The aromatic amine oxidase from Escherichia coli (ECAO) utilizes Cu(II) and 2,4,5- trihdroxyphenylalanine quinone (TPQ) as cofactors in enzymatic catalysis. The TPQ cofactor is clearly identified by a set of characteristic vibrational modes between 1200 and 1700 cm-1 in the resonance Raman (RR) spectrum of the native enzyme. This is the first report of a RR spectrum for an underivatized TPQ cofactor in an enzyme, showing that it is possible to study changes in the cofactor during the natural reaction cycle. The RR spectrum of ECAO closely matches that of a 2-hydroxy-1,4-benzoquinone model compound, particularly in the deprotonated state in aqueous solution. The principal in-phase C=0 symmetric stretching mode of the quinone occurs at 1681 cm-1 in ECAO and at 1666 cm-1 in the model compound and, in both cases, undergoes a downshift of ~25 cm-1 upon substitution of one of the carbonyl oxygens with 180. The overall similarity of the 180 and D shifts in their RR spectra shows that the TPQ cofactor and model compound have the same structure and reactivity, with oxygen exchange occurring at the carbonyl adjacent to the hydroxyl group. Substrate reduction of ECAO under anaerobic conditions leads to a stable semiquinone (λmax at 442 and 468 nm) with a RR spectrum characteristic of an aminesubstituted semiquinone. The intense ring mode at 1647 cm-1 undergoes a shift of -4 cm-1 in enzyme reacted with 15N-containing substrate and a shift of -6 cm-1 upon addition of cyanide, indicating that the semiquinone cofactor is perturbed by formation of the Cu(I)CN complex. Thus, the Cu(I) cofactor appears to be sufficiently close to undergo electrostatic interactions with the semiquinone cofactor in this catalytic intermediate of amine oxidase.
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