THE FREE-RADICAL SITE IN PEA SEEDLING COPPER AMINE OXIDASE PROBED BY RESONANCE RAMAN-SPECTROSCOPY AND GENERATED BY PHOTOLYSIS OF CAGED SUBSTRATE

Resonance Raman spectra were obtained of the free radical site in subs trate reduced anaerobic samples of pea seedling copper amine oxidase ( PSAO). The spectra differ significantly from those reported previously for E. coli copper amine oxidase [Moenne-Loccoz et al, (1995) Biochem istry 34, 7020]. The spectra were found to be independent of substrate (benzylamine, spermidine or methylamine) used to reduce the TOPA quin one cofactor, however, several of the peaks in the Raman spectrum disp layed small shifts on using [(SN)-S-15]benzylamine, proving incorporat ion of the substrate nitrogen atom onto the cofactor radical, Changes in the spectrum mere also observed when measured in D2O solution indic ating a strongly bound proton in the radical. The spectra were indepen dent of pH values between 5 and 9 and are interpreted as showing that the radical exists as a semiiminoquinone radical monoanion, Benzylamin e and phenethylamine have been caged with 2-nitrobenzaldehyde and show n by laser flash photolysis to uncage on a sub-millisecond timescale, Preliminary experiments have shown the formation of the enzyme radical intermediate on laser flash photolysis of 2-nitrobenzyl-caged benzyla mine in the presence of enzyme, This should permit time-resolved reson ance Raman spectral investigations of the catalytic cycle of copper am ine oxidases.