TOPAQUINONE-DEPENDENT AMINE OXIDASES - IDENTIFICATION OF REACTION INTERMEDIATES BY RAMAN-SPECTROSCOPY

Resonance Raman (RR) spectroscopy has proven to be an excellent techni que for providing structural information about the 2,4,5-trihydroxyphe nylalaninequinone (TPQ) cofactor and for identifying the source of oxy gen atoms during the posttranslational synthesis of the cofactor. Thro ugh specific labeling of the C2, C4, and C5 oxygens of TPQ in phenylet hylamine oxidase (PEAO) from Arthrobacter globiformis, we have identif ied the C=O stretch of the C5 carbonyl at 1683 cm(-1) (-27 in O-18) an d the C=O stretch of the C2 carbonyl at 1575 cm(-1) (-21 in O-18). The se vibrational frequencies show that the C-O moiety at C5 has far grea ter double-bond character than at C2 or C4, thereby explaining the exc lusive nucleophilic attack at the C5 position by substrates and substr ate analogs. Bovine serum amine oxidase (BSAO) exhibits a similar v(C= O) mode at 1678 cm(-1) (-22 cm(-1) in O-18). Aniline reacts with the T PQ cofactor of PEAO to form a new derivative (lambda(max) 450 nm) with properties similar to the proposed substrate-imine intermediate in th e catalytic cycle. It retains the C2=O spectral features of the native enzyme and exhibits a new C5=N stretch at 1603 cm(-1) (-29 in N-15). In contrast, methylamine reacts with both PEAO and BSAO under anaerobi c conditions to form a different stable adduct (lambda(max) at 385 nm) with properties closer to the proposed product-imine intermediate in the catalytic cycle. This species has a distinctive RR spectrum with a C=N stretch at 1617 cm(-1) that corresponds to the atoms of the added methylamine (-58 cm(-1) with CD3NH2, -19 cm(-1) with (CH3NH2)-N-15). The lack of D2O dependence of v(C=N) shows that this is a deprotonated imine, which would be more stable toward hydrolysis than the postulat ed protonated imine in the enzymatic reaction. However, the BSAO produ ct imine (from methylamine) does undergo hydrolysis and conversion to semiquinone upon addition of cyanide. It is possible that the inactive form of the product imine is stabilized by deprotonation and flipping of the TPQ ring [Cai, D., Dove, J., Nakamura, N., Sanders-Loehr, J., and Klinman, J. P. (1997) Biochemistry 36, 11472-11478].