BIOCHEMICAL AND SPECTROSCOPIC CHARACTERIZATION OF THE HIGH-MOLECULAR-WEIGHT CYTOCHROME-C FROM DESULFOVIBRIO-VULGARIS HILDENBOROUGH EXPRESSED IN DESULFOVIBRIO-DESULFURICANS G200

The gene of high molecular weight, multiheme cytochrome c (Hmc) from t he sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has been overexpressed in Desulfovibrio desulfuricans G200. The recombina nt protein has been purified. Its molecular weight (65 600), amino aci d composition, and NH2-terminal sequence were found to be identical to those of the wild-type protein. The recombinant protein has been spec troscopically characterized (optical spectrum, EPR, circular dichroism ) and compared to the wild-type protein. We have found 16 hemes per mo lecule by iron analysis and the pyridine hemochrome test. Both high- a nd low-spin features were observed in the EPR spectrum. A detailed spi n quantitation analysis indicates 1 or 2 high-spin hemes and 14 or 15 low-spin hemes per molecule. The redox potentials of the hemes determi ned by voltammetric techniques gave an average of three different valu es, 0, -100, and -250 mV (versus NHE), for the wild-type and the recom binant cytochrome. The low potential values are similar to the values observed for the bis(histidinyl) coordinated hemes of cytochrome c3. A comparison of the arrangement of heme binding sites and coordinated h istidines in the amino acid sequences of cytochrome c3 and Hmc has sho wn that the latter contains four domains, three of which are complete c3-like domains, while the fourth represents an incomplete c3-like dom ain which may contain His-Met coordinated hemes. These data are in agr eement with the detailed study of the number and types of hemes report ed in this paper.