THE ROLE OF LYSINE AND ARGININE RESIDUES AT THE FERREDOXIN-BINDING SITE OF SPINACH GLUTAMATE SYNTHASE

Treatment of ferredoxin-dependent, spinach glutamate synthase with either the arginine-modifying reagent phenylglyoxal or the lysine-modifying reagents N-acetylsuccinimide and dansyl chloride resulted in a significant loss of enzymatic activity when the physiological electron donor, reduced ferredoxin, was used as the electron-donating substrate. In contrast, the reagents caused no inhibition of enzyme activity when the non-physiological reductant, reduced methyl viologen, was used as the electron donor. Formation of an electrostatically-stabilized complex between glutamate synthase and ferredoxin prior to exposure of the enzyme to phenylglyoxal or N-acetylsuccinimide protected the enzyme against the loss of ferredoxin-dependent activity caused by either modifying agent. Treatment of glutamate synthase with either reagent resulted in a loss of ferredoxin-binding capacity, as assayed by affinity chromatography, gel filtration, spectral perturbations and by the ability of the enzyme to form an active cross-linked complex with ferredoxin. Absorbance and circular dichroism spectra indicated that neither of the modifying reagents produced major conformational changes in the enzyme. These results have been interpreted in terms of a ferredoxin-binding site on glutamate synthase, similar to those found on other ferredoxin-dependent chloroplast enzymes, that contains both lysine and arginine residues.