Investigation of Spectroscopic Intermediates during Copper-Binding and TPQ Formation in Wild-Type and Active-Site Mutants of a Copper-Containing Amine Oxidase from Yeast † Joanne E. Dove, Benjamin Schwartz, Neal K. Williams, ‡ and Judith P. Klinman* Departments of Chemistry and Molecular and Cell Biology, UniVersity of California, Berkeley, California 94720 ReceiVed September 24, 1999; ReVised Manuscript ReceiVed January 12, 2000 ABSTRACT: Copper amine oxidases possess the unusual ability to generate autocatalytically their organic cofactor, which is subsequently utilized in turnover. This cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), is formed within the active site of these enzymes by the oxidation of a single tyrosine residue. In Vitro, copper(II) and oxygen are both necessary and sufficient for the conversion of tyrosine to TPQ. In this study, the biogenesis of TPQ has been characterized in an amine oxidase from Hansenula polymorpha expressed as the apo-enzyme in Escherichia coli. With the WT enzyme, optical absorbances which are copper or oxygen dependent are observed and characterized. Active-site mutants are used to investigate further the nature of these spectral species. Evidence is presented which suggests that tyrosine is activated for reaction with oxygen by liganding to Cu(II). In the following paper in this issue [Schwartz, B., Dove, J. E., and Klinman, J. P. (2000) Biochemistry 39, 3699-3707], the initial reaction of precursor protein with oxygen is characterized kinetically. Taken together, the available data suggest a mechanism for the oxidation of tyrosine to TPQ where the role of the copper is to activate substrate. Copper amine oxidases (CAOs) 1 constitute a ubiquitous class of enzymes which catalyze the oxidation of primary amines to the corresponding aldehydes, concomitant with the reduction of dioxygen to hydrogen peroxide (1). These enzymes allow prokaryotes and fungi to use primary amines as the sole source of nitrogen for growth. In plants, hydrogen peroxide generated by CAOs during enzymatic turnover has been shown to be important for cell wall formation and wound healing (2, 3). A role for CAOs in mammals has not been as well-defined, but existing proposals have focused on two possiblities. The initial suggestion was that CAOs function to metabolize biogenic amines (3). Recently, evidence of hydrogen peroxide involvement in cell-signaling pathways (4-6) has raised the possibility of a role for CAOs in cellular homeostasis. CAOs are homodimers with each subunit containing a mononuclear copper and 2,4,5-trihydroxyphenylalanine quino- ne (TPQ or topa quinone) as cofactors (7). Catalysis has been shown to proceed via ping-pong kinetics (1) with the following half reactions: The catalytic mechanism has been extensively studied (8, 9), but much less is known about the biogenesis of the cofactor. TPQ is formed by the oxidation of a tyrosine within the active site (10). Crystal structures of holo-enzymes from various sources have shown that the copper is liganded by three histidines and two waters and that the plane of the TPQ ring is approximately 4-5 Å away from the copper (11- 14). A crystal structure of the apo-enzyme from Arthrobactor globiformis (AGAO) has also been solved, indicating no significant differences in the backbone between the apo- and holo-enzymes. The main structural difference in the apo- enzyme is a rotation of the precursor tyrosine side chain relative to the position of the mature cofactor, such that the tyrosyl oxygen is pointed toward the vacant copper-binding site (13). Under certain conditions, the mature TPQ has also been shown to rotate toward the copper site in a manner similar to the precursor tyrosine. In this case, TPQ can then ligand the copper through its 0-4 oxygen, though this is a catalytically inactive conformation (13, 14). TPQ is generated autocatalytically, requiring only copper and oxygen to initiate the reaction (15, 16). Freeman and co-workers have suggested a mechanism for biogenesis based largely on structural data (13). The mechanism (Scheme 1) † This work was supported by a National Institutes of Health Grant (GM 39296) to J.P.K. J.E.D. was supported by a Training Grant from the National Institutes of Health (GM 08295-10). B. Schwartz was supported by a National Institutes of Health Postdoctoral Fellowship (GM 18813). N.K.W. was supported by an Australian National Health and Medical Research Council C. J. Martin Postdoctoral Fellowship. * To whom correspondence should be addressed. E-mail: klinman@ socrates.berkeley.edu. Phone: (510) 642-2668. Fax: (510) 643-6232. ‡ Current address: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia. 1 Abbreviations: WT, wild-type; HPAO, Hansenula polymorpha amine oxidase; CAO, copper amine oxidase; TPQ, topa quinone or 2,4,5-trihydroxyphenylalanine quinone; AGAO, Arthrobactor globi- formis amine oxidase; PCD, protocatechuate 3,4-dioxygenase; PCA, protocatechuate; HEPES, (N-[2-hydroxyethyl]piperazine-N′-[2-ethane- sulfonic acid]); ICP-AES, inductively coupled plasma-atomic emission spectroscopy; EPR, electron paramagnetic resonance; LMCT band, ligand to metal charge-transfer band; DEANO, diethylamine nitric oxide, sodium salt. TPQ ox + R-NH 3 + f TPQ red + R-CHO (Reductive) TPQ red + O 2 f TPQ ox + H 2 O 2 + NH 4 + (Oxidative) 3690 Biochemistry 2000, 39, 3690-3698 10.1021/bi992225w CCC: $19.00 © 2000 American Chemical Society Published on Web 03/10/2000