A new class of integral membrane oxidoreductase complex was recently discovered in bacteria (Yanyushin et al. Biochemistry, 44, 10037-10045, 2005). The complex was purified from the filamentous green anoxygenic phototroph Chloroflexus aurantiacus. The complex consists of seven subunits, the genes for which were identified using trypsin disgestion fingerprinting, mass spectroscopy and the partially completed genome sequence. Three of the subunits appear to be integral membrane proteins based on hydropathy plots. Two of the subunits contain c-type heme cofactors, based on both heme staining and sequence data. Another subunit has sequence homology at the N-terminal end to a molybdopterin enzyme and the C-terminal end to an FeS protein, although the complex does not contain Mo or pterin cofactors, and the primary sequence is missing key elements of their binding sites. The complex has been named the MFIcc complex. Similar although distinct complexes were isolated from phototrophically and aerobically grown cells, and appear to be coded for by distinct genes for at least most of the subunits. The genes that code for the protein subunits of the complexes are arranged in a cluster on the genome and form a putative operon. What appear to be homologous operons for similar complexes were identified in genomes of a number of nonphotosynthetic bacteria. The presence of genes for this complex are anticorrelated with the presence of genes for the cytochrome bc1 complex, suggesting that the new complex may functionally replace the cytochrome bc1 complex. Chloroflexus aurantiacus almost certainly does not contain a bc1 complex based on both biochemical studies and the lack of genes in its genome that code for the protein subunits. This project involves complete biochemical and energetic characterization of this complex. This includes determination of overall oligomeric state and numbers of each subunit by the use of gel filtration and blue-native and SDS gel electrophoresis, coupled with mass spectroscopy and protein cross-linking studies. Additional information about the redox centers and their midpoint potentials will be obtained using redox potentiometry coupled with UV-Vis and EPR spectroscopies. Quantitative determination of the number and types of cofactors will be carried out using a variety of standard techniques.
Broader Impacts The information obtained on these complexes will provide new insights into novel bacterial bioenergetic mechanisms and the origin and evolution of energy conserving systems. This project will train a graduate student and a postdoctoral fellow in the integrated techniques of biochemistry, molecular biology, biophysical analysis and bioinformatics that are essential parts of modern research in bioenergetic systems. Undergraduate students will also be involved in all aspects of the research.
