Mutation of the five conserved histidines in the endothelial nitric-oxide synthase hemoprotein domain - No evidence for a non-heme metal requirement for catalysis

Five conserved histidine residues are found in the human endothelial nitric -oxide synthase (NOS) heme domain: His-420, His-421, and His-461 are close to the heme, whereas His-146 and His-214 are some distance away. To investi gate whether the histidines form a nonheme iron-binding site, we have expre ssed the H146A, H214A, H420A, H421A, and H461A mutants, The H420A mutant co uld not be isolated, and the H146A and H421A mutants were inactive, The H21 4A mutant resembled the wild-type enzyme in all respects. The H461A mutant had a low-spin heme, but high concentrations of L-Arg and tetrahydrobiopter in led to partial recovery of activity. Laser atomic emission showed that t he only significant metal in NOS other than calcium and iron is zinc, The a ctivities of the NOS isoforms were not increased by incubation with Fe2+, b ut were inhibited by high Fe2+ or Zn2+ concentrations. The histidine mutati ons altered the ability of the protein to dimerize and to bind heme, Howeve r, the protein metal content, the inability of exogenous Fe2+ to increase c atalytic activity, and the absence of evidence that the conserved histidine s form a metal site provide no support for a catalytic role for a non-heme redox-active metal.