title: Nuclear magnetic resonance and chemical modification studies of bovine erythrocyte superoxide dismutase: evidence for zinc-promoted organization of the active site structure.
abstract: Nuclear magnetic resonance (NMR) spectroscopy of the exchangeable protons, tentatively assigned as histidine resonances, of bovine erythrocyte superoxide dismutase in H2O has been found to be a powerful method to study the active site of the enzyme. This technique has been employed in conjunction with chemical modification of the histidine residues using diethylpyrocarbonate (DEP) to show that zinc alone organizes the active site structure. All eight histidines per 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 copper ion. A spectrum very similar to that of the reduced enzyme is generated by addition of one zinc ion per subunit of apoprotein showing that zinc alone restores much of the native structure. This interpretation is supported by the fact that addition of up to 1 mol of zinc per subunit statistically reduces the number of histidine residues that can be modified by DEP until, at Zn: apoprotein ratios greater than or equal to 1, only His-19 reacts. The NMR spectrum of the apo plus 2 Zn2+ protein has additional structure that is briefly discussed.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004047:Diethyl Pyrocarbonate; D004912:Erythrocytes / Q000201:enzymology*; D006639:Histidine / Q000032:analysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D013482:Superoxide Dismutase / Q000097:blood; D015032:Zinc / Q000494:pharmacology
chemical_list: D006639:Histidine; D013482:Superoxide Dismutase; D015032:Zinc; D004047:Diethyl Pyrocarbonate