Kinetic characterization of Desulfovibrio gigas hydrogenase upon selectivechemical modification of amino acid groups as a tool for structure-function relationships

The effect of amino acid residues modification of Desulfovibrio gigas hydro genase on different activity assays is reported. The first method consisted in the modification of glutamic and aspartic acid residues of the enzyme w ith ethylenediamine in order to change the polarity of certain regions of t he protein surface. The second method consisted in the modification of hist idine residues with a Ru complex in order to change the acid-base propertie s of the histidine residues. The implication of these modifications in the enzyme kinetics has been studied by measuring in parallel the activities of para/ortho hydrogen conversion, deuterium/hydrogen exchange and dyes reduc tion with hydrogen. Our experimental data support some hypothesis based on the three-dimensional structure of this enzyme: (a) electrostactic interact ions between the hydrogenase and the redox partner play an essential role i n the kinetics; (b) the histidine ligand and the surrounding acidic residue s of the distal [4Fe4S] cluster form the recognition site of the redox part ner of the hydrogenase; and (c) histidine residues are involved in the hydr on transfer pathway of the hydrogenase. (C) 2000 Elsevier Science B.V. All rights reserved.