BIOSYNTHESIS OF TOPA QUINONE COFACTOR IN BACTERIAL AMINE OXIDASES - SOLVENT ORIGIN OF C-2 OXYGEN DETERMINED BY RAMAN-SPECTROSCOPY

Resonance Raman spectroscopy is an excellent technique for providing s tructural information on the 2,4,5-trihydroxyphenylalanine quinone (TP Q) cofactor in copper-containing amine oxidases. This technique has be en used to investigate the copper- and O-2-dependent biosynthesis of t he TPQ cofactor in phenylethylamine oxidase (PEAO) and histamine oxida se from Arthrobacter globiformis. Incubation of the holoenzyme in (H2O )-O-18 causes frequency shifts at 1684 (-26) cm(-1) in PEAO and at 167 9 (-28) cm(-1) in histamine oxidase, allowing this feature to be assig ned to the C=O stretch of a single carbonyl group at the C-5 position. When apoprotein is reacted with Cu(II) and O-2 in the presence of (HO )-O-218, 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 (HO)-O-218-r egenerated protein is back-exchanged into (HO)-O-216, they can be assi gned to oxygen isotope substitution at the C-2 postion. No isotope shi fts are observed when apoprotein is regenerated with Cu(II) in the pre sence of O-18(2). Thus, it is concluded that the C-2 oxygen atom of TP Q originates from H2O rather than O-2. The isotope dependence of the 1 397-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 doub le bond character, Similar frequency shifts due to O-18 at C-2 are obs erved in the resonance Raman spectra of (H2O)-O-18-regenerated PEAO af ter derivatization of the C-5 carbonyl with either p-nitrophenylhydraz ine (-5 cm(-1) at 480 cm(-1)) or methylamine (-5 cm(-1) at 1301 cm(-1) ). Taken together, these results indica cate 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.