Functional expression of subunit IV of Rhodobacter sphaeroides cytochrome 6-C1 complex and reconstitution of recombinant protein with three-subunit core complex

Abstract

Subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex was over-expressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-RSIV) using the expression vector, pGEX/ RSIV. Maximum yield of soluble active recombinant fusion protein was obtained from cells harvested 3 h after induction of growth at 37 °C in LB medium. Subunit IV was released from the fusion protein by proteolytic cleavage with thrombin. When subjected to SDS-polyacrylamide gel electrophoresis, isolated recombinant sub-unit IV showed one protein band corresponding to subunit IV of R. sphaeroides cytochrome b-c1 complex. Although the isolated recombinant subunit FV is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1000 kDa. The addition of detergent deaggregates the isolated protein, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. When the three-subunit core cytochrome b-c1 complex, purified from RSΔIV-adapted chromatophores containing a fraction of the wild-type cytochrome b-c1 complex activity, was reacted with varying amounts of recombinant subunit IV, the activity increased as the subunit IV concentration increased. Maximum activity restoration was reached when 1 mol of subunit IV/mol of three-subunit core complex was used. The reconstituted cytochrome b-c1 complex is similar to the wild-type complex in molecular size, apparent Km for Q2H2, and inhibitor sensitivity, indicating that recombinant subunit IV is properly assembled into the active cytochrome b-c1 complex. A tryptophan residue in subunit IV was found to be involved in the interaction with the three-subunit core complex.