RESONANCE INVERSE RAMAN SPECTROSCOPY OF FLAVIN COENZYMES (FLAVOENZYMES, LASER, NONLINEAR)

Resonance inverse Raman spectroscopy was employed to study fluorescent flavoenzymes. Flavin vibrations enhanced by the 450 mm (pi) (--->) (pi)* electronic transition were examined using a c.w. Ar('+) laser probe beam and a XeF (351 nm) excimer pumped dye laser (coumarin 540) pump beam.;Raman spectra of p-hydroxybenzoate hydroxylase were taken with substrates: p-hydroxybenzoate, 2,4-dihydroxybenzoate, and p-aminobenzoate; inhibitors: chloride and azide; and an effector, 6-hydroxynicotinate. These ligands shifted 1100-1300 cm('-1) flavin Raman bands due to changes in hydrogen bond strength between ring III of the flavin and the protein. Flavin ring III Raman band intensities increased with inhibitors, and in a ternary complex of substrate, azide and enzyme. This intensity increase indicates a disruption in ring stacking interaction between the flavin and the protein.;Raman spectra of Old Yellow Enzyme were taken with a substrate analogue: cyclic NADPH; with binding anions: azide, chloride, p-methoxybenzaldehyde, guanine triphosphate; and with p-nitrophenol and pentafluorophenol. Binding anions shifted flavin Raman bands assigned to N(3)-C(4)-C(4a)-N(5) vibrational modes. Flavin bands I and II shifted on binding the substrate analogue and anions indicating stabilization of the zwitterion flavin form. Flavin ring III modes shifted in Old Yellow Enzyme-charge transfer complexes indicating changes in hydrogen bonding between the flavin N(3), C(4), N(5) positions and the protein.;Raman spectra of lactate oxidase were taken in HCl/imidazole and PIPES buffers; with substrate analogues, acetate and propionate; and with competitive inhibitors: phosphate, sulfate, and nitrate. Intensities of bands in the 1450-1600 cm('-1) region decreased with substrate analogues and inhibitors due to relaxation of a ring stacking interaction between the flavin ring III and an aromatic amino acid near the flavin active site. Shifts in the 1232 cm('-1) band and resolution changes in the 1450-1600 cm('-1) region were attributed to hydrogen bond reorganization between flavin ring III atoms and the surrounding protein on binding anions and substrate analogues.