RESUMO
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.
Assuntos
Desulfovibrio/enzimologia , Desulfovibrio/genética , Hidrogenase/química , Hidrogenase/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalografia por Raios X , Cisteína/química , Eletroquímica , Espectroscopia de Ressonância de Spin Eletrônica , Hidrogenase/metabolismo , Ferro/química , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Prolina/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Enxofre/químicaRESUMO
The 2.54 A resolution structure of Ni-Fe hydrogenase has revealed the existence of hydrophobic channels connecting the molecular surface to the active site. A crystallographic analysis of xenon binding together with molecular dynamics simulations of xenon and H2 diffusion in the enzyme interior suggest that these channels serve as pathways for gas access to the active site.
Assuntos
Gases/metabolismo , Hidrogenase/química , Hidrogenase/metabolismo , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Desulfovibrio/enzimologia , Hidrogênio/metabolismo , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Xenônio/metabolismoRESUMO
Hydrogenases are proteins which metabolize the most simple of chemical compounds, molecular hydrogen, according to the reaction H2<-->2H+ + 2e-. These enzymes are found in many microorganisms of great biotechnological interest such as methanogenic, acetogenic, nitrogen fixing, photosynthetic or sulfate-reducing bacteria. The X-ray structure of a dimeric [NiFe] hydrogenase together with a wealth of biophysical, biochemical and genetic studies have revealed that the large subunit contains the bimetallic [Ni-Fe] active site, with biologically uncommon CO and CN ligands to the iron, whereas the small subunit contains three iron-sulfur cluster. During catalysis, the nickel atom is most likely responsible for a base-assisted heterolytic cleavage of the hydrogen molecule whereas the iron atom could be redox active. Specific channels are probably required for the transfer of the chemical reaction partners (H2, H+ and e-) between the active site, deeply buried inside the protein, and the molecular surface. The generation of a functional enzyme, including the assembly of the complex catalytic center, requires maturation and involves a large number of auxiliary proteins which have been partly characterized by molecular biology.
