Spectroscopic and kinetic characterization of active site mutants of Desulfovibrio fructosovoransNi-Fe hydrogenase.
Site-directed mutagenesis of amino acid residues proximate to the active site of the Ni-Fe hydrogenase of Desulfovibrio fructosovorans has been done. The different mutants have been analyzed by FTIR spectroscopy and compared with wild type enzyme. The changes observed in the spectra confirm that hydrogen bonds between the CN(-) ligands of the active site's Fe atom and certain neighbor amino acid residues stabilize the active center within the protein matrix. However, kinetic analysis of the mutants indicates that none of the replaced residues have an important role in the catalytic mechanism of the hydrogenase.
DeLacey
Antonio L
AL
Instituto de Catalisis, CSIC, Campus de Cantoblanco, 28049 Madrid, Spain. alopez@icp.csic.es
Fernandez
Victor M
VM
Rousset
Marc
M
Cavazza
Christine
C
Hatchikian
E Claude
EC
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D003486:Cyanides / Q000737:chemistry; D003901:Desulfovibrio / Q000201:enzymology*; D006860:Hydrogen Bonding; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D017186:Titrimetry
D000596:Amino Acids; D003486:Cyanides; D008024:Ligands; C043296:nickel-iron hydrogenase; D006864:Hydrogenase