1. The reconstitution of chlorophyllide biosynthesis by barley etioplast membranes is described. 2. The process is dependent on the addition of NADPH and protochlorophyllide and on illumination, which can be either continuous or intermittent. 3. The reconstituted process involves spectroscopically similar intermediates to the native reaction in whole leaves. 4. Steps in the process are an initial enzymic formation in the dark of a photoactive complex, P638/652 (probably a ternary protochlorophyllide-NADPH-enzyme complex), followed by a very rapid light-dependent hydrogen transfer from the NADPH to the protochlorophyllide giving chlorophyllide, finally releasing the enzyme for repeating the process. 5. A continuous assay for the system regenerating complex P638/652 was devised on the basis of monitoring chlorophyllide formation.6. The pH optimum of the reaction is at 6.9 and Km values for protochlorophyllide and NADPH are 0.46 and 35,uM respectively. 7. The reaction is associated specifically with the etioplast membrane fraction. 8. Activities of the system assayed in vitro are more than adequate to account for rates of chlorophyll formation in vivo.

The effect of cadmium on the biosynthesis of chlorophyll has been investigated in the leaves of dark‐grown seedlings of barley (Hordeum vulture L. cv. Proctor). Cd2+ inhibited the production of chlorophyll by affecting 1) the synthesis of 5‐aminolacvulinic acid and 2) the protoehlorophyllide reductase ternary complex with its substrates. Cd2+ had no effect on the constituent enzymes that catalyse the synthesis of free protoehlorophyllide from 5‐aminolaevulinic acid. The results obtained are consistent with Cd2+ inhibiting the formation of chlorophyll by reacting with essential thiol groups in both the protochlorophyllide reductase protein and the enzyme(s) involved in the light dependent synthesis of 5‐aminolaevulinic acid.

An homology model of protochlorophyllide reductase (POR) from Synechocystis sp. was constructed on a template from the tyrosine-dependent oxidoreductase family. The model showed characteristics appropriate to a globular, soluble protein and was used to generate a structure of the ternary complex of POR, nicotinamide adenine dinucleotide phosphate (NADPH), and protochlorophyllide. The POR ternary model was validated by mutagenesis experiments involving predicted coenzyme-binding residues and by chemical modification experiments. A core tryptophan residue was shown to be responsible for much of the protein's fluorescence. Both quenching of this residue by coenzyme and fluorescence resonance energy transfer (FRET) from the protein to the coenzyme allowed the binding constant of NADPH to be determined. Replacement of this residue by Tyr gave an active mutant with approximately halved fluorescence and a negligible FRET signal, consistent with the role of this residue in energy transfer to the NADPH at the active site and with the model. The mechanism of the enzyme is discussed in the context of the model and semiempirical molecular orbital calculations.

1. Chlorophyll (ide) formation from protochlorophyll (ide) that is normally inactive was demonstrated in etioplast membranes isolated from maize and barlley plants, the process being dependent on intermittent illumination and the addition of NADPH. 2. The addition of NADPH to the membranes was shown to result in the conversion of inactive protochlorophyll (ide) absorbing at about 630 nm into a form(s) with light-absorption maxima at about 640 and 652 nm, both of which disappear when chlorophyll (ide) is formed on illumination. 3. The temperature-dependence of the activation process and its response to a variety of reagents were examined. From these, the conclusion is drawn that -SH groups are involved in the activation but in the active complex these are unavailable for reaction with -SH reagents. 4. Evidence is presented for the occurrence of glucose 6-phosphate dehydrogenase activity within etioplasts and the suggestion is made that the oxidative pentose phosphate pathway can provide the NADPH required for chlorophyll biosynthesis during the early stages of greening.

Putative protochlorophyllide reductase cDNA clones (252 and 113) were isolated from an etiolated-oat (Avena sativa) cDNA library. These were used to indirectly characterize a further clone, p127, isolated from a A-phage gtl cDNA library. The latter (1.15 kb in length) was sequenced, and the derived amino acid sequence was shown to be remarkably similar to that derived from chemical analysis of a CNBr-cleavage fragment of the purified reductase. p127 codes for more than 95 %o of the reductase protein.