IDENTIFICATION OF THE DOMAINS OF NEURONAL NITRIC-OXIDE SYNTHASE BY LIMITED PROTEOLYSIS

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as subs trates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofa ctors. The protein consists of a central calmodulin-binding sequence f lanked on the N-terminal side by a haem-binding region, analogous to c ytochrome P-450, and on the C-terminal side by a region homologous wit h NADPH:cytochrome P-450 reductase. The structure of recombinant rat b rain nitric oxide synthase was analysed by limited proteolyis. The pro ducts were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three f ragments, similar to those in a previous report [Sheta, McMillan and M asters (1994) J. Biol. Chem. 269, 15147-15153]: that of M(r) approx. 1 35 000 (N-terminus Gly-221) resulted from loss of the N-terminal exten sion (residues 1-220) unique to neuronal nitric oxide synthase. The fr agments of M(r) 90 000 (haem region) and 80 000 (reductase region, N-t erminus Ala-728) were produced by cleavage within the calmodulin-bindi ng region. With more extensive trypsin treatment, these species were s hown to be transient, and three smaller, highly stable fragments of M( r) 14 000 (N-terminus Leu-744 within the calmodulin region), 60 000 (N -terminus Gly-221) and 63 000 (N-terminus Lys-856 within the FMN domai n) were formed. The species of M(r) approx. 60 000 represents a domain retaining haem and nitroarginine binding. The two species of M(r) 63 000 and 14 000 remain associated as a complex. This complex retains cy tochrome c reductase activity, and thus is the complete reductase regi on, yet cleaved at Lys-856. This cleavage occurs within a sequence ins ertion relative to the FMN domain present in inducible nitric oxide sy nthase. Prolonged proteolysis treatment led to the production of a pro tein of M(r) approx. 53 000 (N-terminus Ala-953), corresponding to a c leavage between the FMN and FAD domains. The major products after chym otryptic digestion were similar to those with trypsin, although the pa thway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These d ata have allowed us to identify stable domains representing both the a rginine/haem-binding and the reductase regions.