Molecular O2 plays a paradoxical role in aerobic organisms in that it is both essential for energy generation as well as very toxic. Extremely reactive derivatives of O2 are produced during normal metabolism and can cause considerable damage to various cellular components, including DNA, lipids and proteins. In fact, these so-called been implicated in a wide variety of human diseases including cancer, diabetes, Alzheimer's and arthritis. The defense systems against ROS in aerobes have been well established and involve, among others, the enzyme superoxide dismutase and catalase. In contrast, the genome sequences of several anaerobes reveal that they do not contain these enzymes, which suggest that they have alternative pathways. Understanding the molecular basis of the O2 response in anaerobes has direct ramifications for the treatment of diseases caused by anaerobic pathogens. It is expected that the viability of these organisms is crucially dependent on the efficiency of their defense against ROS. Differences between anaerobes and aerobes in the pathways used to scavenger thee species might therefore be exploited as highly specific targets for drug development. The overall objective of this research is to elucidate these pathways in the hyperthermophilic anaerobe, Pyrococcus furiosus, which grows optimally at 100 degrees Centigrade. A novel non-heme iron protein termed neelaredoxin which has superoxide reductase rather than dismutase activity has been purified from this organism. The gene encoding it has been cloned and sequenced, and the crystal structure of the recombinant protein (obtained from Escherichia coli) has been solved to 1.7 A. Homologs of neelaredoxin have been found in all anaerobes examined. The P. furiosus protein is a homotetramer and contains one iron atom per subunit (of 14 kDa), which is bound to the protein in an unprecedented coordination geometry, involving four state and pre-steady state kinetics of the native protein and of mutants in which active site residues have been changed. The structural, vibrational, magnetic, electronic and redox properties of the metal center in this protein and mutants thereof, in the absence and presence of exogenous ligands, will be characterized using a range of complimentary spectroscopic techniques: electron paramagnetic resonance, optical absorption, and variable temperature magnetic circular dichroism, resonance Raman and Fourier transform infra-red. The structure of neelaredoxin, mutants and derivatives with bound exogenous ligands will be determined using crystallography. Finally, genomic and proteomic methods will be used to analyze protein patterns and gene transcripts in P. furiosus grown under various stress conditions to characterize other proteins involved in the O2 detoxification pathway.
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