[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis

[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis

^*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, and ^‡Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel, Commissariat à l’Energie Atomique-Centre National de la Recherche Scientifique, 41, Avenue des Martyrs, 38027 Grenoble CDX 1, France

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved July 23, 1998 (received for review June 1, 1998)

Abstract

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.

Footnotes

↵ † M.R. and Y.M. contributed equally to this work.
↵ § To whom reprint requests should be addressed. e-mail: hatch{at}ibsm.cnrs-mrs.fr.
This paper was submitted directly (Track II) to the Proceedings Office.
Data deposition: The coordinate reported in this paper has been deposited in the Protein Data Bank, Biology Department, Brookhaven National Laboratory, Upton, NY 11973 (PDB ID code 1frf).