Heme and chlorophyll (Chl) share a common biosynthetic pathway up to the branch point where magnesium chelatase and ferrochelatase (FeCH) insert either magnesium for Chl biosynthesis or ferrous iron for heme biosynthesis. A distinctive feature of FeCHs in cyanobacteria is their C-terminal extension, which forms a putative transmembrane segment containing a Chl-binding motif. We analyzed the deltaH324 strain of Synechocystis sp. strain PCC 6803, which contains a truncated FeCH enzyme lacking this C-terminal domain. Truncated FeCH was localized to the membrane fraction, suggesting that the C-terminal domain is not necessary for membrane association of the enzyme. Measurements of enzyme activity and complementation experiments revealed that the deltaH324 mutation dramatically reduced activity of the FeCH, which resulted in highly upregulated 5-aminolevulinic acid synthesis in the deltaH324 mutant, implying a direct role for heme in the regulation of flux through the pathway. Moreover, the deltaH324 mutant accumulated a large amount of protoporphyrin IX, and levels of Chl precursors were also significantly increased, suggesting that some, but not all, of the "extra" flux can be diverted down the Chl branch. Analysis of the recombinant full-length and truncated FeCHs demonstrated that the C-terminal extension is critical for activity of the FeCH and that it is strictly required for oligomerization of this enzyme. The observed changes in tetrapyrrole trafficking and the role of the C terminus in the functioning of FeCH are discussed.

Institute of Microbiology Department of Autotrophic Microorganisms Opatovicky mlyn 379 81 Trebon Czech Republic

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Beck, C. F., and B. Grimm. 2006. Involvement of tetrapyrroles in cellular regulation, p. 223-235.

Boudreau, E., Y. Takahashi, C. Lemieux, M. Turmel, and J. D. Rochaix. 1997. The chloroplast ycf3 and ycf4 open reading frames of PubMed PMC

Camadro, J. M., and P. Labbe. 1988. Purification and properties of ferrochelatase from the yeast PubMed

Castelfranco, P. A., and O. T. G. Jones. 1975. Protoheme turnover and chlorophyll synthesis in greening barley tissue. Plant Physiol. 55485-490. PubMed PMC

Chamovitz, D., G. Sandmann, and J. Hirschberg. 1993. Molecular and biochemical characterization of herbicide-resistant mutants of cyanobacteria reveals that phytoene desaturation is a rate-limiting step in carotenoid biosynthesis. J. Biol. Chem. 26817348-17353. PubMed

Dailey, H. A., and T. A. Dailey. 2003. Ferrochelatase, p. 93-121.

Dailey, T. A., and H. A. Dailey. 2002. Identification of [2Fe-2S] clusters in microbial ferrochelatases. J. Bacteriol. 1842460-2464. PubMed PMC

Dolganov, N. A., D. Bhaya, and A. R. Grossman. 1995. Cyanobacterial protein with similarity to the chlorophyll a/b binding proteins of higher plants: evolution and regulation. Proc. Natl. Acad. Sci. USA 92636-640. PubMed PMC

Funk, C., and W. Vermaas. 1999. A cyanobacterial gene family coding for single-helix proteins resembling part of the light-harvesting proteins from higher plants. Biochem. 389397-9404. PubMed

Gora, M., J. Rytka, and R. Labbe-Bois. 1999. Activity and cellular location in PubMed

Grzybowska, E., M. Gora, D. Plochocka, and J. Rytka. 2002. PubMed

Houghton, J. D., C. L. Honeybourne, K. M. Smith, H. D. Tabba, and O. T. G. Jones. 1982. The use of N-methylprotoporphyrin dimethyl ester to inhibit ferrochelatase in PubMed PMC

Jansson, S. 1999. A guide to the PubMed

Jensen, P. E., L. C. D. Gibson, K. W. Henningsen, and C. N. Hunter. 1996. Expression of the PubMed

Ke, S.-H., and E. L. Madison. 1997. Rapid and efficient site-directed mutagenesis by single-tube “megaprimer” PCR method. Nucleic Acid Res. 253371-3372. PubMed PMC

Komenda, J., V. Reisinger, B. Ch. Müller, M. Dobakova, B. Granvogl, and L. A. Eichacker. 2004. Accumulation of the D2 protein is a key regulatory step for assembly of the photosystem II reaction center complex in PubMed

Kwon, S. J., A. L. de Boer, R. Petri, and C. Schmidt-Dannert. 2003. High-level production of porphyrins in metabolically engineered PubMed PMC

Lee, J., H. J. Lee, M. K. Shin, and W. S. Ryu. 2004. Versatile PCR-mediated insertion or deletion mutagenesis. BioTechniques 36398-399. PubMed

