Carboxyl terminus of inducible nitric oxide synthase. Contribution to NADPH binding and enzymatic activity.

Cloning of a nitric oxide synthase (NOS) from RAW 264.7 mouse macrophages (Xie, Q.-w., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T., and Nathan, C. (1992) Science 256, 225-228) yielded two sets of cDNA: one with a longer coding region of 1144 amino acids, whose sequence matched that of the purified protein, and another with a shorter coding region of 1122 amino acids, in which the last 10 carboxyl-terminal amino acids differed completely from those of the long form. We have now found that the short form lacks NOS activity. To determine the basis of this defect, we prepared recombinant chimeric, deletional, and point mutants of the long and short NOS variants, monitored their expression by immunoblot, and tested their enzymatic activity. By itself, lack of the 22-carboxyl-terminal residues of the long form NOS was scarcely consequential. Mutation of Phe1122, the only aromatic residue within one of the longest conserved regions shared by all NOSs of reported sequence, reduced enzymatic activity by 41%. Deletion of 23 carboxyl-terminal amino acids (including Phe1122) reduced activity by 71%. Further loss of Ile1121, another completely conserved residue, reduced activity by 95%, and with the deletion of the rest of the conserved region, NOS activity was undetectable. Normal dimerization and binding of heme and calmodulin by the short variants militated against distortions of tertiary structure affecting the amino-terminal half or middle portion of the protein. In contrast, the short variants were deficient in binding to NADPH, as predicted by a model of tertiary structure based on that of spinach ferredoxin-NADP+ reductase. This is the first demonstration that the carboxyl terminus of NOS is a functionally critical region.