Animals and plants secrete nitric oxide (NO ) to kill pathogenic microbes. However, pathogens can detoxify NO and disarm this important immune defense. Many microbes product flavohemoglobins (flavoHbs) that can metabolize NO. A Function for flavoHbs in NO detoxification is supported by the sensitivity of flavoHb-deficient microbes to the antibiotic action of NO and by the induction of flavoHB expression by NO. FlavoHb is an ancient member of the widely-distributed family of O2-binding hemoglobins (Hbs) that appear to bear an intrinsic capacity for 'oxidizing' NO to form nitrate. We plan to tes the hypothesis that flavoHbs, and Hbs with associated flavin-containing reductases, function as efficient NO dioxygenases (NODs). The goals of this proposal are to elucidate the enzyme mechanism of the proposed NO dioxygenase function and to elucidate structural adaptations of flavoHbs, and Hbs with associated reductases, which support a NO dioxygenation mechanism and NO detoxification function. 1) Elementary NOD reaction rate constants for E.coli flavoHb are being measured and compared with the corresponding values for mammalian O2 transport Hbs. Microbial flavoHbs, and microbial and human Hbs with their co-expressed reductases, are being examined for NOD activity. 2) Intermediates of the NOD enzyme mechanism are being elucidated using stopped flow, EPR, and infrared spectrophotometry, specific traps, and indicators. 18O isotope labelling is being used to test the proposed 'dioxygenase' mechanism of flavoHb and oxgenated Hbs and to measure the contribution of water O atoms in the mechanism of nitrate formation. 3) Existing flavoHb structures are being used to suggest potenial mechanisms, and site- directed mutants of E. coli flavoHb are being prepared to test the roles of key conserved amino acids and motifs in NOD steady-state, ligan- binding, reduction kinetics and in intermediate stability. 4) Mutant and wild-type E. coli flavoHb crystals are being prepared in various ligand forms for x-ray diffraction analysis to elucidate structure-function relationships. Knowledge of the kinetic, mechanistic and structural requirements of the NOD activity will help define the NOD function of the family (flavo) Hbs and will also provide information from the design of anti-infective NOD inhibitors.
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