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Studies of the electronic and magnetic properties of sulfite reducing enzymes from Desulfovibrio vulgaris (hildenborough)
METAL CLUSTERS
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483
STUDIES OF THE ELECTRONIC AND MAGNETIC PROPERTIES OF SULFITE REDUCING ENZYMES FROM DESULFOVlBRlO VULGARIS (HILDENBOROUGH)
S.-M. L;laLand J. A. Cowan Evan’s Laborato y of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA. The sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) contains both a low molecular weight assimilatory sulfite reductase (SiR, Mr- 23,500) and a large, hexameric dissimilatory enzyme (desulfoviridin, DV, Mr- 224,OOO).Each contains the coupled [Fe4S41-siroheme prosthetic center common to this enzyme class, although their spectral characteristics are distinct owing to differences in the coordination geometry and protein environment at the active site. Highly purified samples of each enzyme display standard EPR characteristics. For the oxidized enzymes: SiR shows a rhombic low-spin hexacoordinate siroheme (S=l/2, g=2.45, 2.38,1.78) and DV shows rhombic high spin pentacoordinate siroheme (S=5/2, g= 6.30,5.63,2.00). The reduced enzymes show characteristic ferredoxin-like Fe4S4 EPR spectra (g=2.04,1.93 for SiR and g=2.07,1.93,1.89 for DV) which differ from spectra obtained with E.coli sulfite reductase 111. EPR spin quantitation, optical spectroscopy and chemical analysis are consistent with a prosthetic center composition of one/two of the coupled units (below) for SiR/DV, respectively [21. There is no evidence for demetallated siroheme or clusters of higher nuclearity [31. Heterogeneity has been observed in the power saturation behaviour of the rhombic g-tensor components from the siroheme ( P1/2 for SiR : gx, 7.10 mW; gy, 7.35mW; gz, 2.7 mW and for DV : gx, 14.4 mW; gy, 12.0 mW, gz, 1.6 mW), which may be ascribed, in part, to the coupling of the two redox centers. Further evidence is provided from PI/~ measurements on the reduced Fe& center. Coupling between the cluster and siroheme is found to be weak in comparision with E. coZisulfite reductase. Reduction in the presence of N02produces new EPR features (g-2 and 41, that reflect intermediates resulting from the first step of nitrite reduction. Mechanistic issues pertaining to the identity of such intermediates in nitrite and sulfite reducing enzymes are the focus of current research activity. COpH
1. 2. 3.
P. A. Janick, L. M. Siegel Biochemistry 21 3538 (1982). B, M. Wolfe, S. M. Lui, J. A. Cowan (manuscript submitted). A. J. Pierik, W. R. Hagen Eur. 1. Biochem. 195 505 (1991).
~042
483
STUDIES OF THE ELECTRONIC AND MAGNETIC PROPERTIES OF SULFITE REDUCING ENZYMES FROM DESULFOVlBRlO VULGARIS (HILDENBOROUGH)
S.-M. L;laLand J. A. Cowan Evan’s Laborato y of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA. The sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) contains both a low molecular weight assimilatory sulfite reductase (SiR, Mr- 23,500) and a large, hexameric dissimilatory enzyme (desulfoviridin, DV, Mr- 224,OOO).Each contains the coupled [Fe4S41-siroheme prosthetic center common to this enzyme class, although their spectral characteristics are distinct owing to differences in the coordination geometry and protein environment at the active site. Highly purified samples of each enzyme display standard EPR characteristics. For the oxidized enzymes: SiR shows a rhombic low-spin hexacoordinate siroheme (S=l/2, g=2.45, 2.38,1.78) and DV shows rhombic high spin pentacoordinate siroheme (S=5/2, g= 6.30,5.63,2.00). The reduced enzymes show characteristic ferredoxin-like Fe4S4 EPR spectra (g=2.04,1.93 for SiR and g=2.07,1.93,1.89 for DV) which differ from spectra obtained with E.coli sulfite reductase 111. EPR spin quantitation, optical spectroscopy and chemical analysis are consistent with a prosthetic center composition of one/two of the coupled units (below) for SiR/DV, respectively [21. There is no evidence for demetallated siroheme or clusters of higher nuclearity [31. Heterogeneity has been observed in the power saturation behaviour of the rhombic g-tensor components from the siroheme ( P1/2 for SiR : gx, 7.10 mW; gy, 7.35mW; gz, 2.7 mW and for DV : gx, 14.4 mW; gy, 12.0 mW, gz, 1.6 mW), which may be ascribed, in part, to the coupling of the two redox centers. Further evidence is provided from PI/~ measurements on the reduced Fe& center. Coupling between the cluster and siroheme is found to be weak in comparision with E. coZisulfite reductase. Reduction in the presence of N02produces new EPR features (g-2 and 41, that reflect intermediates resulting from the first step of nitrite reduction. Mechanistic issues pertaining to the identity of such intermediates in nitrite and sulfite reducing enzymes are the focus of current research activity. COpH
1. 2. 3.
P. A. Janick, L. M. Siegel Biochemistry 21 3538 (1982). B, M. Wolfe, S. M. Lui, J. A. Cowan (manuscript submitted). A. J. Pierik, W. R. Hagen Eur. 1. Biochem. 195 505 (1991).