Inhibition and Oxygen Activation in Copper Amine Oxidases

Eric M. Shepard *‡ and David M. Dooley *†

‡ Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, United States

† Office of the President, University of Rhode Island, Kingston, Rhode Island 02881, United States

Acc. Chem. Res., Article ASAP

DOI: 10.1021/ar500460z

Publication Date (Web): April 21, 2015

Copyright © 2015 American Chemical Society

*Voice, 406-994-6162; fax, 406-994-5407; e-mail, eshepard@montana.edu., *Voice, 401-874-2444; fax, 401-874-7149; e-mail, davedooley@ds.uri.edu.

Biography

Eric M. Shepard graduated from Rocky Mountain College with a B.Sc. degree in chemistry (1999). He received his Ph.D. (2006) in biochemistry from Montana State University in the laboratory of David Dooley, where he was supported by a NSF IGERT fellowship. He is currently a Senior Research Scientist in the laboratory of Joan Broderick where he studies [FeFe]-hydrogenase H-cluster biosynthesis.

Biography

David M. Dooley graduated from the University of California, San Diego, with a B.A. in chemistry (1974) and received his Ph.D. (1979) in chemistry from Caltech in the laboratory of Harry Gray. He was Professor of Chemistry at Amherst College before becoming Head of the Department of Chemistry and Biochemistry at Montana State University in 1993. He then became Provost and Vice President for Academic Affairs at Montana State and now serves as the President for the University of Rhode Island. His research interests encompass copper–oxygen chemistry in metalloproteins.

Abstract

Conspectus

Copper-containing amine oxidases (CuAOs) use both copper and 2,4,5-trihydroxyphenylalanine quinone (TPQ) to catalyze the oxidative deamination of primary amines. The CuAO active site is highly conserved and comprised of TPQ and a mononuclear type II copper center that exhibits five-coordinate, distorted square pyramidal coordination geometry with histidine ligands and equatorially and axially bound water in the oxidized, resting state. The active site is buried within the protein, and CuAOs from various sources display remarkable diversity with respect to the composition of the active site channel and cofactor accessibility. Structural and mechanistic factors that influence substrate preference and inhibitor sensitivity and selectivity have been defined. This Account summarizes the strategies used to design selective CuAO inhibitors based on active site channel characteristics, leading to either enhanced steric fits or the trapping of reactive electrophilic products. These findings provide a framework to support the future development of candidate molecules aimed at minimizing the negative side effects associated with drugs containing amine functionalities. This is vital given the existence of human diamine oxidase and vascular adhesion protein-1, which have distinct amine substrate preferences and are associated with different metabolic processes. Inhibition of these enzymes by antifungal or antiprotozoal agents, as well as classic monoamine oxidase (MAO) inhibitors, may contribute to the adverse side effects associated with drug treatment. These observations provide a rationale for the limited clinical value associated with certain amine-containing pharmaceuticals and emphasize the need for more selective AO inhibitors.

This Account also discusses the novel roles of copper and TPQ in the chemistry of O2activation and substrate oxidation. Reduced CuAOs exist in a redox equilibrium between the Cu(II)–TPQAMQ (aminoquinol) and Cu(I)–TPQSQ (semiquinone). Elucidating the roles of Cu(I), TPQSQ, and TPQAMQ in O2 activation, for example, distinguishing inner-sphere versus outer-sphere electron transfer mechanisms, has been actively investigated since the discovery of TPQSQ in 1991 and has only recently been clarified. Kinetics and spectroscopic studies encompassing metal substitution, stopped-flow and temperature-jump relaxation methods, and oxygen kinetic isotope experiments have provided strong support for an inner-sphere electron transfer step from Cu(I) to O2. Data for two enzymes support a mechanism wherein O2 prebinds to a three-coordinate Cu(I) site, yielding a [CuII(η1-O2–1)]+ intermediate, with H2O2 generated from ensuing rate-determining proton coupled electron transfer from TPQSQ. While kinetics data from the cobalt-substituted yeast enzyme indicated that O2 is reduced through an outer-sphere process involving TPQAMQ, new findings with a bacterial CuAO demonstrate that both the Cu(II) and Co(II) forms of the enzyme operate via parallel mechanisms involving metal–superoxide intermediates. Structural observations of a coordinated TPQSQ–Cu(I) complex in two CuAOs supports previous indications that Cu(II)/(I) ligand substitution chemistry may be mechanistically relevant. Substantial evidence indicates that rapid and reversible inner-sphere reduction of O2at a three-coordinate Cu(I) site occurs, but the existence of a coordinated semiquinone in some AOs suggests that, in these enzymes, an outer-sphere reaction between O2 and TPQSQ may also be possible, since this is expected to be energetically favorable compared with outer-sphere electron transfer from TPQAMQ to O2.

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