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

Author
item CHEN, Y-L - IOWA STATE UNIV., AMES,IA
item PARK, S - IOWA STATE UNIV., AMES,IA
item THORNBERG, R - IOWA STATE UNIV., AMES,IA
item Tabatabai, Louisa
item KINTENAR, A - IOWA STATE UNIV., AMES,IA

Submitted to: Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/21/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Brucella abortus Cu-Zn superoxide dismutase (SOD) is an enzyme that forms hydrogen peroxide from an oxygen radical ion called superoxide anion. This study describes the characterization of the catalytic site of the recombinant form of superoxide dismutase. The catalytic site was studied using proton nuclear magnetic resonance (NMR) and heavy nitrogen NMR of the seven histidine residues which are presumed to be at the catalytic site of the enzyme. The results of this study show that the catalytic site of the Brucella SOD is similar to the catalytic site of the well-studied mammalian erythrocyte SOD.

Technical Abstract: Prokaryotic CU-ZN superoxide dismutases (SODs) are rare and poorly characterized compared to their eukaryotic counterparts. To better characterize the structure of the prokaryotic enzyme, an NMR investigation of Brucella abortus Cu-Zn SOD in the reduced form was undertaken. The enzyme studied was a recombinant form, expressed in E. coli. The enzyme initially lacked a full complement of Cu and Zn ion. After demetallation and remetallation with a stoichiometric amount of Cu and Zu ion, the specific activity of the recombinant B. abortus Cu-Zn SOD was comparable to the specific activity of the bovine enzyme. The *15N and *1H resonances of seven active site histidine imidazole rings were assigned using two-dimensional NMR methods. A self-consistent set of unclear Overhauser effects between imidazole ring protons was observed, which was in agreement with the predictions of a model based on the X-ray crystallographic structure of the oxidized bovine enzyme. These observations strongly suggest that the structure of the active site of the prokaryotic enzyme is similar to that of the eukaryotic enzyme. Differences in the observed and predicted nuclear Overhauser effects could be ascribed to differences in the oxidization state of the Cu ion (Cu[I] in the reduced B. abortus enzyme and Cu[II] in the oxidized bovine enzyme) as much as they could to the different origins of the enzymes.