Characterisation of the assembly pathway of the pea NADPH : protochlorophyllide (Pchlide) oxidoreductase (POR), with emphasis on the role of its substrate, Pchlide

The homologous import and membrane association of a key enzyme for chloroph yll biosynthesis, the NADPH:protochlorophyllide (Pchlide) oxidoreductase (P OR, EC 1.6.99.1) into pea chloroplasts was investigated in vitro. The co-fa ctor, NADPH, decreased binding of the precursor protein (pPOR) to the envel ope membranes in the presence of ATP, The decrease of the binding reaction with NADPH was not observed with the precursor of the small subunit of Rubi sco (pSS), To investigate possible substrate-dependency for the import reaction, inter nal Pchlide concentrations in the plastids were raised by either an additio n of delta -aminolevulinic acid to isolated plastids or etiolation of the s eedlings prior to plastid isolation. Increased amounts of plastid-bound Pch lide gave no observable differences in POR import. The capacity of POR and 11 different POR mutants, carrying charged-to-alani ne scanning substitutions, to form a catalytically active POR-Pchlide-NADPH complex and to associate with the thylakoid membranes in a protease-resist ant way were tested. Wild-type POR, as well as the mutants with charge subs titutions in the N-terminal region of the protein, exhibited higher catalyt ic activity than the POR mutants carrying substitutions in the C-terminal r egion. Formation of a catalytically active complex did not, however, increa se the association efficiency onto the thylakoids. We can, therefore, postu late that the import of pea POR into pea chloroplasts was not substrate-dep endent, nor did formation of catalytically active complexes stimulate or in hibit the membrane association reaction of POR.