title: Influence of heme-thiolate in shaping the catalytic properties of a bacterial nitric-oxide synthase.
abstract: Nitric-oxide synthases (NOS) are heme-thiolate enzymes that generate nitric oxide (NO) from L-arginine. Mammalian and bacterial NOSs contain a conserved tryptophan (Trp) that hydrogen bonds with the heme-thiolate ligand. We mutated Trp(66) to His and Phe (W66H, W66F) in B. subtilis NOS to investigate how heme-thiolate electronic properties control enzyme catalysis. The mutations had opposite effects on heme midpoint potential (-302, -361, and -427 mV for W66H, wild-type (WT), and W66F, respectively). These changes were associated with rank order (W66H < WT < W66F) changes in the rates of oxygen activation and product formation in Arg hydroxylation and N-hydroxyarginine (NOHA) oxidation single turnover reactions, and in the O(2) reactivity of the ferrous heme-NO product complex. However, enzyme ferrous heme-O(2) autoxidation showed an opposite rank order. Tetrahydrofolate supported NO synthesis by WT and the mutant NOS. All three proteins showed similar extents of product formation (L-Arg → NOHA or NOHA → citrulline) in single turnover studies, but the W66F mutant showed a 2.5 times lower activity when the reactions were supported by flavoproteins and NADPH. We conclude that Trp(66) controls several catalytic parameters by tuning the electron density of the heme-thiolate bond. A greater electron density (as in W66F) improves oxygen activation and reactivity toward substrate, but decreases heme-dioxy stability and lowers the driving force for heme reduction. In the WT enzyme the Trp(66) residue balances these opposing effects for optimal catalysis.
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
authors.forename: Luciana
authors.initials: L
authors.lastname: Hannibal
authors.forename: Ramasamy
authors.initials: R
authors.lastname: Somasundaram
authors.forename: Jesús
authors.initials: J
authors.lastname: Tejero
authors.forename: Adjele
authors.initials: A
authors.lastname: Wilson
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006900:Hydroxylation; D020125:Mutation, Missense; D009249:NADP / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction
chemical_list: D001426:Bacterial Proteins; D006418:Heme; D009249:NADP; D019001:Nitric Oxide Synthase