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...