Kinetic characterization of transient free radical intermediates in reaction of lysine 2,3-aminomutase by EPR lineshape analysis

Abstract

Radicals have been detected in the steady states of most of the adenosylcobalamin-dependent reactions. Although in most cases the structures of these radicals have not been fully established, the thiyl-protein radical in B 12 -dependent ribonucleotide reductase has been characterized. Radical intermediates in monooxygenase reactions—such as those of cytochrome P450s and methane monooxygenase—have been postulated. However, the assignment of radical mechanisms is controversial in these cases because of the indirect nature of the evidence for the participation of organic radicals. The most direct evidence for a radical mechanism is the observation of transient radicals by electron paramagnetic resonance spectroscopy (EPR). In these cases, the question of kinetic competence can be addressed experimentally by rapid-mix freeze-quench EPR spectroscopy. This technique has allowed the kinetic competence of the thiyl-protein radical in the B l2 -dependent ribonucleotide reductase to be proven through studies in the past. The method described in the chapter should be generally applicable to the evaluation of kinetic competence for any enzymatic radical that can be labeled with deuterium or carbon-13 to perturb the EPR spectrum. Increasing numbers of such radicals are being observed in the rapidly expanding world of enzymatic radicals.