9723608 Hooper NH2OH is oxidized (NH2OH + H2O -> HNO2 + 4e- + 4H+) by the enzyme hydroxylamine oxidoreductase, "HAO", the most complex hemoprotein known, which contains 7 c-hemes and 1 substrate-binding heme P460 per 63 kDa subunit. The latter is a novel tyrosine-derivatized c-heme. At least two pairs of the hemes interact electronically and one of the pairs forms the active site. A second hydroxylamine-oxidizing enzyme, "cytochrome P460", has one metal center which is a lysine-derivatized c-heme and it, together with a cytochrome P-460 from M.capsulatus, will be studied. A crystal structure for HAO has recently appeared which allows one to propose and test distinct catalytic mechanisms involving Fe-N or C-N intermediates, identify and probe important prosthetic groups and amino acids in the active site, and investigate structural elements responsible for the magnetic interactions previously observed between metal centers. The focus of this project is on HAO and cytochromes P460. Approaches include peptide analysis, DNA and protein sequencing, kinetics, suicide substrates, potentiometry, Optical-, EPR-, Mossbauer-, resonance Raman-, and ENDOR- spectroscopies and X-ray crystallographic analysis. In soils, Nitrosomonas helps oxidize ammonia to the form of nitrogen needed by crops. In flooded soils fertilizer nitrogen can also be lost by conversion by Nitrosomonas to the gas, nitrous oxide (N20), a greenhouse gas and a catalyst of ozone destruction. The process studied here is also the limiting step in the removal of ammonia in waste water treatment. Finally, Nitrosomonas will also catalyze degradation of many chlorinated pollutants. An understanding of the biochemistry of the organism involved could lead to more efficient use of fertilizer-nitrogen, decreased production of nitrous oxide, improvements in the waste water treatment, and improved on-site bioremediation of contaminated soils.
