Cryoreduction EPR and 13C, 19F ENDOR study of substrate-bound substates and solvent kinetic isotope effects in the catalytic cycle of cytochrome P450cam and its T252A mutant

Abstract

We recently used cryoreduction EPR/ENDOR techniques to show that a substrate can modulate the properties of both the monooxygenase active-oxygen intermediates and of the proton-delivery network which encompasses them. In the present report we use Q-band pulsed 19F ENDOR (Mims 3-pulse sequence) to examine the substrate binding geometries of camphor, through use of the 5,5'--difluorocamphor, and 13C ENDOR to examine the binding of 5-methylenyl camphor labeled with 13C at C11. These probes are examined in multiple states of the catalytic cycle of P450cam and its T252A mutant. As part of this investigation we further report a new cryoreduction reaction, the reduction of a ferroheme to the EPR-visible Fe(I) state, and use it to probe the substrate binding to the EPR-silent ferroheme state. Finally we report the solvent kinetic isotope effect on the decay of the camphor complex of the hydroperoxo-ferric intermediate, the first such measurement on an individual step within the P450cam reaction cycle. Following reduction of oxyferrous-P450cam, this step is the rate-limiting step in camphor hydroxylation, and its solv-KIE of 1.8 at 190 K establishes that it involves activation of the hydroperoxo moiety by transfer of the 'second' proton of catalysis. We suggest that the finding that the heme pocket can exist in multiple substates, including multiple substrate binding locations, even in P450cam, along with the established possibility that the hydroperoxo-ferriheme intermediate can react with substrate, may explain the formation of multiple products by P450s.

Active intermediates in heme monooxygenase reactions as revealed by cryoreduction/annealing, EPR/ENDOR studies

“…The most detailed studies were carried out with cytochrome P450cam [20, 41, 56] and gsNOS [25] which form moderately stable oxycomplexes. This reaction cycle for P450cam is likely to be shared by most member of the P450 family, and this protein commonly is considered as prototypical [2].…”

“…During extended annealing at ∼ 180K the EPR signal of 5 disappears and is replaced by a signal from the primary product state of camphor hydroxylation 7 , a non-equilibrium conformer of the complex of ferric P450cam with 5-exo hydroxycamphor. The decay of 5 slows down by factor of sKIE ∼ 2 in deuterated solvent [56], which indicates that the rate-limiting step for reaction of this species involves activation of the hydroperoxy moiety by transfer of the “second “ proton of catalysis. The primary product 7 converts to a relaxed conformer during annealing at ∼ 220 K; at low protein concentration the bound 5-exo hydroxycamphor then dissociates at temperatures above 240K.…”

“…Cryoreduction/annealing experiments confirmed this supposition. As formed cryoreduction of the ternary complexes of ferrous oxy wt and T252A P450 with 5-methylenyl camphor produced 5 with similar EPR and ENDOR properties, both formed the epoxide upon annealing product [41, 56]. However, the primary product state 7 trapped during annealing the wt 5 has the epoxide product bound to the ferriheme, as shown by the presence of strongly–coupled, non-exchangeable proton ENDOR signals, and thus implies that 6 is the intermediate that epoxidizes 5-methylenyl camphor (Scheme 2).…”

Mechanistic studies on versatile metal-assisted hydrogen peroxide activation processes for biomedical and environmental incentives

“…This species is perceived to be responsible for the thermodynamically demanding activation of C-H bonds [52,[63][64][65][66][67][68][69][70]. However, in a study involving the application of H2O2, the homolytic mode of O-O bond scission seemed to prevail [71].…”

5-Fluorolysine as alternative substrate of lysine 5,6-aminomutase: A computational study

“…Fluorine ( 19 F: I = ½, natural abundance 100%, c = 26.54 [33]) has favorable parameters to act as a probe for EPR and ENDOR [34][35][36][37][38][39][40] identification of radical intermediates by introducing hyperfine splittings. Electron withdrawing effect of fluorine is helpful to stabilize radicals.…”