Hydrogen-deuterium exchange in yeast Cu2,Zn2-superoxide dismutase and apo-superoxide dismutase

Abstract

Keywords: Yeast Cu2,Zn2-superoxide dismutase, apo-superoxide dismutase, hydrogendeuterium exchange, metal-dependent structural transition, E X 2 -m e c h a n i s mThe role of copper and zinc for the structural properties of yeast Cu2,Zn2-SOD was studied by measuring the hydrogen-deuterium exchange in peptide groups of Cu:,Zn:-SOD, Cu:,E2-SOD, E:,Zn2-SOD and apo-SOD. Apo-SOD displays remarkably rapid exchange kinetics as compared to Cu2,Zn2-SOD, indicating a less stable structural organization after metal removal. At neutral and alkaline pD the exchange rates are in agreement with the EX2 mechanism of exchange; the exchange rates at low pD suggest a structural transition. In Cu2,Zn2-SOD this transition occurs at pD about 4.5, and in apo-SOD at pD about 6.2, 1.7 pD units higher. Binding of either copper or zinc to apo-SOD results in reductions of the exchange rate, which is further reduced after full reconstitufion to Cu_~,Zn.,-SOD, demonstrating that copper and zinc are equally important for the structural stability of the protein molecule. I N T R O D U C T I O NCu_,,Znz-SOD from yeast is a member of a highly conserved group of enzymes found in the cytosol of eucaryotic cells, believed to act as scavengers of toxic superoxide ions, O~, formed during oxidative metabolism. Yeast Cu2,Zn2-SOD has a molecular weight of 31,900, and it is composed of two identical subunits each containing a bimetallic Cu-Zn-center, which is essential for the catalytic mechanism (8,15,22). The X-ray crystal structure of bovine erythrocyte Cu2,Zn2-SOD at 2A resolution shows that each subunit consists of an eight stranded [3-bartel of Greek key topology, suggesting a very rigid structure. The copper is liganded to four histidines in a distorted square plane, and the zinc is liganded to three histidines and an aspartic acid in a tetrahedral geometry (28). 'H-NMR studies indicate close similarity in the metal binding sites of bovine erythrocyte and yeast Cu2,Zn2-SOD (8), although a metal bridging histidine in the bovine enzyme is apparently not liganded to zinc in the yeast enzyme (3).It is now well established that copper is essential for the catalysis of superoxide dismutation, during which copper is alternately oxidized and Abbreviations: Cu_,,E2-SOD = di-copper superoxide dismutase; E2,Zn2-SOD = di-zinc superoxide dismutase; SOD = superoxide dismutase.Springer-Verlag