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The free radical site in pea seedling copper amine oxidase probed by resonance Raman spectroscopy and generated by photolysis of caged substrate
Author(s) -
Johnson Steven A.,
Bisby Roger H.,
Tavender Susan M.,
Parker Anthony W.
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00045-2
Subject(s) - benzylamine , chemistry , photochemistry , flash photolysis , amine oxidase , resonance raman spectroscopy , substrate (aquarium) , raman spectroscopy , amine gas treating , copper , photodissociation , organic chemistry , kinetics , physics , oceanography , quantum mechanics , reaction rate constant , optics , geology
Resonance Raman spectra were obtained of the free radical site in substrate 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) Biochemistry 34, 702]. The spectra were found to be independent of substrate (benzylamine, spermidine or methylamine) used to reduce the TOPA quinone cofactor, however, several of the peaks in the Raman spectrum displayed small shifts on using [ 15 N]benzylamine, proving incorporation of the substrate nitrogen atom onto the cofactor radical. Changes in the spectrum were also observed when measured in D 2 O solution indicating a strongly bound proton in the radical. The spectra were independent of pH values between 5 and 9 and are interpreted as showing that the radical exists as a semiiminoquinone radical monoanion. Benzylamine and phenethylamine have been caged with 2‐nitrobenzaldehyde and shown 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 benzylamine in the presence of enzyme. This should permit time‐resolved resonance Raman spectral investigations of the catalytic cycle of copper amine oxidases.