.

Original languageEnglish
Pages (from-to)39864-39871
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number43
Publication statusPublished - 26 Oct 2001

Keywords

  • MILK XANTHINE DEHYDROGENASE
  • FLAVIN SEMIQUINONE
  • CYTOCHROME CD(1)
  • BINDING-SITE
  • L-ARGININE
  • HEME
  • REDUCTION
  • BRAIN
  • HYDROXYARGININE
  • OXYGENATION

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