Observation of a Flavin Semiquinone in the Resting State of Monoamine Oxidase B by Electron Paramagnetic Resonance and Electron Nuclear Double Resonance Spectroscopy^,

Abstract

Monoamine oxidase (MAO) plays an essential role in the regulation of various neurotransmitter and xenobiotic amines. Inhibitors of MAO have been employed in the treatment of depression and as adjuncts in Parkinson's disease therapy. X-Band and Q-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic techniques are employed to characterize a signal assigned as a stable red anionic semiquinone radical in the resting state of MAO B. It is shown that the radical signal is not affected during substrate (either benzylamine or phenylethylamine) turnover, by anaerobic incubation with substrate, or by covalent modification of the active site flavin cofactor in the catalytically active dimer. Upon denaturation, however, the semiquinone absorbances and EPR signals are lost. Photoreduction of the native enzyme in the presence of ethylenediaminetetraacetate generates an EPR signal that is not the same as that obtained in the resting state and shows different proton ENDOR signals. These results suggest that the two flavin prosthetic groups that exist in catalytically active monoamine oxidase B are physically distinct.

High-Level Expression of Human Liver Monoamine Oxidase B in Pichia pastoris

“…Further reduction by the addition of a total of 1.2 mol of dithionite per mole of enzyme results in the formation of the flavin hydroquinone, with little or no semiquinone form remaining (EPR data not shown). These data show that a semiquinone form is not required for enzyme activity as suggested by DeRose et al (21). This radical form in bovine liver MAO B probably originates from reactive oxygen species that are known to form on the disruption of mitochondria (45), although further work is required to document this suggestion.…”

“…The expressed enzyme exhibits many of the kinetic and structural properties of the well-studied bovine liver enzyme. The properties of the recombinant enzyme suggest the observed flavin radical in the resting form of the bovine enzyme observed previously (20,21) has no catalytic significance, in contrast to previous suggestions, and probably arises as an artifact of preparation. Using high-resolution mass spectral techniques, we found that the posttranslational modifications observed in bovine liver MAO B (N-terminal acetylation and covalent FAD incorporation) are also found in the recombinant human liver MAO B.…”

“…The presence of variable levels of anionic semiquinone in bovine liver MAO B was first demonstrated by Raman spectroscopy (20) and later also observed by EPR spectroscopy of the "resting" form of the enzyme (21). DeRose et al (21) suggested this semiquinone form of the enzyme may have mechanistic significance in support of the proposed aminium cation radical mechanism (42).…”

A Stable Tyrosyl Radical in Monoamine Oxidase A

“…Previous EPR studies have not revealed the presence of the tyrosyl radical reported here (15,31). However, the conditions employed in these studies were not optimized for the detection of the additional features shown in Fig.…”

Flavoenzyme catalysed oxidation of amines: roles for flavin and protein-based radicals

“…An organic radical species was originally reported in EPR spectra of resting (i.e. flavin in the oxidized state) bovine liver MAO B [13,14], but more recent studies have confirmed the lack of a protein-based radical in highly purified forms of recombinant MAO A and MAO B in the resting state [15,16]. The lack of an observed radical species in the resting states of MAO A and MAO B therefore was seen to seriously question the validity of this proposed mechanism for the MAO enzymes.…”