The reduction of chlorophyllide b and its analogue zinc pheophorbide b in etioplasts of barley (Hordeum vulgare L.) was investigated in detail. In intact etioplasts, the reduction proceeds to chlorophyllide a and zinc pheophorbide a or, if incubated together with phytyldiphosphate, to chlorophyll a and zinc pheophytin a, respectively. In lysed etioplasts supplied with NADPH, the reduction stops at the intermediate step of 7 1 -OHchlorophyll(ide) and Zn-7 1 -OH-pheophorbide or Zn-7 1 -OH-pheophytin. However, the final reduction is achieved when reduced ferredoxin is added to the lysed etioplasts, suggesting that ferredoxin is the natural cofactor for reduction of chlorophyll b to chlorophyll a. The reduction to chlorophyll a requires ATP in intact etioplasts but not in lysed etioplasts when reduced ferredoxin is supplied. The role of ATP and the significance of two cofactors for the two steps of reduction are discussed.

During senescence of flowering plants, only breakdown products derived from chlorophyll a were detected although b disappears, too (Matile et al., 1996, Plant Physiol 112: 1403-1409). We investigated the possibility of chlorophyll b reduction during dark-induced senescence of barley (Hordeum vulgare L.) leaves. Plastids isolated from senescing leaves were lysed and incubated with NADPH. We found 7(1)-hydroxy-chlorophyll a, 7(1)-hydroxy-chlorophyllide a, and, after incubation with Zn-pheophorbide b, also Zn-7(1)-hydroxy-pheophorbide a, indicating activity of chlorophyll(ide) b reductase. The highest activity was found at day 2 of senescence when chlorophyll breakdown reached its highest rate. Chlorophyllase reached its highest activity under the same conditions only at days 4-6 of senescence. Based on the chlorophyll b reductase activity of plastids at day 2.5 of senescence (=100%), the bulk of activity (83%) was found in the thylakoids and only traces (5%) in the envelope fraction. Chlorophyll b reduction is considered to be an early and obligatory step of chlorophyll b breakdown.

Barley (Hordeum vulgare L.) etioplasts were isolated, and the pigments were extracted with acetone. The extract was analyzed by HPLC. Only protochlorophyllide a and no protochlorophyllide b was detected (limit of detection 6 1% of protochlorophyllide a). Protochlorophyllide b was synthesized starting from chlorophyll b and incubated with etioplast membranes and NADPH. In the light, photoconversion to chlorophyllide b was observed, apparently catalyzed by NADPH:protochlorophyllide oxidoreductase. In darkness, reduction of the analogue zinc protopheophorbide b to zinc 7 I -hydroxy-protopheophorbide a was observed, apparently catalyzed by chlorophyll b reductase. We conclude that protochlorophyllide b does not occur in detectable amounts in etioplasts, and even traces of it as the free pigment are metabolically unstable. Thus the direct experimental evidence contradicts the idea by Reinbothe et al. (Nature 397 (1999) 80^84) of a protochlorophyllide b-containing light-harvesting complex in barley etioplasts.z 1999 Federation of European Biochemical Societies.

Enzyme activity of chlorophyll(ide) b reductase is present in etioplasts. Recently the conversion of chlorophyllide b to chlorophyll a via 7'-hydroxychlorophyll a was demonstrated in barley etioplasts. We used zinc pheophorbide b for a detailed investigation of the reduction of the 7-formyl group to the 7l-hydroxy compound in intact barley etioplasts. The reaction proceeded likewise before esterification and after esterification with phytyl diphosphate. The metal-free pheophorbide b, that is not accepted by chlorophyll synthase for esterification, is reduced to 71-hydroxypheophorbide a to a small extent. The zinc (13'S)-pheophorbide b is at least equally well accepted for reduction as the epimer with the 13'R configuration of natural chlorophyll b. The reaction requires NADPH or NADH, although the latter is less effective. ATP is not required for the first step to the 7I-hydroxy compound. The significance of chlorophyll b reduction for acclimation from shade to sun leaves and for chlorophyll degradation is discussed.

The chemical reduction of the formyl group of pheophorbide b with sodium cyanoborohy­ dride in methanol leads to 71-methoxy-and 71-hydroxy-pheophorbide a. The same reaction with zinc pheophorbide b yields in addition zinc pheophorbide a. This was characterized by mass and 1H -NMR spectroscopy. Infiltration of zinc pheophorbides a and b and of zinc 71-hydroxy-pheophorbide a into etiolated oat leaves yielded phytylated products. The best yield in the esterification was obtained with 71-hydroxy-pheophorbide a. Analysis of the products revealed the formation of zinc pheophytin a from all infiltrated compounds. The significance for the transformation of chlorophyll b into chlorophyll a is discussed.