Cytochromes b are an important class of hemoproteins involved in electron transfer reactions. The former functions primarily as an essential electron transfer component in the pathways of fatty acid desaturation, cholesterol biosynthesis and drug metabolism. The form found in erythrocytes is involved in the pathway for methemoglobin reduction. A deficiency in this pathway predisposes the organism to methemoglobinemia, a pathological condition which can cause systems from cyanosis to mental retardation. We have synthesized and overexpressed a gene that codes for the outer mitochondrial membrane cytochrome b5 from rat liver, and recently shown that its E 1\2 is approximately 100 mV more negative than that observed for the microsomal or erythrocyte proteins. Interestingly, the E 1/2 of NADH-cytochrome b5 reductase is -88 mV, and -102 mV in the presence of NAD. This suggests that the mitochondrial protein is likely to react differently with the NADH-cytochrome b5 reductase typical of the pathways mentioned above. The PI intends to accomplish the following; 1. Develop the methodology for the bacterial expression of 13C-heme enriched b5 by combining the now elucidated biosynthetic pathway of heme and the special properties built in our expression system of the mitochondrial cytochrome b5. Expression of the cytochrome b5 gene turns on heme synthesis, which is then incorporated in the overexpressed polypeptide, thus simplifying the isolation and purification of heme. This methodology will not only benefit the research of the PI, but it will also be useful to other researchers interested in NMR of heme proteins, since the isotopically labelled heme can be removed from cytochrome b5 in order to reconstitute other proteins with it, or to use it in model compound studies. II. Elucidate the role that heme propionates in cytochrome b5 play in binding to physiological partner proteins such as cytochromes c and P-450. To these ends, cytochrome b5 with 13C labelled heme will be used to extract information such as binding sites, stoichiometries and binding constants, which will be useful for the understanding of electron transfer reactions. The complexes that b5 forms with ubiquitous Ca2= and Mg2+ ions, which have recently been implicated in modulating the E 1/2 value of b5, by binding to the exposed heme propionates, will also be studied by 13C and 113 Cd NMR spectroscopies. III. Use site directed mutagenesis, NMR spectroscopy X-ray crystallographic and electrochemical techniques to study the major factors believed to control the reduction potential of bis-His ligated cytochromes b: These factors are; a) Accessibility of water to the hydrophobic heme environment. b) Coulombic interactions between charged residues close the heme and the positive charge on the ferric heme. c) Degree of protonation of axial histidyl imidazole Ndelta, which results in a stronger or weaker Fe-N bond. d) Geometrical arrangement of axial histidyl imidazole planes which can be influenced by the hydrogen bond network around the axial ligand. This information will be useful for the detailed understanding of structure function in the bis-His ligated cytochromes b, and to researchers interested in the area of molecular "maquettes "18,19, a novel class of simplified versions of metalloproteins involved in redox catalysis and in energy conversion.