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.

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

“…This information supplements the NMR studies of Cu2,Zn2-SOD and apo-SOD (9,14,24), which only revealed considerable differences in the active site region, and the ultracentrifugation studies of yeast apo-SOD (l 0) which showed a Iog(kot) decrease of the sedimentation coefficient from 3.0 S to 2.5 S upon metal removal, suggesting a general loosening of the protein structure. This is consistent with the observation for bovine apo-SOD, that this protein denatures at a temperature 45 ~ lower than the holo-enzyme (23).…”

“…reduced (15), whereas zinc is believed to be responsible for correct conformation around the active site (9,24).…”

Mutations in copper-zinc superoxide dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein.

“…For example, binding of zinc ions to the zinc site of the apoprotein causes the protein to adopt a native-like structure, even in the absence of Cu2+ (38), and to increase the overall stability of the protein (36). In addition, removal of zinc ions from WT CuZnSOD to form the zinc-free derivative, Cu"ESOD, causes the SOD activity to become pH dependent (32).…”

Mapping Cu(II) Binding Sites in Prion Proteins by Diethyl Pyrocarbonate Modification and Matrix-assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometric Footprinting

“…DEPC can also react with other nucleophilic residues including sulfhydryl, arginyl, and tyrosyl residues, as well as with ␣-and ⑀-amino groups (40). However, in contrast to arginine, tyrosine, and amino acids with sulfhydryl side chains, the imidazole ring of histidine is known as a copper binding site, and many studies have demonstrated that metal coordination to histidine residues of peptides or proteins protects these amino acids from DEPC modification (41)(42)(43)(44). Since DEPC-modified histidines increase in mass of 72.06 Da for each mono-carbethoxylation of the pyrrole type of nitrogen in the imidazole ring in exposed residues (di-substituted adducts are only formed at high DEPC concentrations; not presented), resulting adducts can be detected and enumerated by MALDI-MS analysis (45)(46)(47).…”