title: Identification of Amino Acids at the Catalytic Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase.
abstract: An in silico model of the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942, and information about active sites in related enzymes, had identified Cys148, Met149, Met306, Asp163, and Arg351 as amino acids likely to be involved in either nitrate binding, prosthetic group binding, or catalysis. Site-directed mutagenesis was used to alter each of these residues, and differences in enzyme activity and substrate binding of the purified variants were analyzed. In addition, the effects of these replacements on the assembly and properties of the Mo cofactor and [4Fe-4S] centers were investigated using Mo and Fe determinations, coupled with electron paramagnetic resonance spectroscopy. The C148A, M149A, M306A, D163N, and R351Q variants were all inactive with either the physiological electron donor, reduced ferredoxin, or the nonphysiological electron donor, reduced methyl viologen, as the source of electrons, and all exhibited changes in the properties of the Mo cofactor. Charge-conserving D163E and R351K variants were also inactive, suggesting that specific amino acids are required at these two positions. The implications for the role of these five conserved active-site residues in light of these new results and previous structural, spectroscopic, and mutagenesis studies for related periplasmic nitrate reductases are discussed.
authors: Department of Chemistry and Biochemistry, Texas Tech University , Lubbock, Texas 79409-1061, United States.
authors: Anurag P
authors: AP
authors: Srivastava
authors: Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287-1604, United States.
authors: James P
authors: JP
authors: Allen
authors: Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia , Athens, Georgia 30602-2556, United States.
authors: Brian J
authors: BJ
authors: Vaccaro
authors: Department of Chemistry and Biochemistry, Texas Tech University , Lubbock, Texas 79409-1061, United States.
authors: Masakazu
authors: M
authors: Hirasawa
authors: Department of Chemistry and Biochemistry, Texas Tech University , Lubbock, Texas 79409-1061, United States.
authors: Suzanne
authors: S
authors: Alkul
authors: Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia , Athens, Georgia 30602-2556, United States.
authors: Michael K
authors: MK
authors: Johnson
authors: Department of Chemistry and Biochemistry, Texas Tech University , Lubbock, Texas 79409-1061, United States.|Center for Biotechnology and Genomics, Texas Tech University , Lubbock, Texas 79409-3132, United States.
authors: David B
authors: DB
authors: Knaff
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D008982:Molybdenum / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009565:Nitrate Reductases / Q000737:chemistry* / Q000235:genetics; D046940:Synechococcus / Q000201:enzymology*
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D005288:Ferredoxins; D008982:Molybdenum; D009565:Nitrate Reductases