The role of tryptophan in the reaction catalyzed by spinach ferredoxin-dependent nitrite reductase

Abstract

Treatment of nitrite reductase with an 8–10-fold excess of N-bromosuccinimide (NBS) for 16–24 h modifies slightly less than 1 mol of tryptophan per mol of enzyme and eliminates approx. 80% of the activity of the enzyme, whether the physiological electron donor, reduced ferredoxin, or the non-physiological donor, reduced methyl viologen, is used as a source of electrons. NBS treatment does not result in any detectable change in the secondary structure of the enzyme and does not alter the oxidation-reduction properties of the siroheme and [4Fe-4S] cluster prosthetic groups of the enzyme. NBS has little or no effect on the affinity of substrate binding by nitrite reductase. These data suggest the possibility that one of the eight tryptophan residues known to be present in spinach nitrite reductase may play a direct role in the electron transfer reaction catalyzed by the enzyme. Data presented below suggest that this tryptophan may be proximal to both the ferredoxin-binding site and the siroheme group.