In the chlorophyll (Chl) biosynthesis pathway the formation of protochlorophyllide is catalyzed by Mg-protoporphyrin IX methyl ester (MgPME) cyclase. The Ycf54 protein was recently shown to form a complex with another component of the oxidative cyclase, Sll1214 (CycI), and partial inactivation of the ycf54 gene leads to Chl deficiency in cyanobacteria and plants. The exact function of the Ycf54 is not known, however, and further progress depends on construction and characterization of a mutant cyanobacterial strain with a fully inactivated ycf54 gene. Here, we report the complete deletion of the ycf54 gene in the cyanobacterium Synechocystis 6803; the resulting Δycf54 strain accumulates huge concentrations of the cyclase substrate MgPME together with another pigment, which we identified using nuclear magnetic resonance as 3-formyl MgPME. The detection of a small amount (~13%) of Chl in the Δycf54 mutant provides clear evidence that the Ycf54 protein is important, but not essential, for activity of the oxidative cyclase. The greatly reduced formation of protochlorophyllide in the Δycf54 strain provided an opportunity to use (35)S protein labeling combined with 2D electrophoresis to examine the synthesis of all known Chl-binding protein complexes under drastically restricted de novo Chl biosynthesis. We show that although the Δycf54 strain synthesizes very limited amounts of photosystem I and the CP47 and CP43 subunits of photosystem II (PSII), the synthesis of PSII D1 and D2 subunits and their assembly into the reaction centre (RCII) assembly intermediate were not affected. Furthermore, the levels of other Chl complexes such as cytochrome b 6 f and the HliD- Chl synthase remained comparable to wild-type. These data demonstrate that the requirement for de novo Chl molecules differs completely for each Chl-binding protein. Chl traffic and recycling in the cyanobacterial cell as well as the function of Ycf54 are discussed.

ChELSI Institute Department of Chemical and Biological Engineering University of Sheffield Sheffield UK

Department of Molecular Biology and Biotechnology University of Sheffield Sheffield UK

Department of Molecular Biology and Biotechnology University of SheffieldSheffield UK; ChELSI Institute Department of Chemical and Biological Engineering University of SheffieldSheffield UK

Department of Molecular Biology and Biotechnology University of SheffieldSheffield UK; Sir William Dunn School of Pathology University of OxfordOxford UK

Institute of Microbiology Centre Algatech Academy of Sciences of the Czech Republic Třeboň Czech Republic

Institute of Microbiology Centre Algatech Academy of Sciences of the Czech RepublicTřeboň Czech Republic; Faculty of Science University of South BohemiaČeské Budějovice Czech Republic

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The biogenesis and maintenance of PSII: Recent advances and current challenges

Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins

Psb34 protein modulates binding of high-light-inducible proteins to CP47-containing photosystem II assembly intermediates in the cyanobacterium Synechocystis sp. PCC 6803

Evolution of Ycf54-independent chlorophyll biosynthesis in cyanobacteria

The Ribosome-Bound Protein Pam68 Promotes Insertion of Chlorophyll into the CP47 Subunit of Photosystem II