Interactions between spinach ferredoxin-nitrite reductase and its substrates. Evidence for the specificity of ferredoxin.
Reduced ferredoxin can serve as electron donor in the 6-electron reduction of nitrite to ammonia catalyzed by spinach nitrite reductase. We have examined interactions between nitrite reductase and its substrates, ferredoxin and nitrite, with emphasis upon protein-protein interactions between ferredoxin and nitrite reductase. Ferredoxin, of the proteins tested, is the most effective in retarding low ionic strength inactivation of nitrite reductase. The interaction appears to be electrostatic, and the apparent Kd, calculated from the concentration dependence of ferredoxin protection, is about 1 microM in 2 mM Tris. Chemical modification of carboxyl residues of ferredoxin resulting in a change of charge reduces its reactivity with both ferredoxin:NADP+ oxidoreductase and nitrite reductase, indicating the importance of charge-charge interactions. Cross-linking studies provided no evidence for a ternary complex containing the oxidoreductase and nitrite reductase but indicated that the two enzymes will compete for ferredoxin, possibly using the same site (or overlapping sites) on the ferredoxin. A complex containing ferredoxin:NADP+ oxidoreductase, ferredoxin, and cytochrome c was detected, indicating that ferredoxin has different binding sites for cytochrome c and ferredoxin:NADP+ oxidoreductase. Active cross-linked complexes of ferredoxin and nitrite reductase were obtained and were less sensitive to low ionic strength inactivation than free reductase and had decreased ferredoxin-supported nitrite reductase activity. The evidence presented of protein-protein interactions between ferredoxin and nitrite reductase indicates that ferredoxin is indeed the specific physiological electron donor to the reductase.
D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D003574:Cytochrome c Group / Q000378:metabolism; D004563:Electrochemistry; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009572:Nitrite Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010944:Plants / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity
D003574:Cytochrome c Group; D005288:Ferredoxins; D009573:Nitrites; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases; D005022:Ethyldimethylaminopropyl Carbodiimide