Coleman, Rachael Elizabeth2021-12-202022-09-102021-08Coleman_cornellgrad_0058F_12630http://dissertations.umi.com/cornellgrad:12630https://hdl.handle.net/1813/110527141 pagesNitrification is the process in which ammonia (NH3) is ultimately converted to nitrate (NO3-) by ammonia-oxidizing microorganisms. In NH3-oxidizing bacteria (AOB), anammox bacteria, and comammox bacteria the enzyme hydroxylamine oxidoreductase (HAO) is able to convert hydroxylamine (NH2OH) to nitric oxide (NO). What makes HAO unique is the presence of a P460 cofactor, which has been observed in only one other known protein family: cytochrome (cyt) P460. The P460 cofactor is a modified c-heme based cofactor that contains a post-translational modification in the form of a cross-link from the peptide backbone onto the porphyrin macrocycle itself. HAO uses a Tyr residue from a neighboring subunit to bind to the porphyrin macrocycle twice, whereas cyt P460 uses a lysine residue to bind to the γ meso carbon once. In cyt P460, the cross-link is absolutely essential for catalysis and a portion of this thesis addresses that the cross-link does not alter the electronic structure of this system in a catalytically meaningful way. Instead, the cross-link plays a vital role in positioning the heme relative to a second-sphere glutamate residue that serves the purpose of deprotonating the bound NH2OH. Although the cross-link of cyt P460 is absolutely essential for activity, the mechanism of its formation is unknown, but was reported to occur through an autocatalytic mechanism. The second half of this thesis addresses how the cross-linkof cyt P460 forms. We found that by excluding oxygen during protein expression results in a cross-link-deficient, catalytically incompetent protein, but treatment of this this protein with hydrogen peroxide (H2O2) results in a fully cross-linked, catalytically competent cyt P460. Additional studies showed that the fold of cyt P460 promotes cross-link formation, and which residues are important for this post-translational modification to form are also addressed.enBioinorganiccross-linkCytochrome P460HemesNitrificationdissertation or thesishttps://doi.org/10.7298/qbrt-0t51