The overall objective of this research is to elucidate the role of the supernumeral subunit, subunit IV (Mr = 14,384), in the cytochrome bc1 complex from a photosynthetic bacterium Rhodobacter sphaeroides. Previous studies involving site-directed mutagenesis followed by gene complementation (in vivo reconstitution) have established that subunit IV is essential for the catalytic activity, Q-binding, and structural integrity of the complex. However, judging from the 3-D structure model of this bacterial bc1 complex, built with coordinates from their mitochondrial counter parts (cytochrome b, cytochrome c1, the iron-sulfur protein, and subunit VII), direct participation of subunit IV in the catalytic (Q-binding) sites of the complex seems unlikely. In this project, the PI wishes to continue to use in vitro reconstitution, in concert with biophysical, biochemical, and structural analysis, to probe the mode of interaction between subunit IV and the core bc1 complex. The specific aims of this project are: (a) to identify amino acid residues of subunit IV involved in interaction with the three-subunit core complex; (b) to identify amino acid residues of cytochrome b involved in interaction with subunit IV; (c) to investigate the effect of subunit IV on the protein conformation and thermostability of the cytochrome bc1 complex by circular dichroism and differential scanning calorimetry; (d) to investigate the protein:lipid interactions in three- and four- subunit complexes; (e) to examine the Q sequestering role of subunit IV using synthetic Q- derivatives; and (f) to explore the possible association of supernumeral subunit function with extra segments in the bacterial core subunits. The research described in this project will increase our understanding of the structure-function and mechanisms of action of the Rhodobacter sphaeroides photosynthetic bacterial bc1 complex. The knowledge gained will provide a better understanding of energy transduction mechanisms in the photosynthetic apparatus. This may lead to the design of methods to improve photosynthetic efficiency and effective herbicide design. It will also enhance our understanding of mitochondrial bioenergetics.
