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 investigate whether the histidines form a non-heme iron-binding site, we have expressed the H146A, H214A, H420A, H421A, and H461A mutants. The H420A mutant could not be isolated, and the H146A and H421A mutants were inactive. The H214A mutant resembled the wild-type enzyme in all respects. The H461A mutant had a low-spin heme, but high concentrations of L-Arg and tetrahydrobiopterin led to partial recovery of activity. Laser atomic emission showed that the only significant metal in NOS other than calcium and iron is zinc. The activities of the NOS isoforms were not increased by incubation with Fe(2+), but were inhibited by high Fe(2+) or Zn(2+) concentrations. The histidine mutations altered the ability of the protein to dimerize and to bind heme. However, the protein metal content, the inability of exogenous Fe(2+) to increase catalytic activity, and the absence of evidence that the conserved histidines form a metal site provide no support for a catalytic role for a non-heme redox-active metal.