A Resonance Raman Study on a Reaction Intermediate of Pseudomonas L-Phenylalanine Oxidase (Deaminating and Decarboxylating)

Abstract

Resonance Raman (RR) spectra of purple intermediates of L-phenylalanine oxidase (PAO) with non-labeled and isotopically labeled phenylalanines as substrates, i. e., [1-¹³C], [2-¹³C], [ring-U-¹³C₆], and [¹⁵N]phenylalanines, were measured with excitation at 632.8 nm within the broad absorption band around 540 nm. The spectra obtained resemble those of purple intermediates of u-amino acid oxidase (DAO). The isotope effects on the 1, 665cm^-1 band with [¹⁵N] or [2-¹³C]phenylalanine indicate that the band is due to the C=N stretching mode of an imino acid derived from phenylalanine, ^{i. e.}, α-imino-β-phenylpropionate. The intense band at 1, 389cm^-1 is contributed to by the CO₂^- symmetric stretching and C-CO₂^- stretching modes of α-imino-β-phenylpropionate. The 1, 602cm^-1 band, which does not shift upon isotopic substitution of phenylalanine, corresponds to the 1, 605cm^-1 band of DAO purple intermediates and was assigned to a vibrational mode associated with the C(10α)=C(4α)-C(4)=O moiety of reduced flavin. These results confirm that PAO purple intermediates consist of the reduced enzyme and an imino acid derived from a substrate, and suggest that the plane defined by C(10a)=C(4a)-C(4)=0 of reduced flavin and the plane containing H₂N⁺=C-CO₂^- of an imino acid are arranged in close contact to each other, generating a charge-transfer interaction.