Oxidase activity was enriched in extracts of the developing, lignifying xylem of Sitka spruce, Leyland cypress, Lawson cypress and Wych elm obtained by a procedure that selects cell-wall-associated glycoproteins. All of the xylem extracts were able to oxidise the monolignol, coniferyl alcohol, and had a greater affinity for coniferyl alcohol than ABTS. The oxidases were strongly inhibited by Cu-chelators and had inhibition profiles broadly similar to catechol oxidase-type polyphenol oxidases. Analysis of the oxidation products of coniferyl alcohol generated in the presence of the spin trap POBN, by electron paramagnetic resonance spectroscopy, showed that a spin adduct of parameters a(N) = 15.65-15,74 G and a(H) = 2.73-2.78 G had been generated which can be most readily assigned to a POBN adduct of the β- carbon-centred free radical of coniferyl alcohol. Electron paramagnetic resonance spectra of the extracts from the four species contained signals representative of Cu2+ and a free radical species of g value ~ 2.0036. The intensity of the free radical signal was diminished by addition of coniferyl alcohol but was restored upon aeration. The Cu2+ signals were similarly altered by this treatment, but diminution was less marked. Cycles of depletion then recovery of the free radical signal could be obtained by addition of aliquots of coniferyl alcohol followed by aeration which strongly suggests that the free radical is directly involved in the oxidation mechanism. We propose that coniferyl alcohol oxidase operates via a bound free radical which is re-oxidised via a charge relay mechanism involving bound Cu2+ ions and molecular oxygen.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Plant Science