The Metalloenzymology of Hydroxylamine Oxidation by Ammonia Oxidizing Bacteria and Archaea
Majer, Sean Helmuth
Anthropogenic influences on environmental N-fluxes, particularly the accelerated production of NxOy species, have established a pressing demand to understand how ammonia oxidizing bacteria (AOB) and archaea (AOA) carry out controlled N-oxide transformations. Specifically, these organisms utilize a host of enzymes that support transition metal active sites capable of extracting reducing equivalents from ammonia-oxidation to nitrite (NO – ), a process called2 nitrification. Of particular interest are those enzymes which catalyze the oxidation of hydroxylamine (NH2OH) to nitric oxide (NO) and nitrous oxide (N2O). Cytochrome P460, a unique c-heme protein that is ubiquitous among AOB, selectively oxidizes NH2OH to N2O. Enzymes that selectively oxidize NH2OH are predicted to be operative in the emerging clade of AOA, but their identities remain elusive to the research community. This thesis describes the work we have undertaken towards a critical understanding cytochrome P460 and its catalysis. Structure-function relationships are emphasized, including the participation of the secondary coordination sphere during catalysis and essential post-translational modifications to the proenzyme for NH2OH-oxidation. A distal base is revealed to be necessary for cytochrome P460 NH2OH-oxidase activity, and a flexible loop within may be responsible for kinetic barriers in catalysis. This thesis also investigates the mechanism of formation for the unique lysine-porphyrin cross-link of cytochrome P460. The heme P460 cofactor is matured in an oxygen-dependent pathway which may proceed through a heme peroxo-species. The curiously stable ferric nitrosyl intermediate of cytochrome P460, as well as its spectroscopic characterization, is discussed in the context of enzyme reactivity. Corroborating what has been observed for other ferric nitrosyl species, the electronic ground-state of this species is best described as an admixture of Fe(II)–NO+ and Fe(III)–NO•. However, the perturbations of synchronous heme ruffling and meso-carbon substitution may effect the electronic structure in a yet unknown way. Finally, the thesis will present a number of findings regarding our efforts to identify an enzyme competent for catalytic NH2OH-oxidation from AOA. Open thoughts on AOA metabolism, its players, and other protein candidates outside of primary metabolism which are worth investigating are discussed at length, as well as candid observations on the state of the literature surrounding this emerging field of AOA biochemistry.
bioinorganic chemistry; cytochrome P460; hydroxylamine oxidation; nitrification; spectroscopy
Lancaster, Kyle M.
Crane, Brian; Lin, Hening
Chemistry and Chemical Biology
Ph. D., Chemistry and Chemical Biology
Doctor of Philosophy
dissertation or thesis