CHARACTERIZATION OF HEME-DEFICIENT NEURONAL NITRIC-OXIDE SYNTHASE REVEALS A ROLE FOR HEME IN SUBUNIT DIMERIZATION AND BINDING OF THE AMINO-ACID SUBSTRATE AND TETRAHYDROBIOPTERIN

Neuronal nitric-oxide (NO) synthase contains FAD, FMN, heme, and tetra hydrobiopterin as prosthetic groups and represents a multifunctional o xidoreductase catalyzing oxidation of L-arginine to L-citrulline and N O, reduction of molecular oxygen to superoxide, and electron transfer to cytochromes. To investigate how binding of the prosthetic heme moie ty is related to enzyme activities, cofactor, and L-arginine binding, as well as to secondary and quaternary protein structure, we have puri fied and characterized heme-deficient neuronal NO synthase. The heme-d eficient enzyme, which had preserved its cytochrome c reductase activi ty, contained FAD and FMN, but virtually no tetrahydrobiopterin, and e xhibited only marginal NO synthase activity. By means of gel filtratio n and static light scattering, we demonstrate that the heme-deficient enzyme is a monomer and provide evidence that heme is the sole prosthe tic group controlling the quaternary structure of neuronal NO synthase . CD spectroscopy showed that most of the structural elements found in the dimeric holoenzyme were conserved in heme-deficient monomeric NO synthase. However, in spite of being properly folded, the heme-deficie nt enzyme did bind neither tetrahydrobiopterin nor the substrate analo g N-G-nitro-L-arginine. Our results demonstrate that the prosthetic he me group of neuronal NO synthase is requisite for dimerization of enzy me subunits and for the binding of amino acid substrate and tetrahydro biopterin.