Model Complexes for Reactive Intermediates in Cytochrome P450 Nitric Oxide Reductase (P450nor)

Nitric oxide (NO) is involved in many important biological processes in humans, but the overproduction of NO has detrimental consequences for human health. In contrast, denitrifying organisms efficiently detoxify NO. Fungi produce NO during anaerobic respiration, and detoxify NO by reduction to nitrous oxide (N2O) using a Cytochrome P450-type nitric oxide reductase (P450nor). The active site of this enzyme contains a ferric heme coordinated to a proximal cysteinate ligand. In the proposed mechanism, the ferric heme binds NO and is reduced to a ferrous HNO complex. It is speculated that this intermediate is protonated to a formally iron(IV)-NHOH species as the critical intermediate for NO reduction.To elucidate whether an iron(IV)-NHOH type intermediate is catalytically competent for N-N bond formation, I prepared the first model complex for such a species using a bis-picket fence porphyrin as supporting ligand. Using resonance Raman and Mossbauer spectroscopy I was able to show that the electronic structure of out model is best described as an iron(III)-NHOMe(radical) species, and not an iron(IV) complex or a porphyrin radical species. This complex has a half live of 68 minutes at –80oC. This species reacts with NO gas to form a ferric species, however N2O is not formed. Future studies will determine if an N-N bond was formed by analysis of the N-containing products. Next, we investigated whether ferrous HNO complexes are catalytically competent intermediates in P450nor. We synthesized iron(II)-RNO complexes, where the N-atom is substituted with an alkyl group (R = iPr, Ph) rather than a H-atom. The reaction of these complexes with NO results in an iron(II)-NO complex, rather than N-N bond formation. The addition of a neutral thiol ligand trans to the RNO ligand could activate these complexes toward N-N bond formation. Finally, a new method to synthesize ferric heme-nitrosyl complexes was developed, which allowed us to determine key factors that result in denitrosylation of ferric heme-nitrosyl complexes. This opens up the door to study the P450nor reaction of an iron(III)-NO complex with a hydride to form a ferrous HNO complex.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/137122/1/asmcquar_1.pd