STRUCTURAL CHARACTERIZATION OF THE ACTIVE-SITE OF BRUCELLA-ABORTUS CU-ZN SUPEROXIDE-DISMUTASE - A N-15 AND H-1-NMR INVESTIGATION

Prokaryotic Cu-Zn superoxide dismutases (SODs) are rare and poorly cha racterized compared to their eukaryotic counterparts. To better charac terize the structure of the prokaryotic enzyme, an NMR investigation o f Brucella abortus Cu-Zn SOD in the reduced form was undertaken. The e nzyme studied was a recombinant form, expressed in Escherichia coli. T he enzyme initially lacked a full complement of Cu and Zn ion. After d emetallation and remetallation with a stoichiometric amount of Cu and Zn ion, the specific activity of the recombinant B. abortus Cu-Zn SOD was comparable to the specific activity of the bovine enzyme. The N-15 and H-1 resonances of seven active site histidine imidazole rings wer e assigned using two-dimensional NMR methods. A self-consistent set of nuclear Overhauser effects between imidazole ring protons was observe d, which was in agreement with the predictions of a model based on the X-ray crystallographic structure of the oxidized bovine enzyme (Taine r, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., & Richardson , D. C. (1982) J. Mol. Biol. 160, 181-217). These observations strongl y suggest that the structure of the active site of the prokaryotic enz yme is similar to that of the eukaryotic enzyme. Differences in the ob served and predicted nuclear Overhauser effects could be ascribed to d ifferences in the oxidation state of the Cu ion (Cu(I) in the reduced B. abortus enzyme and Cu(II) in the oxidized bovine enzyme), as much a s they could to the different origins of the enzymes. The NMR data wer e also compared to a similar H-1 NMR study of the human enzyme (Bertin i, I., Capozzi, F., Luchinat, C., Piccioli, M., & Viezzoli, M. S. (199 1) fur. J. Biochem. 197, 691-697). The pattern of nuclear Overhauser e ffects and the chemical shifts of corresponding resonances were very s imilar in H-1 NMR spectra of the human and B. abortus enzymes. Signifi cant differences in the chemical shifts or exchange behavior of a few resonances indicated differences in the environments of several histid ines in the active sites of reduced B. abortus and human Cu-Zn SODs. T his is consistent with the presence of a number of insertions and dele tions in the loop regions that make up the active site as indicated by amino acid sequence alignment studies. The tautomeric and protonation states of the active site histidines were also determined in this stu dy, and the results were in agreement with previous studies. The reson ances of nitrogen atoms coordinated to metal ions were found to fall b etween those of protonated and unprotonated nitrogens on histidine imi dazoles.