Roles of the heme proximal side residues tryptophan409 and tsyptophan421 of neuronal nitric oxide synthase in the electron transfer reaction

摘要

Nitric oxide synthase (NOS) has an oxygenase domain with a thiol-coordinated heme active side similar to cytochrome P450. In contrast to cytochrome P450, however, conserved aromatic amino acids are situated in the heme proximal side of NOS. For example, in endothelial NOS (eNOS), the indole-ring nitrogen of Trp180 hydrogen-binds to the thiol of Cys186, the internal axial ligand to the heme. And, the aromatic side chain of Trp192 forms a bridge between this residue and the protein. Trp180 and Trp192 of eNOS correspond to Trp409 and Trp421 of neuronal NOS (nNOS), respectively. In order to understand the roles of the aromatic amino acids in catalysis, we generated Trp409His, Trp409Leu, Trp421His and Trp421Leu mutants of nNOS and determined their catalytic parameters. The Trp409Leu mutant was very poorly expressed in E. coli and was easily denatured during purification procedures. The NO formation activities of the Trp409His and Trp421Leu mutants were 11 and 25 mu mol/min per mu mol heme, respectively, and are lower than that (44 mu mol/min per mu mol heme) of the wild type. The activity (46 mu mol/min per mu mol heme) of the Trp421His mutant was comparable to that of the wild-type enzyme. However, NADPH oxidation rates of Trp421His (230 mu mol/min per mu mol heme) and Trp421Leu (104 mu mol/min per mu mol heme) in the presence of L-Arg were much larger than those observed for the wild type (65 mu mol/min per mu mol heme) and the Trp409His mutant (43 mu mol/min per mu mol heme). The cytochrome c reduction rate of the Trp421His mutant was 6-fold larger than that of the wild type. The heme reduction rate with NADPH for the Trp421His mutant (0.09 min(-1)) was much lower than that (1.0 min(-1)) of the wild type. Taken together, it appears that Trp421 may be involved in inter-domain/inter-subunit electron transfer reactions. (C) 2000 Elsevier Science B.V. All rights reserved. [References: 32]