The goal of this project is to determine the basic mechanisms of two heme proteins: cytochrome c oxidase (CcO) and nitric oxide synthase (NOS). CcO is the terminal enzyme in the electron transfer chain. It is membrane bound and has the dual function of reducing O2 to H2O and translocating protons across the inner mitochondrial membrane. The enzyme plays an essential role in mammalian physiology owing to the dependence of essentially all vital organs on aerobic metabolism. The understanding of its catalytic process is incomplete and its mechanism of proton translocation is unknown. The structures of intermediates in the catalytic cycle will be determined and the role of Tyr 244 as a key redox element during enzyme turnover will be studied. New models of proton translocation and energy storage will be tested and refined. NOS is the enzyme that generates nitric oxide (NO) from arginine and oxygen. NO has been found to play many important physiological roles such as a neurotransmitter, a vasodilator and a cytotoxic agent. Thus, mechanisms of its generation and control are of utmost medical importance. It has been discovered that the NO generated by the enzyme significantly inactivates the enzyme by coordination to the heme making the mechanism of its generation and release critical for understanding the catalytic process. It is hypothesized that the strength of the bond between the thiolate and the heme iron atom on the proximal side regulates the NO self inhibition. New experiments will be done to test this hypothesis including a comparison of the Fe-S, the Fe-NO and the NO vibrational modes among the three isoforms. Site directed mutants that may affect the NO self inhibition will also be studied. To determine the catalytic mechanism of the enzyme the resonance Raman spectrum of the oxygen intermediates will be identified and followed during catalytic turnovers. Inhibition mechanisms of the various isoforms of the enzyme will also be studied.
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