The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. Therefore, R. sphaeroides provides an ideal system for studying supernumerary subunit function. In this study, various recombinant mutant subunit IV's were generated and in vitro reconstituted with 3-subunit core complex to identify the regions or individual amino acid residues of subunit IV required for interaction with the core complex. The effect of subunit IV on the stability of the complex was investigated with differential scanning calorimetry (DSC). The effect of the subunit IV on the pre-steady state kinetics of reduction of bc1 complex by ubiquinol was investigated by using stopped-flow reaction analyzer. Superoxide generation in the pre-steady reaction was measured by monitoring the chemiluminescence of MCLA-superoxide adducts.The transmembrane helix region (86-109) is found essential for assembly of subunit IV into the complex. Residues 77-85 of subunit IV are also critical for the interaction between subunit IV and 3-subunit core complex based on the reconstitutive activities of the N-terminal truncation mutants. In the region of 77-85, residues Y81, R82, Y83, and R84 are identified as critical residues, and the positively charged side-chains at R82 and R84 are essential for their functions. The subunit IV is found being able to increase the thermo-stability of the complex and having the function of reducing electron leakage from the complex.Department of Biochemistry and Molecular Biolog
