Control of electron transfer in neuronal NO synthase

The nitric oxide synthases (NOSs) are dimeric flavocytochromes consisting o f an oxygenase domain with cytochrome P450-like Cys-ligated haem, coupled t o a diflavin reductase domain, which is related to cytochrome P450 reductas e. The NOSs catalyse the sequential mono-oxygenation of arginine to N-hydro xyarginine and then to citrulline and NO. The constitutive NOS isoforms (cN OSs) are regulated by calmodulin (CaM), which binds at elevated concentrati ons of free Ca2+, whereas the inducible isoform binds CaM irreversibly. One of the main structural differences between the constitutive and inducible isoforms is an insert of 40-50 amino acids in the FMN-binding domain of the cNOSs. Deletion of the insert in rat neuronal NOS (nNOS) led to a mutant e nzyme which binds CaM at lower Ca2+ concentrations and which retains activi ty in the absence of CaM. In order to resolve the mechanism of action of Ca M activation we determined reduction potentials for the FMN and FAD cofacto rs of rat nNOS in the presence and absence of CaM using a recombinant form of the reductase domain. The results indicate that CaM binding does not mod ulate the reduction potentials of the flavins, but appears to control elect ron transfer primarily via a large structural rearrangement, We also report the creation of chimaeric enzymes in which the reductase domains of nNOS a nd flavocytochrome P450 BM3 (Bacillus megaterium III) have been exchanged. Despite its very different flavin redox potentials, the BM3 reductase domai n was able to support low levels of CaM-dependent NO synthesis, whereas the NOS reductase domain did not effectively substitute for that of cytochrome P450 BM3.