Monoamine oxidases (MAO) A and B are ϳ60-kDa outer mitochondrial membrane flavoenzymes catalyzing the degradation of neurotransmitters and xenobiotic arylalkyl amines. Despite 70% identity of their amino acid sequences, both enzymes exhibit strikingly different properties when exposed to thiol-modifying reagents. Human MAO A and MAO B each contain 9 cysteine residues (7 in conserved sequence locations).
Monoamine oxidases (MAO)1 A and B are outer mitochondrial membrane enzymes whose function is to catalyze the oxidative deamination of neurotransmitters (serotonin, dopamine, and norepinephrin), dietary amines (phenylethylamine), and arylalkylamine-containing drugs used in numerous therapies (1). Human enzymes are pharmacological targets for antidepressants and neuro-protective agents. Although MAO A and MAO B have similar catalytic activities, they differ in substrate specificities and tissue distribution (1, 2). Exploitation of subtle differences in MAO A and MAO B structures is desirable to establish isoform-specific drugs.Human MAO A and MAO B are encoded by separate genes and share a sequence homology of ϳ70% (3). Activity of either enzyme is sensitive to thiol-modifying reagents (4, 5), and cysteine residues have been implicated in the catalytic mechanism of MAO based on the results of site-directed mutagenesis (6, 7), thiol titration (8), and inhibitor binding (9) experiments. Human MAO A and MAO B each contain 9 cysteine residues with 7 of them in highly conserved positions (Scheme 1; A165/ B156, A201/B192, A306/B297, A321/B312, A374/B365, A398/ B389, and A406/B397) (3).2 In each enzyme, one conserved cysteine residue (Cys 406 in MAO A and Cys 397 in MAO B) is linked in a thioether bond to the 8␣-methylene of FAD (10, 11), therefore leaving 8 cysteine residues either as free thiols or in disulfide bonds. Gomes et al. (8) reported that 2 cysteine residues in bovine liver MAO B are protected from reaction with thiol probes by substrates, concluding that these 2 residues are located to the active site and essential for enzyme catalytic activity. The proximity of Cys 365 to the catalytic site in bovine MAO B was proposed by Zhong and Silverman (9) by demonstrating this residue to be modified on inhibition of bovine liver MAO B with N-cyclopropyl-␣-methylbenzylamine. Mutagenesis experiments by Wu et al. (6) (6,7,9), are located on the surface of the molecule.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).To address these uncertainties that exist in the literature * This work was supported by National Institutes of Health Grant GM-29433. The mass spectrometry e...