Essential histidyl residues of ferredoxin-NADP+ oxidoreductase revealed by diethyl pyrocarbonate inactivation

Abstract

Diethyl pyrocarbonate inhibited diaphorase activity of ferredoxin-NADP+ oxidoreductase [purified from spinach chloroplasts] with a 2nd-order rate constant of 2 mM-1 .cntdot. min-1 at pH 7.0 and 20.degree. 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 tryosyl 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 .apprx. 2 histidine residues were blocked by the reagent. In differential-labeling experiments with NADP+ as protective agent, diaphorase inactivation resulted from blocking of only 1 histidyl residue per mole of emzyme. Modified reductase did not bind pyridine nucleotides. Modification of the flavoprotein in the presence of NADP+, i.e., with full preservation of diaphorease activity, resulted in a significant impairment of cytochrome c reductase activity, with a 2nd-order rate constant for inactivation of .apprx. 0.5 mM-1 .cntdot. min-1. Reversal by hydroxylamine and spectroscopic data indicated that this 2nd residue was also a histidine. Ferredoxin afforded only slight protection against this inhibition. 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 2nd histidine might be located in the electron pathway between FAD and ferredoxin. Two different types of essential histidyl residues can be distinguished in ferredoxin-NADP+ oxidoreductase. One of them appears to be related with the nucleotide binding site, presumably behaving as a positive counterpart for the anionic molecule of NADP+. The 2nd, less reactive, histidine residue may be involved in the electron transport between ferredoxin and the flavin moiety.