By definition, aerobic organisms, both prokaryotic and eukaryotic, require molecular oxygen for energy conservation. This is a mixed blessing, however, as extremely reactive oxygen derivatives are produced during normal metabolism that can damage all cellular components. These so-called reactive oxygen species (ROS) have been implicated in a wide variety of chronic and infectious human diseases, including cancer, Alzheimer's disease, arthritis and AIDS, yet ROS are also used as a defense system against pathogens and in signal transduction pathways. Understanding the responses of microbes to oxygen has direct ramifications for the treatment of diseases caused by anaerobic pathogens. In 1999 we proposed that anaerobes have a novel response to ROS in which a non-heme iron protein termed superoxide reductase (SOR) played a key role. SOR was characterized from the hyperthermophilic anaerobe, Pyrococcus furiosus, and over the prior funding period it has been established using structural and spectroscopic approaches that SOR is uniquely suited to catalyze superoxide reduction. Using DNA microarrays to all 2065 ORFs in the complete P. furiosus genome, it was shown that the genes encoding SOR and related proteins are all expressed at significant levels in the absence of any oxidative shock. P. furiosus is therefore continuously 'armed' and ready to deal with ROS exposure. This is a first line of defense, however, as DNA microarray analyses show that the complete response to oxidative stress requires the induction of a large number of novel proteins (encoded by conserved/hypothetical genes), some of which are also induced by growth at sub-optimal temperatures. In the proposed research, the novel stress-regulated proteins, together with SOR and related reductases and oxidases, will be characterized with respect to their regulation, multiprotein complex formation, and catalytic functions using immunological, biochemical and structural analyses. A variety of complementary spectroscopic techniques, including EPR, ENDOR, MCD, resonance Raman, FTIR and X-ray absorption, will be utilized to probe the catalytic function of specific members of the stress-related pathways, with particular emphasis on SOR. The results will provide completely new insights into the stress responses of anaerobes, and provide strategies for determining the function of uncharacterized hypothetic algenes that typically account for half of a microbial genome.
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