Cloning, sequencing and functional expression in Escherichia coli of dmc gene encoding periplasmic tetra heme cytochrome c3 from Desulphovibrio desulphuricans M6

A small soluble protein, periplasmic tetraheme cytochrome c(3), was purified from Desulphovibrio desulfuricans M6 and its gene, dmc, was cloned and the complete nucleotide sequence determined. The purity index of purified cytochrome c(3) was 3.3 and the molecular weight was determined as 14.5 kDa by SDS-PAGE. It was found that the 387 bp of dmc gene encoded 21 amino acids of hydrophobic signal peptide and 107 residues of apoprotein. The nucleotide sequence and the predicted amino acid sequence of dmc showed 76% and 83% identities to those of 13 kDa cytochrome c(3) from D. desulphuricans ATCC 27774, respectively. dmc gene was functionally expressed in aerobically grown Escherichia coli BL-21(DE3) by co-expressing eight ccm genes which were reported to be involved in cytochrome c maturation. The molecular weight of overexpressed holocytochrome c(3) was identical to that of the original protein. Visible spectrum of dithionite-reduced form exhibited typical characteristics of c-type cytochromes. In addition, the redox potential was measured to -340 mV by cyclic voltammetry. (C) 2001 Academic Press.