This proposal involves characterizing the structures and chemistry of two very different types of heme proteins. The first of these is yeast cytochrome c peroxidase (CcP), which occurs naturally in yeast mitochondria and is a prototypical peroxidase. It is a -34KD ferriheme enzyme whose cellular function is to use reducing equivalents from its natural redox partner, cytochrome c (cytc), to decompose hydrogen peroxide. In this role it acts as a cytotoxic protective agent, participates in long-distance electron transfer and may also be important for oxidative stress signaling. The second group of proteins that we plan to study are a group of heme-based biological oxygen sensors. These include the FixLs from Bradyrhizobium japonicum (BjFixL) and Sinorhizobium meliloti (SmFixL) and the Direct Oxygen Sensor protein from E. coli (EcDos). The FixL proteins regulate expression of the nif and fix operons in their respective bacteria, which, in turn, control the biosynthesis of all proteins needed for nitrogen fixation. The EcDos protein is thought to participate in the aerobic/anaerobic switch in E. coli. All three of these proteins contain a central domain structure consisting of a PAS-heme binding domain (sensing domain) linked to a catalytic domain (kinase for the FixLs; phosphodiesterase for EcDos). While these three sensors are of bacterial origin, it has recently been noted that molecular events triggering human renal fibrosis, resulting from hypoxia involves a protein with heme-based oxygen sensing linked to protein kinase catalysis, similar to the FixLs (Norman, J. T., Clark, J. M,., and Garcia. P. L., """"""""Hypoxia Promotes Fibrogenesis in Human Renal Fibroblasts,"""""""" (2000) Kidney Int., 58, 2351-2366). All four of the heme proteins that we intend to study (CcP, FixLs, EcDos) are already being expressed and studied in our laboratory. The goals for both protein types are the same. We propose an integrated effort to study their function, structure and dynamics. The goal is to elucidate how they function on a molecular basis, and what structural features are critical to that function. We shall proceed using modern protein engineering methods combined with x-ray crystallography, kinetics, photothermal methods, equillibrium dynamics methods and NMR spectroscopy.
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