Chlorophyll a Formation in the Chlorophyll bReductase Reaction Requires Reduced Ferredoxin

Abstract

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.

Update on the biochemistry of chlorophyll breakdown

“…It encodes a chloroplast-localized protein that contains an iron-sulfur cluster and a FAD-binding pocket. Recombinant HCAR catalyzed reduction of C7-hydroxymethyl Chl a to Chl a in the presence of reduced ferredoxin, in agreement with its proposed biochemical properties (Scheumann et al 1998). Interestingly, HCAR has high sequence homology to cyanobacterial divinyl reductases, which catalyze reduction of the C8 vinyl moiety of divinyl-protochlorophyllide to an ethyl group during Chl biosynthesis.…”

“…The two enzymes catalyzing these consecutive reactions, Chl b reductase and hydroxymethyl Chl a reductase (HCAR), have different biochemical properties; while the latter is a ferredoxin-dependent, stroma-localized enzyme, Chl b reductase localizes to the thylakoid membrane and requires NADPH as electron source (Ito et al 1996;Scheumann et al 1998Scheumann et al , 1999. Genes encoding both enzymes have recently been identified Meguro et al 2011).…”

In Situ Conversion of Protochlorophyllideb to Protochlorophyllide a in Barley

“…By analogy, this enzyme activity was found to also convert the nonesterified precursor of Chl b, Chlide b, as well as pyrochlorophyllide b and the magnesium-free pheophorbide b into the respective Chl a and 7-hydroxy compounds (23)(24)(25)(26).…”

“…In a recent paper, Scheumann et al (21) even generally questioned the existence of Pchlide b, but at the same time demonstrated that barley etioplasts rapidly convert exogenously added zinc protopheophorbide b (ZnPPb) to ZnPPa. This prompted us to conclude that Chl(ide) b reductase, presumably responsible for this conversion (22)(23)(24)(25)(26), could also metabolize the endogenously occurring Pchlide b to Pchlide a.…”

“…As a second method, we quantified ZnPPa and ZnPPb binding by HPLC (26). However, also by these pigment measurements, no difference in ZnPPa and ZnPPb binding could be seen (Fig.…”