THE CATALYTIC MECHANISM OF SUPEROXIDE DISMUTASE BASED ON CRYSTALLOGRAPHIC STUDIES OF THE REDUCED ENZYME AND INHIBITOR COMPLEXES. Keith S. Wilson¹, Marta Ferraroni², Pier Luigi Orioli³, Wojciech R. Rypniewski¹ & Stefano Mangani². ¹European Molecular Biology Laboratory, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany; ²Department of Chemistry, University of Siena, Pian dei Mantellini 44, I-53100 Siena, Italy; ³Department of Chemistry, University of Florence, via G. Capponi 7, I-50121 Firence, Italy.

A new crystal form of bovine superoxide dismutase is studied in which the copper ion is reduced to Cu(I), as in the course of the dismutation reaction. The structure has an unusually high solvent content, with V_M = 4.5 Å/dal, from which the solvent content can be estimated at 73%. Crystal structures have been solved of the unliganded enzyme and in complex with inhibitors, azide and thiocyanide. The results are used in the study of the catalytic mechanism of SOD. The new crystal form of reduced SOD confirms our earlier observation that the imidazole of His61 is not protonated upon reduction of copper, that the coordination of the metal ions is maintained and that the protons necessary for the reaction can be efficiently provided directly from the solvent. In addition, the crystal structure of reduced SOD and its complexes with anions are compared with the structure of the analogous adducts of oxidised SOD. The azide complex with reduced SOD and that with oxidised SOD fit with the mechanism proposed by Osman & Basch in which a stable complex between Cu(II) SOD and superoxide is formed and an outersphere electron transfer occurs in the first part of the cycle. Once Cu is reduced, it gives back one electron to the bound superoxide which is released as hydroperoxide anion. Different results obtained by NMR and other spectroscopic techniques suggest that the detailed reaction mechanism could differ depending on conditions.