title: Structural characterization of the active site of Brucella abortus Cu-Zn superoxide dismutase: a 15N and 1H NMR investigation.
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 Escherichia coli. The enzyme initially lacked a full complement of Cu and Zn ion. After demetallation 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 15N and 1H resonances of seven active site histidine imidazole rings were assigned using two-dimensional NMR methods. A self-consistent set of nuclear 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 (Tainer, J.A., Getzoff, E. D., Beem, K. M., Richardson, J.S., & Richardson, D.C. (1982) J. Mol. Biol. 160, 181-217). 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 oxidation 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. The NMR data were also compared to a similar 1H NMR study of the human enzyme (Bertini, I., Capozzi, F., Luchinat, C., Piccioli, M., & Viezzoli, M. S. (1991) Eur. J. Biochem. 197, 691-697). The pattern of nuclear Overhauser effects and the chemical shifts of corresponding resonances were very similar in 1H NMR spectra of the human and B. abortus enzymes. Significant differences in the chemical shifts or exchange behavior of a few resonances indicated differences in the environments of several histidines in the active sites of reduced B. abortus and human Cu-Zn SODs. This is consistent with the presence of a number of insertions and deletions 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 study, and the results were in agreement with previous studies. The resonances of nitrogen atoms coordinated to metal ions were found to fall between those of protonated and unprotonated nitrogens on histidine imidazoles.
authors.affiliation: Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA.
authors.forename: Y L
authors.initials: YL
authors.lastname: Chen
authors.forename: S
authors.initials: S
authors.lastname: Park
authors.forename: R W
authors.initials: RW
authors.lastname: Thornburg
authors.forename: L B
authors.initials: LB
authors.lastname: Tabatabai
authors.forename: A
authors.initials: A
authors.lastname: Kintanar
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002003:Brucella abortus / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006859:Hydrogen; D007093:Imidazoles / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D013045:Species Specificity; D013054:Spectrophotometry, Atomic; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007093:Imidazoles; D008667:Metalloproteins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; C029899:imidazole; D006859:Hydrogen; D013482:Superoxide Dismutase