Resonance Raman spectra of the 2,4-dinitrophenylhydrazine derivatives of bovine plasma amine oxidase [amine:oxygen oxidoreductase (deaminating) (copper-containing), EC 1.4.3.6] have been measured. Detailed comparisons to the spectra of the corresponding derivatives of methoxatin (pyrroloquinolinequinone), pyridoxal, and other aldehydes and diones provide further evidence that covalently bound methoxatin or a closely similar derivative is the organic cofactor in copper-containing amine oxidases.
COOHCopper-containing amine oxidases [amine:oxygen oxidoreductase (deaminating) (copper-containing), EC 1.4.3.6] are a widespread and diverse class of enzymes. They can be isolated from microorganisms, plants, and mammals (1, 2). A variety of primary amines, including mono-, di-, and polyamines, can serve as substrates, depending on the enzyme source. In addition to copper, these amine oxidases also contain an organic cofactor that is tightly bound, probably covalently, to the enzyme; recent experiments have established a subunit:copper:cofactor stoichiometry of 2:2:1 for most amine oxidases (1-8). Extensive spectroscopic and ligand binding studies have defined the basic structure of the copper sites (1, 2, 9-15). The presence of an organic cofactor was first inferred from the sensitivity of amine oxidases to carbonyl reagents-e.g., phenylhydrazine, semicarbazide, and hydroxylamine (16). Despite considerable effort, the organic cofactor has not been conclusively identified. Many different techniques have been brought to bear on this problem, but the results have often been ambiguous or even conflicting. For many years the most likely candidate for the cofactor was pyridoxal or a closely related derivative (1, 2, 17-24), but strong evidence against pyridoxal had also been reported (1,2,6,(25)(26)(27)(28). Knowles and Yadav (2) have summarized concisely and analyzed the available data in a recent review. In 1984 two groups independently presented spectroscopic (absorbance and fluorescence) and chromatographic evidence that methoxatin (pyrroloquinolinequinone, PQQ), structure 1, was the organic factor in copper-containing amine oxidases (29,30). Previously methoxatin had been identified as a coenzyme only in prokaryotic organisms (31). Methoxatin contains electrophilic carbonyl groups and displays one-and two-electron redox chemistry (31). In this context it should be noted that free radicals have been detected in some amine oxidase reactions (32) and that sequential one-electron steps may be involved in the reoxidation of substrate-reduced amine oxidases by°2 (33-37). Several properties of methoxatin that are relevant to its potential functions in amine metabolism have already been 1 delineated. For example, data pertaining to redox potentials, carbonyl chemistry, mechanisms of reactions with amines, and metal ion complexation are available for methoxatin and various analogues (38-41).The identification of the organic cofactor as methoxatin is potentially very important to understanding the mechanisms and functions ...