Manganese Horseradish Peroxidase And Cytochrome P450: Probes Into The Intermediates Of Oxidized Heme Proteins.

Manganese substituted horseradish peroxidase (A), was oxidized by p-nitroperoxybenzoic acid to produce B with a visible absorption Soret at 412 nm and g = 5.06, 2.04 signals in the electron paramagnetic resonance spectrum. Compound B reacted with thiosalicylic acid to form disulfide and A. Compound B was characterized by oxidative and reductive titration. By oxidative and reductive titration with p-nitroperoxybenzoate and thiosalicylate respectively, compound B was oxidized by two electrons and was proposed to be a Manganese (IV) species with a free radical located on an amino acid side chain. Reaction of A with mercaptan and dioxygen resulted in formation of D with a visible absorption Soret at 407 nm and a g = 4.01 EPR signal. Compound D reacted further with mercaptan producing resting state A and disulfide. One equivalent of thiosalicylate reacted with O(,2) and A to form D and three equivalents reacted with D to produce A. Oxygenation of Mn('+2)HRP also produced D. On the basis of these results D was proposed to be a Mn('+3)HRP(O(,2)('-)) or a Mn('+2)HRP(O(,2)) complex. Cytochrome P-450 reacted with hydroperoxyhexane to form 1,2-hexane diol. P-450 mediated conversion of (R)-, (S)-, and racemic 2-deuterio-1-hydroperoxyhexanes to the corresponding diols were accompanied by epimerization at C-2 and a kinetic isotope effect of k(,H)/k(,D) = 2.06. Doubly labelled oxygen-18 labelled hydroperoxyhexane reacted analogously in the presence of unlabelled peroxide to form doubly labelled and unlabelled diols. Oxygen was transferred to the iron atom, then a C-2 hydrogen atom was abstracted nonstereospecifically. The resulting intermediates interacted to transfer (.)OH to the carbon centered radical nonstereospecifically. Epoxidation of E-1-deuteriopropene in H(,2)O by the Cytochrome P-450 LM(,2) reconstituted system produced propene d(,0). Oxidation of E-1-deuterio-1-butene resulted in 12% ethyloxirane, and 88% trans-3-deuterio-2-ethyloxirane. Similar results were observed by oxidation of unlabelled propene and 1-butene in D(,2)O. This phenomenon was unique to these substrates. The exchange was due to reversible and stereoselective deprotonation of an oxametallacycle by a basic side chain forming an iron bound carbene complex intermediate.Ph.D.BiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127959/2/8702730.pd