Nuclear magnetic resonance and chemical modification studies of bovine erythrocyte superoxide dismutase: evidence for zinc-promoted organization of the active site structure

Abstract

NMR spectroscopy of the exchangeable H+, tentatively assigned as histidine resonances, of bovine erythrocyte superoxide dismutase in H2O is a powerful method to study the active site of the enzyme. This technique was employed in conjunction with chemical modification of the histidine residues using diethylpyrocarbonate (DEP) to show that Zn along organizes the active site structure. All 8 histidines/subunit of apoenzyme react with DEP. The accessibility of these residues to solvent is borne out by the broad, featureless NMR spectrum of the apoprotein. In the holoenzyme only His-19, which is exposed to solvent, can be modified with DEP. The reduced holoenzyme shows a well-resolved NMR spectrum compared with the oxidized form in which the lines are broadened by the paramagnetic Cu ion. A spectrum very similar to that of the reduced enzyme is generated by addition of 1 Zn2+/subunit of apoprotein showing that Zn alone restores much of the native structure. This interpretation is supported by the fact that addition of up to 1 mol of Zn/subunit statistically reduces the number of histidine residues that can be modified by DEP until, at Zn: apoprotein ratios .gtoreq. 1, only His-19 reacts. The NMR spectrum of the apo plus 2 Zn2+ protein has additional structure that is briefly discussed.