116 Chapter 4

The heme-thiolate enzymes cytochromes P450, chloroperoxidase, and nitric oxide synthase all activate dioxygen to oxidize substrates. In each of these enzymes, there is a conserved hydrogen bonding network around the proximal thiolate ligand. These hydrogen bond donors come predominantly from backbone amide groups and help to tune the electronics of the heme center. However, in nitric oxide synthase one of these three hydrogen-bond donating groups comes from the side chain of a tryptophan residue, making nitric oxide synthases unique. Three mutant forms of the nitric oxide synthase from Geobacillus stearothermophilus were expressed in E. coli. These mutants each have a single point mutation, converting this native tryptophan residue to a histidine, phenylalanine, or tyrosine. The reactivity of each the wild type enzyme and the three new mutants were tested using stopped-flow mixing coupled with UV-visible absorption spectroscopy and the Griess Assay. Autoxidation rates measured by stopped-flow suggest that the Tyr and Phe mutants do indeed have significantly more negative reduction potentials, but that the His mutant is particularly slow to oxidize. The Griess Assay