Collaborative Research: Biochemical and Biophysical Characterization of Cytochromes b5 from Outer Mitochondrial Membrane

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Membrane-anchored, mammalian cytochromes b5 are located in the endoplasmic reticulum (microsomal or Mc cyt b5) and the outer membranes of mitochondria (OM cyt b5). Although Mc cytochromes b5 have been identified from a number of mammalian sources, the only OM cyt b5 that has been positively identified to date originates from rat liver. The three-dimensional structures of rat OM cyt b5 and the Mc cytochromes b5 are very similar. Nonetheless, rat OM cyt b5 has a much lower reduction potential and much higher stability toward chemical and thermal denaturation than the Mc cytochromes b5. Furthermore, hemin in rat OM cyt b5 is kinetically trapped at physiological temperatures. From amino acid sequence alignments, crystal structure comparisons, and molecular dynamics simulations, several amino acid residues can be identified as potential determinants of the unusual biophysical properties of rat OM cyt b5. Consequently, a systematic study will be conducted in which these residues in the rat OM protein are replaced with the corresponding residues in the bovine Mc isoform. These studies will be performed with the expectation of decreasing the stability and kinetic barriers for hemin release of rat OM cyt b5. A complementary study will be carried out in which the stability of bovine Mc cyt b5 will be increased by incorporating the stabilizing features found in the rat OM protein. It is also important to establish whether the biophysical properties of rat OM cyt b5 are restricted to this protein, or rather are common to mitochondrial cytochromes b5. Consequently, the gene coding for human testis cyt b5, a protein very likely to be the human analogue of rat OM cyt b5, will be synthesized, placed in a vector suitable for high level expression and characterized for its biophysical properties.