Direct demonstration of the presence of coordinated sulfate in the reaction pathway of Arabidopsis thaliana sulfite oxidase using 33S labeling and ESEEM spectroscopy

摘要

Sulfite oxidase from Arabidopsis thaliana has been reduced at pH = 6 with sulfite labeled with ³³S (nuclear spin I = 3/2), followed by reoxidation by ferricyanide to generate the Mo(V) state of the active center. To obtain information about the hyperfine interaction (hfi) of ³³S with Mo(V), continuous wave EPR and electron spin echo envelope modulation (ESEEM) experiments have been performed. The interpretation of the EPR and ESEEM spectra was facilitated by a theoretical analysis of the nuclear transition frequencies expected for the situation of the nuclear quadrupole interaction being much stronger than the Zeeman and hyperfine interactions. The isotropic hfi constant of ³³S determined in these experiments was about 3 MHz, which demonstrates the presence of coordinated sulfate in the sulfite-reduced low-pH form of the plant enzyme.