SpeakerDr. John Dawson, Chemistry & Biochemistry, University of South Carolina. Host: Alison Butler
Date and LocationWednesday November 18, 2015 4:00pm to 5:00pm
Generation and Reactivity of Transient Cytochrome P450 Oxygen-Containing Intermediates
John H. Dawson,a Daniel P. Collins,a D. M. Indika Bandara,a Anuja Modi,a Shengxi Jin,a Thomas A. Bryson,a Eric D. Coulter,a Zanna Beharry,a and David P. BalloubaDepartment of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208 USA; firstname.lastname@example.org and bDepartment of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109 USA
Cytochrome P450 is a versatile heme-containing oxygenase that transfers an O-atom from dioxygen to a wide range of organic substrates. Also an oxygenase, nitric oxide synthase first hydroxylates arginine to an N-hydroxyimine intermediate and then, in a second O-atom transfer step, forms citrulline and NO. Both ferric enzymes first bind substrate, are reduced and then bind dioxygen to give the oxyferrous state. Addition of a second electron yields a peroxo-ferric intermediate, protonation of which generates a hydroperoxoferric state; lose of water then forms a ferryl [oxo-iron(IV)] porphyrin radical (Compound I). Although the latter is thought to be the ultimate oxidant, the preceeding two species have been proposed as secondary oxidants. T252A P450-CAM, hydroxylates camphor to a very limited extent, indicating it does not form Compound I. However, it still accepts electrons from NADH to give hydrogen peroxide, presumably via the peroxo/hydroperoxoferric intermediates. Thus, T252A P450-CAM is the ideal mutant to test whether either of these two species are capable of O-atom transfer. We have prepared a series of camphor analogues and related compounds with reactive functional groups for O-atom transfer to investigate the mechanism of dioxygen activation by P450 and NOS. We have used this approach to investigate sulfoxidation, thioether S-dealkylation, deformylation and N-hydroxyimine denitrosation. Results of these studies, with emphasis on the involvement of a second active P450 oxidant in O-atom transfer, will be presented.
We are also investigating the generation of transient intermediates in the reaction cycle of P450-CAM. While monitoring the reaction of ferric cytochrome P450-CAM with substituted (Cl, CH3, OCH3) perbenzoic acids using rapid scan stopped flow spectroscopy, an intermediate appears en route to Compound I. We have proposed that this moiety is an acylperoxo-ferric heme adduct, which would then undergo O-O bond cleavage, releasing the substituted benzoic acid as a leaving group, to generate Compound I. Singular value decomposition analysis of stopped flow data for the formation of this intermediate shows that the energy of its Soret absorption peak is sensitive to the electron donor properties of the aromatic ring perbenzoic acid substituents. A linear Hammett correlation plot is seen for the energy of the Soret peak vs. the Hammett ρ constant. This correlation requires that the substituents remain as part of the ligand bound to the heme iron, providing direct evidence that the intermediate is indeed a ferric-acylperoxo derivative. Linear Hammett correlation plots are also seen for both the rate of intermediate formation as well as for its conversion to Compound I. It is proposed that the electron donating/withdrawing properties of the substituents affect the acidity of the O-OH for binding to form the acylperoxo intermediate as well as the propensity of the substituted benzoic acid to serve as the leaving group during O-O bond cleavage to yield Compound I.
Funding: NIH GM 26730. We thank Steve Sligar for the T252A over expression system.