Preparation and characterization of the water-soluble heme-binding domain of cytochrome c1 from the Rhodobacter sphaeroides bc1 complex.

The ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodobacter sphaeroides consists of four subunits. One of these subunits, cytochrome c1, is the site of interaction with cytochrome c2, a periplasmic protein. In addition, the sequences of the fbcC gene and of the cytochrome c1 subunit that it encodes suggest that the protein should be located on the periplasmic side of the cytoplasmic membrane and that it is anchored to the membrane by a single membrane-spanning alpha-helix located at the carboxyl-terminal end of the polypeptide. Site-directed mutagenesis of the fbcC gene was used to alter the codon for Gln228 to a stop codon. This results in the production of a truncated version of the cytochrome c1 subunit that lacks the membrane anchor at the carboxyl terminus. The bc1 complex fails to assemble properly as a result of this mutation, but the Rb. sphaeroides cells expressing the altered gene contain a water-soluble form of cytochrome c1 in the periplasm. The water-soluble cytochrome c1 was purified and characterized. The amino-terminal sequence is identical with that of the membrane-bound subunit, indicating the signal sequence is properly processed. High pressure liquid chromatography gel filtration chromatography indicates it is monomeric (28 kDa). The heme content and electrochemical properties are similar to those of the intact subunit within the complex. Flash-induced electron transfer kinetics measured using whole cells demonstrated that the water-soluble cytochrome c1 is competent as a reductant for cytochrome c2 within the periplasmic space. These data show that the isolated water-soluble cytochrome c1 retains many of the properties of the membrane-bound subunit of the bc1 complex and, therefore, will be useful for further structural and functional characterization.