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, two related peptides of mol.wts. 35000/37000 and 34000/35000 respectively and showing properties expected of the reductase have been identified, whereas the same technique with barley (Hordeum vulgare) extracts resulted in labelling a single peptide of mol.wt. 38000. 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 favouring a ternary complex as the structure for the photoactive enzyme--substrates intermediate.