Covalent labelling of the NADPH: protochlorophyllide oxidoreductase from etioplast membranes with [3H]N-phenylmaleimide

Abstract

[3H]N-Phenylmaleimide has been used to covalently label in a specific manner the substrate-protected thiol groups of the enzyme protochlorophyllide reductase. In membrane preparations from oat (Avena sativa) and runner-bean (Phaseolus vulgaris) seedlings, 2 related peptides of MW 35,000/37,000 and 34,000/35,000, respectively, and showing properties expected of the reductase were identified; the same technique with barley (Hordeum vulgare) extracts resulted in labeling a single peptide of MW 38,000. Evidence is presented that both NADPH and protochlorophyllide are required for protection of the essential thiol groups on the reductase in oat extracts, a situation favoring a ternary complex as the structure for the photoactive enzyme-substrates intermediate.