PURIFICATION, CHARACTERIZATION AND GENE SEQUENCE-ANALYSIS OF A NOVEL CYTOCHROME-C CO-INDUCED WITH REDUCTIVE DECHLORINATION ACTIVITY IN DESULFOMONILE-TIEDJEI DCB-1
Tm. Louie et al., PURIFICATION, CHARACTERIZATION AND GENE SEQUENCE-ANALYSIS OF A NOVEL CYTOCHROME-C CO-INDUCED WITH REDUCTIVE DECHLORINATION ACTIVITY IN DESULFOMONILE-TIEDJEI DCB-1, Archives of microbiology, 168(6), 1997, pp. 520-527
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.