PURIFICATION, CHARACTERIZATION AND GENE SEQUENCE-ANALYSIS OF A NOVEL CYTOCHROME-C CO-INDUCED WITH REDUCTIVE DECHLORINATION ACTIVITY IN DESULFOMONILE-TIEDJEI DCB-1

The sulfate-reducing bacterium, Desulfomonile tiedjei DCB-1, conserves energy for growth from reductive dechlorination of 3-chlorobenzoate v ia halorespiration. To understand this respiratory process better, we examined electron carriers from different cellular compartments of D. tiedjei. A 50-kDa cytochrome from the membrane fraction was found to b e co-induced with dechlorination activity. This inducible cytochrome w as extracted from the membrane fractions by Tris-HCl buffer containing ammonium sulfate at 35% saturation and was purified to electrophoreti c homogeneity by phenyl superose, Mono Q, and hydroxyapatite chromatog raphy. The purified cytochrome had a high-spin absorption spectrum. In a pH titration experiment, the absorption spectrum of the inducible c ytochrome shifted to low spin at pH 13.2. The midpoint potential of th e inducible cytochrome at pH 7.0 was -342 mV. The NH2-terminal amino a cid sequence of the inducible cytochrome was determined and was used t o obtain inverse PCR products containing the sequence of the gene enco ding the inducible cytochrome. The ORF was 1398 bp and coded for a pro tein of 52.6 kDa. Two c-type heme-binding domains were identified in t he COOH-terminal half of the protein. A putative signal peptide of 26 residues was found at the NH2-terminal end. The protein sequence was n ot found to have substantial sequence similarity to any other sequence in GenBank. We conclude that this is a c-type cytochrome substantiall y different from previously characterized c-type cytochromes.