A glutamate is the essential proton transfer gate during the catalytic cycle of the [NiFe] hydrogenase.
Kinetic, EPR, and Fourier transform infrared spectroscopic analysis of Desulfovibrio fructosovorans [NiFe] hydrogenase mutants targeted to Glu-25 indicated that this amino acid participates in proton transfer between the active site and the protein surface during the catalytic cycle. Replacement of that glutamic residue by a glutamine did not modify the spectroscopic properties of the enzyme but cancelled the catalytic activity except the para-H(2)/ortho-H(2) conversion. This mutation impaired the fast proton transfer from the active site that allows high turnover numbers for the oxidation of hydrogen. Replacement of the glutamic residue by the shorter aspartic acid slowed down this proton transfer, causing a significant decrease of H(2) oxidation and hydrogen isotope exchange activities, but did not change the para-H(2)/ortho-H(2) conversion activity. The spectroscopic properties of this mutant were totally different, especially in the reduced state in which a non-photosensitive nickel EPR spectrum was obtained.
Dementin
Sébastien
S
Unité de Bioénergétique et Ingénierie des Protéines, Institut de Biologie Structurale et Microbiologie, Marseille, France.
Burlat
Bénédicte
B
De Lacey
Antonio L
AL
Pardo
Alejandro
A
Adryanczyk-Perrier
Géraldine
G
Guigliarelli
Bruno
B
Fernandez
Victor M
VM
Rousset
Marc
M
D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003901:Desulfovibrio / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D018698:Glutamic Acid / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared; D013997:Time Factors; D014961:X-Ray Diffraction
D011522:Protons; D001224:Aspartic Acid; D018698:Glutamic Acid; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen