Oxidation-reduction properties of the ferredoxin-linked glutamate synthase from spinach leaf.

Oxidation-reduction titrations have been conducted to determine the midpoint potential (Em) values of the three electron-carrying prosthetic groups of the ferredoxin-linked glutamate synthase isolated from spinach leaves. Titrations using electron paramagnetic resonance (EPR) signals to monitor the oxidation state of the [3Fe-4S]+,0 cluster found in the enzyme, indicated the presence of a single n = 1 component with Em = -170 mV at pH 7.7. Titrations using absorbance changes in the visible region to monitor the oxidation states of the FAD and FMN groups present in the enzyme could be fit to a single n = 2 Nernst curve with Em = -180 mV at pH 7.7. The magnitude of the absorbance change observed during this titration accounts for all of the FMN and FAD found in the enzyme, indicating that the two flavins are either isopotential or differ in Em by less than about 30 mV. Neither optical nor EPR titrations gave any evidence for the presence of stable flavin free radicals. These results represent the first characterization of the redox properties of the prosthetic groups of a ferredoxin-dependent glutamate synthase.