Identification of Lys116 as the target of N‐ethylmaleimide inactivation of ferredoxin:NADP+ oxidoreductase

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Abstract
Oxidized ferredoxin:NADP+ oxidoreductase (FNR) was slowly and irreversibly inactivated by N-ethylmaleimide. Complete protection against inactivation was afforded by saturating concentrations of NADP+. In the presence of NADPH, a rapid inhibition of the enzyme ensued; however, this inhibition was found to be reversible. In the tryptic map of the flavoprotein, modified with N-ethyl[2,3-14C]maleimide in oxidizing conditions, a unique radioactive peptide was found. Its sequence comprised residues 110-117 of the enzyme: Lys116 was shown to be the residue alkylated by N-ethylmaleimide. It is noteworthy that the same residue of FNR was found to be modified by 5-dimethylaminoaphthalene-1-sulfonyl(dansyl) chloride at the putative NADP(H)-binding site [Cidaria, D., Biondi, P. A., Zanetti, G. & Ronchi, S. (1985) Eur. J. Biochem. 146, 295-299]. Furthermore, the data reported here demonstrate that the sulfhydryl groups of FNR are not involved in enzyme inactivation by N-ethylmaleimide.