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 resembl e that of NADPH-cytochrome P450 reductase (CPR), with the exception of a fe w regions. One such region is the C terminus; all NOS isoforms are 20-40 am ino acids longer than CPR, forming a "tail" that is absent in CPR. To inves tigate its function, we removed the 21-amino acid C-terminal tail from muri ne macrophage inducible NOS (MOS) holoenzyme and from a flavin domain const ruct. Both the truncated holoenzyme and reductase domain exhibited cytochro me c reductase activities that were 7-10-fold higher than the nontruncated forms. The truncated holoenzyme catalyzed NO formation approximately 20% fa ster than the intact form. Using stopped-flow spectrophotometry, we demonst rated 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-termin al tail. The heme-nitrosyl complex, formed in all NOS isoforms during NO ca talysis, is 8-fold less stable in truncated iNOS. Although both CPR and int act NOS can exist in a stable, one electron-reduced semiquinone form, neith er the truncated holoenzyme nor the truncated flavin domain demonstrate suc h 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.