RUI: Characterization of Reactive Intermediates in the Cytochrome P450 Pathway

University Of North Carolina Greensboro, Greensboro NC

Investigators

Abstract

This award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports Professor Gregory Raner at the University of North Carolina at Greensboro to directly observe and characterize intermediates in two distinct catalytic pathways of cytochrome P450 by taking advantage of rapid mixing and data acquisition techniques. The first objective is to generate and spectrally characterize two related intermediates in the reaction of P450BM3-F87G with oxygen atom donors. The second objective is to identify transient intermediates in the peroxo-pathway for P450BM3-F87G. The stopped-flow and freeze-quench EPR methods for rapid mixing and monitoring of the spectroscopic properties of the enzyme will be used to achieve both specific objectives. The oxygen atom donors, m-chloroperoxybenzoic acid and iodosobenzene will be combined with the F87G mutant of P450BM3 and over the time course of several milliseconds to several minutes, spectra will be acquired. In addition, samples will be rapidly frozen at various time points after mixing, and analyzed using electron paramagnetic resonance spectroscopy in order to provide supporting experimental details relating to the identity of intermediates formed in the reactions. Reaction mixtures will be prepared in which an aldehyde substrate will react with P450BM3-F87G in the presence of hydrogen peroxide, resulting in deformylation of the aldehyde, with chemical modification of the heme cofactor in the enzyme. Analogous freeze-quench EPR experiments will also be carried out. Successful attainment of the research objectives will provide critical details concerning the catalytic mechanism of cytochrome P450 concerning relationships between electronic structure and activity, which may lead to the more efficient use of cytochrome P450 enzymes in biotechnology. The proposed study will equip undergraduate and Masters students with the valuable research skills at the interface of chemistry and biology.