Molecular wires comprising a Ru(II)-or Re(I)-complex head group, an aromatic tail group, and an alkane linker reversibly inhibit the activity of the copper amine oxidase from Arthrobacter globiformis (AGAO), with K i values between 6 M and 37 nM. In the crystal structure of a Ru(II)-wire:AGAO conjugate, the wire occupies the AGAO active-site substrate access channel, the trihydroxyphenylalanine quinone cofactor is ordered in the ''off-Cu'' position with its reactive carbonyl oriented toward the inhibitor, and the ''gate'' residue, Tyr-296, is in the ''open'' position. Head groups, tail-group substituents, and linker lengths all influence wire-binding interactions with the enzyme.diimine ͉ topaquinone ͉ metalloenzyme ͉ active site C opper and quinone containing amine oxidases (EC 1.4.3.6) catalyze the oxidative deamination of primary amines to the corresponding aldehydes with concomitant generation of ammonia and hydrogen peroxide.Each subunit of these homodimeric enzymes contains a deeply buried active site comprised of a single type II (''non-blue,'' square-pyramidal) copper atom and an organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (topaquinone or TPQ) (1, 2). The finding that the human vascular adhesion protein (HVAP-1) is a copper amine oxidase (CuAO) has heightened interest in the mechanism and inhibition of these enzymes (3). With the potential for therapeutic applications, research has focused on elucidation of the factors that govern inhibitor sensitivity and selectivity.We are exploring the potential of channel-blocking metaldiimine wire complexes to function as highly selective inhibitors of CuAOs. We chose phenylethylamine oxidase from Arthrobacter globiformis for initial study, owing to its ease of expression and purification as a C-terminal Strep-tag II fusion protein (4). Our choice of metal-diimine wires was based on the results of extensive investigations of their conjugates with cytochrome P450cam, which have revealed structural features of conformational states that likely are involved in steps of the catalytic cycle of the enzyme (5-9). Similar molecular wires have been used in attempts to measure the reduction potentials of deeply buried protein cofactors; indeed, in experiments of relevance here, a diethylaniline-tipped triphenylene wire coupled to a gold electrode allowed electrochemical characterization of Arthrobacter globiformis amine oxidase (AGAO) cofactor TPQ (10). Binding of the wire in the active-site channel was not established independently but could be inferred from the efficiency of electron tunneling from the electrode to the buried cofactor.We have designed and synthesized a series of highly potent channel-blocking inhibitors of AGAO. The crystal structure of a Ru-wire:AGAO conjugate clearly demonstrates that the wire resides in the active-site channel; it also reveals key aspects of active-site topology and conformational mobility. Furthermore, variations in binding in response to changes in wire sensitizer, substrate, and linker compositions have led to particula...
