The C terminus of mouse macrophage inducible nitric-oxide synthase attenuates electron flow through the flavin domain.
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.
Roman
L J
LJ
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Miller
R T
RT
de La Garza
M A
MA
Kim
J J
JJ
Siler Masters
B S
BS
D000818:Animals; D004579:Electron Transport; D004926:Escherichia coli; D005415:Flavins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008264:Macrophages; D051379:Mice; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005415:Flavins; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse