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Resonance Raman spectroscopy is an excellent technique for providing structural information on the 2,4, 5-trihydroxyphenylalanine quinone (TPQ) cofactor in copper-containing amine oxidases. This technique has been used to investigate the copper- and O2-dependent biosynthesis of the TPQ cofactor in phenylethylamine oxidase (PEAO) and histamine oxidase from Arthrobacter globiformis. Incubation of the holoenzyme in H218O causes frequency shifts at 1684(-26) cm-1 in PEAO and at 1679(-28) cm-1 in histamine oxidase, allowing this feature to be assigned to the C=O stretch of a single carbonyl group at the C-5 position. When apoprotein is reacted with Cu(II) and O2 in the presence of H218O, the resultant holoproteins show increased shifts of -3 to -6 cm-1 in a number of other vibrational modes, particularly at 411 and 1397 cm-1. Because these small shifts persist when the H218O-regenerated protein is back-exchanged into H216O, they can be assigned to oxygen isotope substitution at the C-2 postion. No isotope shifts are observed when apoprotein is regenerated with Cu(II) in the presence of 18O2. Thus, it is concluded that the C-2 oxygen atom of TPQ originates from H2O rather than O2. The isotope dependence of the 1397-cm-1 mode allows it to be assigned to the C O moiety at the C-2 position, with its low frequency being indicative of only partial double bond character. Similar frequency shifts due to 18O at C-2 are observed in the resonance Raman spectra of H218O-regenerated PEAO after derivatization of the C-5 carbonyl with either p-nitrophenylhydrazine (-5 cm-1 at 480 cm-1) or methylamine (-5 cm-1 at 1301 cm-1). Taken together, these results indicate that the TPQ cofactor in the native enzyme has substantial electron delocalization between the C-2 and C-4 oxygens and that only the C-5 oxygen has predominantly C=O character.

Electron paramagnetic resonance (EPR), circular dichroism (CD), and optical absorption spectroscopies have been used to investigate the copper-dependent autoxidation process generating the 6-hydroxydopa (topa) quinone cofactor in the recombinant phenethylamine oxidase from Arthrobacter globiformis. The cupric ion bound to the copper/topa quinone-less, inactive enzyme is first reduced to Cu(I), as inferred from the spectroscopic features observed under strictly anaerobic conditions. Cu(I) is also detectable chemically with a Cu(I)-specific chelating agent, bathocuproinedisulfonate. Introduction of a limited amount of oxygen then leads to the formation of a paramagnetic species (g = 2.004) that is stable for over several to 10 min but vanishes swiftly upon addition of sufficient oxygen. Strikingly, the hyperfine EPR structure of the organic radical is almost identical with that of the topa semiquinolamine observed in the copper/topa quinone-containing, active enzyme anaerobically reduced with substrate. Concomitant with the generation of topa quinone exhibiting characteristic optical absorption and CD bands under fully aerobic conditions, the bound copper finally shows EPR signals typical of nonblue type II Cu(II) and optical absorption around 700 nm with negative CD above 700 nm. None of these spectral changes are evoked in the binding of Cu(II) to the Tyr382-->Phe mutant enzyme, indicating that the precursor Tyr382 to topa quinone participates in the initial reduction of bound copper and serves as the origin of the transiently formed semiquinone radical. The prosthetic cupric ion plays an essential role, by changing its redox state, in the oxidative modification of the tyrosyl phenol ring, leading to topa quinone.

The gene coding for histamine oxidase has been cloned and sequenced from a Coryneform bacterium Arthrobacter globiformis. The deduced amino acid sequence consists of 684 residues with a calculated molecular mass of 75,109 daltons and shows a high overall identity (58%) with that of phenethylamine oxidase derived from the same bacterial strain. Although the sequence similarities are rather low when compared with copper amine oxidases from other organisms, the consensus Asn-Tyr-Asp/Glu sequence, in which the middle Tyr is the precursor to the quinone cofactor (the quinone of 2,4,5-trihydroxyphenylalanine, topa) covalently bound to this class of enzymes, is also conserved in the histamine oxidase sequence. To identify the quinone cofactor, an overexpression plasmid has been constructed for the recombinant histamine oxidase. The inactive enzyme purified from the transformed Escherichia coli cells grown in a copper-depleted medium gained maximal activity upon stoichiometric binding of cupric ions. Concomitantly with the enzyme activation by copper, a brownish pink compound was generated in the enzyme, which was identified as the quinone of topa by absorption and resonance Raman spectroscopies of the p-nitrophenylhydrazine-derivatized enzyme and found at the position corresponding to the precursor Tyr (Tyr-402). Therefore, the copper-dependent autoxidation of a specific tyrosyl residue operates on the formation of the topa quinone cofactor in this enzyme, as recently demonstrated with the precursor form of phenethylamine oxidase (Matsuzaki, R., Fukui, T., Sato, H., Ozaki, Y., and Tanizawa, K. (1994) FEBS Lett. 351, 360-364).

The quinone of 2,4,5-trihydroxyphenylalanine (topa), recently identified as the covalently bound redox cofactor in copper amine oxidases, is encoded by a specific tyrosine codon. To elucidate the mechanism of its formation, the recombinant phenylethylamine oxidase of Arthrobacter globiformis has been overproduced in Escherichia coli and purified in a Cu(2+)-deficient form. The inactive precursor enzyme thus obtained was dramatically activated upon incubation with Cu2+, concomitantly with the formation of the topa quinone at the position corresponding to Tyr382, occurring in the tetrapeptide sequence highly conserved in this class of enzymes. The topa quinone was produced only under aerobic conditions, but its formation required no external enzymatic systems. These findings demonstrate the Cu(2+)-dependent autooxidation of a specific tyrosyl residue to generate the topa quinone cofactor.

The gene of Arthrobacter globiformis encoding a quinoprotein, phenylethylamine oxidase, has been cloned and sequenced. In the deduced amino acid sequence comprising 638 residues is a tetrapeptide sequence, Asn-Tyr-Asp-Tyr, which has been found to be highly conserved in other copper amine oxidase. Mutation of the former Tyr (Tyr-382) of the recombinant enzyme into Phe resulted in the complete loss of catalytic activity and disappearance of the quinone compound that is specifically detected in the wild-type enzyme, suggesting that Tyr-382 is the precursor to the covalently-bound cofactor, most probably topa quinone. Furthermore, the expression of the active, quinone-containing enzyme in Escherichia coli cells was markedly dependent on the presence of Cu2+ ions in the culture medium, and the inactive, Cu2(+)-deficient enzyme produced without Cu2+ ions could be converted to the active quinone form by reconstitution with Cu2+ ions.