Monoamine Oxidase Contains a Redox-active Disulfide

Abstract

Mitochondrial monoamine oxidases A and B (MAO Flavin-containing mitochondrial monoamine oxidases A and B (MAO A and MAO B)1 catalyze the oxidative deamination of neurotransmitters, such as dopamine, serotonin, and noradrenaline in the central nervous system and peripheral tissues. The enzymes share 73% sequence homology and follow the same kinetic and chemical mechanism but have different substrate and inhibitor specificities (1). Inhibitors of these enzymes are medically important antidepressants, but the rational design of new inhibitor drugs is hampered by the lack of the active site structure and by remaining controversies in the catalytic mechanism.Chemical modification experiments provide evidence that a histidine residue (2, 3) is essential for the catalysis. There is also strong evidence that two cysteine residues are present in the active site of MAO (3-9). The inhibition of MAO by sulfhydryl reagents, first observed in 1945 (10), is well established (3-6), but a role for essential cysteine residues in the catalytic mechanism has not been identified. In the chemical mechanism, there is still controversy about whether MAO-catalyzed oxidative deamination proceeds via a radical mechanism, hydride transfer, or oxidation of a carbanion intermediate. In the most extensively tested hypothesis (11), the transfer of one electron from the amine to the enzyme (presumably to the flavin) is followed by the cleavage of the ␣-carbon-hydrogen bond to produce the amino radical. The substrate radical either passes a second electron to the flavin or combines with an unknown active site radical to give a covalent adduct that decomposes to the imine. However, no flavosemiquinone has ever been detected in the catalytic cycle (12-15). When flavosemiquinone is generated by reduction with dithionite, the weak epr signal observed (about half that expected) suggested that there might be coupling of FAD radical with an unknown protein radical (14). If such a putative non-FAD radical can be generated by reduction by dithionite, the number of electrons required to reduce the enzyme should be more than the two necessary for the FAD alone. The data presented in this paper demonstrate the presence of a second redox-active group in addition to the flavin in the active site of MAO. EXPERIMENTAL PROCEDURESEnzymes and Reagents-Human liver MAO A heterologously expressed in yeast and MAO B from beef liver were purified as previously reported (16,17). The concentration of each sample was determined from the oxidized minus reduced extinction coefficient at 456 nm of 10,800 M Ϫ1 cm Ϫ1 for MAO A and 10,300 M Ϫ1 cm Ϫ1 for MAO B. Enzyme activity was determined spectrophotometrically using kynuramine (1 mM) for MAO A and benzylamine (3 mM) for MAO B.Kynuramine dihydrobromide, benzylamine, and D-amphetamine hydrochloride were purchased from Sigma. 4,4Ј-Dipyridyl disulfide (DPDS) (Aldrich) was recrystallized once from ethanol. All the experiments were carried out at 20°C in 50 mM sodium phosphate buffer, pH 7.2, containing 0.01% Brij-35.4,4Ј-Dithio(b...

Structural Comparison of Human Monoamine Oxidases A and B

“…MAO B is inactivated at a faster rate (1 ⁄2 ϭ ϳ3 h) when an 800-fold excess of NEM was used at 25°C. All of the enzyme inactivations exhibit pseudo-first-order kinetic behavior, with the exception of MAO A inactivation at 25°C, which is biphasic, in agreement with previous reports (13).…”

“…A recent study (13) on the stoichiometry of reduction of bovine MAO B or of recombinant human liver MAO A by sodium dithionite led to the conclusion that each enzyme contains a redox active disulfide group, which was suggested to function catalytically in shuttling reducing equivalent between the amine substrate and the FAD. This suggestion has been questioned because anaerobic titration of either enzyme requires stoichiometric levels of substrate to reduce the enzymebound flavin coenzyme (14,15).…”

Substrates but Not Inhibitors Alter the Redox Potentials of Monoamine Oxidases

“…2. After thiol modification, only two electrons were required for full reduction and no semiquinone formation could be detected (7). In this modified enzyme, the slope of the redox plot was 1 as expected for two-electron transfer to both enzyme and dye.…”

Mechanisms underlying striatal vulnerability to 3‐nitropropionic acid

“…As cysteines are known to be essential for MAO activity (Wu et al. 1993; Sablin and Ramsay 1998), it is possible that DA‐quinones produced by 3‐NPA are inhibiting MAO activity forming ‘quino‐MAO‐adducts’. Moreover, quinones of both DA and L‐DOPA were shown to inhibit tryptophan hydroxylase and modify the protein to a redox‐active quino‐tryptophan hydroxylase (Kuhn and Arthur 1998, 1999).…”