INSIGHT INTO VANADIUM CHEMISTRY IN VANADIUM HALO-PEROXIDASES: X-RAY STRUCTURES OF MUTANTS AND FUNCTIONAL DERIVATIVES OF CHLOROPEROXIDASE FROM CURVULARIA INAEQUALIS

A. Messerschmidt¹, S. de Macedo-Ribeiro¹, W. Hemrika², R. Renirie², R. Wever²

¹Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18 A, D-82152 Martinsried, Germany
²$ E.C. Slater Institute, Department of Biochemistry, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands

Keywords: vanadate, peroxide binding, apo-form, sulfate-binding

The x-ray structures presented are all of the chloroperoxidase from Curvularia inaequalis both from natural and recombinant material. The mutants are single-site mutations to alanine of the catalytic His404, the vanadium protein ligand His496 and of Asp292, which forms a salt bridge to Arg490, a residue which is involved in the compensation of the negative charge of the vanadate group. Furthermore, x-ray structures of the apo-form from natural as well as recombinant material will be described. They show that instead of the vanadate at least partially a sulfate anion is bound originating from the crystallization buffer. In the 2.24 A resolution structure of the functional peroxide derivative [1] the peroxide is bound to the vanadium in an h²-fashion after the release of the apical oxygen ligand. A mechanism of the catalytic cycle has been proposed based on the x-ray structures and kinetic data. Vanadium haloperoxidases form a structurally related class of enzymes as documented by high amino acid sequence similarities in regions of residues providing the vanadium histidine ligand, the vanadate-interacting residues and the catalytic histidine. They are also related to a special group of phosphatases.

Acknowledgements. W.H., R.R. and R.W. thank the Netherlands Foundation for Chemical Research (SON), the Netherlands Organization for Scientific Research (NWO) and the Netherlands Technology Foundation (STW) for financial support. S. de M.-R. is grateful for financial help by the Fundacao Para a Ciencia e Tecnologia (FCT), grant Praxis XXI/BD/4050/94.

1. A. Messerschmidt, L.Prade and R. Wever, Biol. Chem., 378, 309 (1997).