A CONSERVED TRYPTOPHAN AT THE FERREDOXIN-BINDING SITE OF FERREDOXIN-NITRITE OXIDOREDUCTASE

Treatment of spinach leaf ferredoxin-dependient nitrite reductase with N-bromosuccinimide (NBS), under conditions where slightly less than 1 mol of tryptophan is modified per mole of nitrite reductase, inhibits the catalytic activity of the enzyme by ca. 80% without any effect on substrate binding or other enzyme properties. Complex formation betwe en nitrite reductase and ferredoxin completely protects the enzyme aga inst this inhibition. Transient kinetic measurements show that the sec ond-order rate constant for reduction of NBS-modified nitrite reductas e by reduced ferredoxin is approximately fourfold larger than that obs erved for the native, unmodified enzyme. Also, reduction of NBS-modifi ed nitrite reductase by the 5-deazariboflavin radical shows a differen t kinetic pattern than that observed with the native enzyme, suggestin g that tryptophan modification increases access of the radical to the low-potential [4Fe-4S] cluster of the enzyme, decreases the accessibil ity to the siroheme group of the enzyme, or both. The tryptophan that is modified has been identified as the absolutely conserved W92. A met hionine, M73, that is also modified by NBS, has been identified. The f erredoxin-binding site on spinach nitrite reductase thus appears to in clude W92 and perhaps M73, in addition to the previously identified R3 75, R556, and K436. (C) 1998 Academic Press.