Interaction of ferredoxin-linked nitrite reductase with ferredoxin

The native, ferredoxin-linked, form ( M r = 85 000) of nitrite reductase (ferredoxin:nitrite oxidoreductase, EC 1.7.7.1) forms a complex with ferredoxin that can be detected either by enzyme binding to a ferredoxin-Sepharose 4B affinity column or by spectral changes produced when the two proteins are mixed. The K d for ferredoxin binding, estimated to be 6.3·10 −7 M, was increased to 1.1·10 −5 M in the presence of nitrite, indicating lower affinity for ferredoxin binding in the ternary nitrite-ferredoxin-enzyme complex. The native enzyme could be split into a modified form ( M 4 = 61 000) that retains activity with the non-physiological electron donor, methyl viologen, but has lost most of the native enzyme's ferredoxin-linked activity and an M r = 24 000 fragment (coupling protein). The coupling protein, but not the modified nitrite reductase, binds to a ferredoxin-Sepharose 4B affinity column. These results are discussed in terms of a ferredoxin-binding domain located on the coupling protein portion of the native enzyme.