Essential histidyl residues of ferredoxin-NADP+ oxidoreductase revealed by diethyl pyrocarbonate inactivation.
Diethyl pyrocarbonate inhibited diaphorase activity of ferredoxin-NADP+ oxidoreductase with a second-order rate constant of 2 mM-1 X min-1 at pH 7.0 and 20 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity could be restored by hydroxylamine, and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Derivatization of tyrosyl residues was also evident, although with no effect on the diaphorase activity. Both NADP+ and NADPH protected the enzyme against inactivation, suggesting that the modification occurred at or near the nucleotide binding domain. The reductase lost all of its diaphorase activity after about two histidine residues had been blocked by the reagent. In differential-labeling experiments with NADP+ as protective agent, it was shown that diaphorase inactivation resulted from blocking of only one histidyl residue per mole of enzyme. Modified reductase did not bind pyridine nucleotides. Modification of the flavoprotein in the presence of NADP+, i.e., with full preservation of diaphorase activity, resulted in a significant impairment of cytochrome c reductase activity, with a second-order rate constant for inactivation of about 0.5 mM-1 X min-1. Reversal by hydroxylamine and spectroscopic data indicated that this second residue was also a histidine. Ferredoxin afforded only slight protection against this inhibition. Conversely, carbethoxylation of the enzyme did not affect complex formation with the ferrosulfoprotein. Redox titration of the modified reductase with NADPH and with reduced ferredoxin suggested that the second histidine might be located in the electron pathway between FAD and ferredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)
D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors; D005287:Ferredoxin-NADP Reductase / Q000032:analysis* / Q000037:antagonists & inhibitors; D005288:Ferredoxins / Q000378:metabolism; D005561:Formates / Q000494:pharmacology*; D006639:Histidine / Q000032:analysis*; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis*; D009249:NADP / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
D005288:Ferredoxins; D005561:Formates; D009711:Nucleotides; D006639:Histidine; D009249:NADP; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D008058:Dihydrolipoamide Dehydrogenase; D004047:Diethyl Pyrocarbonate