Affinity labeling of spinach ferredoxin-NADP+ oxidoreductase with periodate-oxidized NADP+

Periodate-oxidized NADP+ (dialdehyde-NADP+) inactivated soluble ferredoxin-NADP+ oxidoreductase and combined covalently to the enzyme. This inactivation was first order with respect to dialdehyde-NADP+ and followed saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inactivator. NADP+ afforded complete protection against inactivation, while spinach ferredoxin was uneffective. In the presence of exogenous ferredoxin and illuminated thylakoids, the nucleotide analog functioned as a coenzyme for the reductase, although with rather lower efficiency than NADP+. It also acted as a competitive inhibitor with respect to NADPH in diaphorase activity. Incorporation of radioactivity from periodate-oxidized [3H]NADP+ gave a stoichiometry of 0.85 mol of reagent/mol of reductase, indicating that the modification of a single residue in the flavoprotein is responsible for the loss of enzymatic activity.