[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] hydrog enase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S](2+,1+) clusters has been investigate d by using site-directed mutagenesis. Proline 238 of Desulfovibrio fru ctosovorans [NiFe] hydrogenase, which occupies the position of a poten tial ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme w ere investigated in terms of enzymatic activity, EPR and redox propert ies of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic s tudies 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 sign ificant alteration of the spectroscopic and redox properties of the tw o native [4Fe-4S] clusters and the NiFe center occurred. The significa nt decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutan t as compared with the wild-type enzyme. The implications of the resul ts for the role of the high-potential [3Fe-4S] cluster in the intramol ecular electron transfer pathway are discussed.