The C terminus of mouse macrophage inducible nitric-oxide synthase attenuates electron flow through the flavin domain

Linda J. Roman, R. Timothy Miller, Melissa A. De La Garza, Jung Ja P. Kim, Bettie Sue Siler Masters

Research output: Contribution to journalArticle

64 Scopus citations

Abstract

The sequences of nitric-oxide synthase (NOS) flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR), with the exception of a few regions. One such region is the C terminus; all NOS isoforms are 20 - 40 amino acids longer than CPR, forming a 'tail' that is absent in CPR. To investigate its function, we removed the 21-amino acid C-terminal tail from murine macrophage inducible NOS (iNOS) holoenzyme and from a flavin domain construct. Both the truncated holoenzyme and reductase domain exhibited cytochrome c reductase activities that were 7 - 10-fold higher than the nontruncated forms. The truncated holoenzyme catalyzed NO formation approximately 20% faster than the intact form. Using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins and from the flavin to the heme domain is 2 - 5-fold faster in the absence of the C-terminal tail. The heme-nitrosyl complex, formed in all NOS isoforms during NO catalysis, is 5-fold less stable in truncated iNOS. Although both CPR and intact NOS can exist in a stable, one electron-reduced semiquinone form, neither the truncated holoenzyme nor the truncated flavin domain demonstrate such a form. We propose that this C-terminal tail curls back to interact with the flavin domain in such a way as to modulate the interaction between the two flavin moieties.

Original languageEnglish (US)
Pages (from-to)21914-21919
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number29
DOIs
StatePublished - Jul 21 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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