[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis.
The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.
Rousset
M
M
Unité de Bioénergétique et Ingéniérie des Protéines, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, 31, Chemin Joseph Aiguier, 13402 Marseille CDX 20, France.
Montet
Y
Y
Guigliarelli
B
B
Forget
N
N
Asso
M
M
Bertrand
P
P
Fontecilla-Camps
J C
JC
Hatchikian
E C
EC
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D003901:Desulfovibrio / Q000201:enzymology* / Q000235:genetics*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007501:Iron / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013455:Sulfur / Q000737:chemistry
D008024:Ligands; D011994:Recombinant Proteins; D013455:Sulfur; D011392:Proline; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D003545:Cysteine