Monoamine oxidase inhibitors were among the first antidepressants to be discovered and have long been used as such. It now seems that many of these agents might have therapeutic value in several common neurodegenerative conditions, independently of their inhibition of monoamine oxidase activity. However, many claims and some counter-claims have been made about the physiological importance of these enzymes and the potential of their inhibitors. We evaluate these arguments in the light of what we know, and still have to learn, of the structure, function and genetics of the monoamine oxidases and the disparate actions of their inhibitors.

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Monoamine oxidase B (MAO B) is a mitochondrial outermembrane flavoenzyme that is a well-known target for antidepressant and neuroprotective drugs. We determined the structure of the human enzyme to 3 A resolution. The enzyme binds to the membrane through a C-terminal transmembrane helix and apolar loops located at various positions in the sequence. The electron density shows that pargyline, an analog of the clinically used MAO B inhibitor, deprenyl, binds covalently to the flavin N5 atom. The active site of MAO B consists of a 420 A(3)-hydrophobic substrate cavity interconnected to an entrance cavity of 290 A(3). The recognition site for the substrate amino group is an aromatic cage formed by Tyr 398 and Tyr 435. The structure provides a framework for probing the catalytic mechanism, understanding the differences between the B- and A-monoamine oxidase isoforms and designing specific inhibitors.

Incubation of the apoB2 subunit of Escherichia coli ribonucleotide reductase with Fe2+ and O2 produces native B2, which contains the tyrosyl radical-dinuclear iron cluster cofactor required for nucleotide reduction. The chemical mechanism of this reconstitution reaction was investigated by stopped-flow absorption spectroscopy and by rapid freeze-quench EPR (electron paramagnetic resonance) spectroscopy. Two novel intermediates have been detected in the reaction. The first exhibits a broad absorption band centered at 565 nanometers. Based on known model chemistry, this intermediate is proposed to be a mu-peroxodiferric complex. The second intermediate exhibits a broad absorption band centered at 360 nanometers and a sharp, isotropic EPR signal with g = 2.00. When the reaction is carried out with 57Fe2+, this EPR signal is broadened, demonstrating that the intermediate is an iron-coupled radical. Variation of the ratio of Fe2+ to B2 in the reaction and comparison of the rates of formation and decay of the intermediates to the rate of formation of the tyrosyl radical (.Y122) suggest that both intermediates can generate .Y122. This conclusion is supported by the fact that both intermediates exhibit an increased lifetime in a mutant B2 subunit (B2-Y122F) lacking the oxidizable Y122. Based on these kinetic and spectroscopic data, a mechanism for the reaction is proposed. Unlike reactions catalyzed by heme-iron peroxidases, oxygenases, and model complexes, the reconstitution reaction appears not to involve high-valent iron intermediates.

