Nitric Oxide Synthase: Catalytic Model for Reductase Function

Abstract

The NOS reductase unit, comprising three C terminal domains, is closely related to homologs studied intensively for many years. Several groups proposed ping‐pong and two state ping‐pong models for NADPH P450 reductase (e.g., Sem and Kasper, 1994). These proposals were adapted to NOS (Wolthers and Schimerlik, 2002) and shown to describe the kinetics of NADPH cytochrome c reductase activity at low substrate concentrations.We previously constructed a King‐Altman 'Ligand Restricted Conformational Shuttle' (LRCS) model based on an FMN domain shuttle in which cytochrome c interacts with FMN when the FMN binding domain is dissociated from the reductase complex. Here we show that this model explains details of NADPH cytochrome c reductase activity at high and low substrate concentrations without extra inhibitory weak binding sites. This includes potent substrate inhibition at high concentrations of cytochrome c as well as pseudo ping‐pong behavior at low substrate concentrations. The major effect of calmodulin binding on nNOS and eNOS reductase activities is to increase dissociation of the FMN binding domain, making it accessible to cytochrome c for electron transfer. Because of its size and specific interaction with the FMN binding domain, cytochrome c is a good model for NOS catalytic electron transfer. DCIP and ferricyanide reduction do not require similar large scale conformational change.