The three-dimensional structure of recombinant human monoamine oxidase A (hMAO A) as its clorgyline-inhibited adduct is described. Although the chain-fold of hMAO A is similar to that of rat MAO A and human MAO B (hMAO B), hMAO A is unique in that it crystallizes as a monomer and exhibits the solution hydrodynamic behavior of a monomeric form rather than the dimeric form of hMAO B and rat MAO A. hMAO A's active site consists of a single hydrophobic cavity of Ϸ550 Å 3 , which is smaller than that determined from the structure of deprenyl-inhibited hMAO B (Ϸ700 Å 3 ) but larger than that of rat MAO A (Ϸ450 Å 3 ). An important component of the active site structure of hMAO A is the loop conformation of residues 210 -216, which differs from that of hMAO B and rat MAO A. The origin of this structural alteration is suggested to result from long-range interactions in the monomeric form of the enzyme. In addition to serving as a basis for the development of hMAO A specific inhibitors, these data support the proposal that hMAO A involves a change from the dimeric to the monomeric form through a Glu-151 3 Lys mutation that is specific flavin ͉ neurotransmitter ͉ membrane protein ͉ antidepressant target H uman monoamine oxidase A (hMAO A) is an outer mitochondrial membrane-bound flavoenzyme that catalyzes the oxidation of the neurotransmitters serotonin, dopamine, and norepinephrine. Recent studies have demonstrated that a deficiency or low level of expression of this enzyme results in a phenotype of aggressive behavior (1, 2). The elucidation of the 3D structures of human MAO B (hMAO B) (3, 4) (72% sequence identity with hMAO A) and of rat MAO A (rMAO A) (5) (92% sequence identity with hMAO A with no insertions or deletions) has provided insights into the structure and mechanism of these pharmacologically important enzymes. There are several functional properties of hMAO A that differentiate it from rMAO A, despite their high level of sequence identity. hMAO A has been shown to exhibit a 10-fold lower affinity (IC 50 ) than rMAO A for the specific irreversible inhibitor clorgyline (6). Comparisons of the influence of a Phe-208 3 Ile mutation on MAO A from human (7) and rat (8) also show differential effects on activities and sensitivities to irreversible inhibition. Functional differences between hMAO A and rMAO A have been implicated in comparison with their respective sensitivities to phentermine inhibition (9). These differences in properties between hMAO A and rMAO A suggest structural differences exist for these two enzymes.With the development of a high-level expression system for hMAO A in our laboratory (10) and successes with the structural elucidation of hMAO B (3, 4), a collaborative program was established to elucidate the structure of hMAO A by x-ray crystallography. Here, we report the structures of two hMAO A crystal forms and demonstrate structural differences between hMAO A and rMAO A as well as hMAO B. Our data indicate that the considerable literature on MAO A-inhibitor development by using rat models m...

The R2 subunit of Escherichia coli ribonucleotide reductase (RNR) contains a stable tyrosyl radical (•Y122) diferric cluster cofactor. Earlier studies on the cofactor assembly reaction detected a paramagnetic intermediate, X, that was found to be kinetically competent to oxidize Y122. Studies using rapid freeze-quench (RFQ) Mössbauer and EPR spectroscopies led to the proposal that X is comprised of two high spin ferric ions and a S = 1/2 ligand radical, mutually spin coupled to give a S = 1/2 ground state (Ravi, N.; Bollinger, J. M., Jr.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8007−8014). An extension of RFQ methodology to Q-band ENDOR spectroscopy using 57Fe has shown that one of the irons has a very nearly isotropic hyperfine tensor (A(FeA) = −[74.2(2), 72.2(2), 73.2(2)] MHz) as expected for FeIII, but that the other iron site displays considerable anisotropy (A(FeB) = +[27.5(2), 36.8(2), 36.8(2)] MHz), indicative of substantial FeIV character. Reanalysis of the Mössbauer data using these results leads to isomer shifts of δ(FeA) = 0.56(3) mm/s and δ(FeB) = 0.26(4) mm/s. Based on the hyperfine anisotropy of FeB plus the reduced isomer shift, X is now best described as a spin-coupled FeIII/FeIV center without a radical, but with significant spin delocalization onto the oxygen ligand(s).

Monoamine oxidase B (MAO-B)is an outer mitochondrial membrane-bound enzyme that catalyzes the oxidative deamination of arylalkylamine neurotransmitters and has been a target for a number of clinically used drug inhibitors. The 1.7-Å structure of the reversible isatin-MAO-B complex has been determined; it forms a basis for the interpretation of the enzyme's structure when bound to either reversible or irreversible inhibitors. 1,4-Diphenyl-2-butene is found to be a reversible MAO-B inhibitor, which occupies both the entrance and substrate cavity space in the enzyme.

Structures of human monoamine oxidase B (MAO B) in complex with safinamide and two coumarin derivatives, all sharing a common benzyloxy substituent, were determined by X-ray crystallography. These compounds competitively inhibit MAO B with Ki values in the 0.1-0.5 microM range that are 30-700-fold lower than those observed with MAO A. The inhibitors bind noncovalently to MAO B, occupying both the entrance and the substrate cavities and showing a similarly oriented benzyloxy substituent.