Liu, Z., H. Yan, K. Wang, T. Kuang, J. Zhang, L. Gui, X. An, and W. Chang. 2004. Crystal structure of spinach major light harvesting complex at 2.72Å resolution. Nature 428287-292. PubMed

Matsumoto, F., T. Obayashi, Y. Sasaki-Sekimoto, H. Ohta, K. Takamiya, and T. Masuda. 2004. Gene expression profiling of the tetrapyrrole metabolic pathway in PubMed PMC

Mauzerall, D., and S. Granick. 1956. The occurrence and determination of δ-aminolevulinic acid and porphobilinogen in urine. J. Biol. Chem. 219435-446. PubMed

Meskauskiene, R., M. Nater, D. Goslings, F. Kessler, R. den Camp, and K. Apel. 2001. FLU: a negative regulator of chlorophyll biosynthesis in PubMed PMC

Müller, B., and L. A. Eichacker. 1999. Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll PubMed PMC

Ohgari, Y., M. Sawamoto, M. Yamamoto, H. Kohno, and S. Taketani. 2005. Ferrochelatase consisting of wild-type and mutated subunits from patients with a dominant-inherited disease, erythropoietic protoporphyria, is an active but unstable dimer. Hum. Mol. Genet. 14327-334. PubMed

Olsson, U., A. Billberg, S. Sjövall, S. Al-Karadaghi, and M. Hansson. 2002. In vivo and in vitro studies of PubMed PMC

Papenbrock, J., H.-P. Mock, E. Kruse, and B. Grimm. 1999. Expression studies in tetrapyrrole biosynthesis: inverse maxima of magnesium chelatase and ferrochelatase activity during cyclic photoperiods. Planta 208264-273.

Papenbrock, J., H.-P. Mock, R. Tanaka, E. Kruse, and B. Grimm. 2000. Role of magnesium chelatase activity in the early steps of the tetrapyrrole biosynthetic pathway. Plant Physiol. 1221161-1169. PubMed PMC

Papenbrock, J., S. Mishra, H. Mock, E. Kruse, E. Schmidt, A. Petersmann, H. Braun, and B. Grimm. 2001. Impaired expression of the plastidic ferrochelatase by antisense RNA synthesis leads to a necrotic phenotype of transformed tobacco plants. Plant J. 2841-50. PubMed

Porra, R. J., W. A. Thompson, and P. E. Kriedemann. 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophyll

Rieble, S., and S. I. Beale. 1991. Purification of glutamyl-transfer RNA reductase from PubMed

Rippka, R., J. Deruelles, J. B. Waterbury, M. Herman, and R. Y. Stanier. 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 1111-61.

Rosevear, P., T. VanAken, J. Baxter, and S. Ferguson-Miller. 1980. Alkyl glycoside detergents: a simpler synthesis and their effects on kinetic and physical properties of cytochrome c oxidase. Biochemistry 194108-4115. PubMed

Sobotka, R., J. Komenda, L. Bumba, and M. Tichy. 2005. Photosystem II assembly in CP47 mutant of PubMed

Srivastava, A., and S. I. Beale. 2005. Glutamyl-tRNA reductase of PubMed PMC

Suzuki, T., T. Masuda, D. P. Singh, F.-C. Tan, T. Tsuchiya, H. Shimada, H. Ohta, A. G. Smith, and K. Takamiya. 2002. Two types of ferrochelatase in photosynthetic and nonphotosynthetic tissues of cucumber. J. Biol. Chem. 2774731-4737. PubMed

Suzuki, T., T. Masuda, H. Inokuchi, H. Shimada, H. Ohta, and K. Takamiya. 2000. Overexpression, enzymatic properties and tissue localization of a ferrochelatase of cucumber. Plant Cell Physiol. 41192-199. PubMed

Tanaka, R., and A. Tanaka. 2007. Tetrapyrrole biosynthesis in higher plants. Annu. Rev. Plant Biol. 58321-346. PubMed

Tichy, M., and W. Vermaas. 2000. Combinatorial mutagenesis and pseudorevertant analysis to characterize regions in loop E of the CP47 protein in PubMed

van Lis, R., A. Atteia, L. A. Nogaj, and S. I. Beale. 2005. Subcellular localization and light-regulated expression of protoporphyrinogen IX oxidase and ferrochelatase in PubMed PMC

Vavilin, D. V., and W. F. J. Vermaas. 2002. Regulation of the tetrapyrrole biosynthetic pathway leading to heme and chlorophyll in plants and cyanobacteria. Physiol. Plant 1159-24. PubMed

Wu, C. K., H. A. Dailey, J. P. Rose, A. Burden, V. M. Sellers, and B. C. Wang. 2001. The 2-Å structure of human ferrochelatase, the terminal enzyme of heme biosynthesis. Nat. Struct. Biol. 8156-160. PubMed

Xu, H., D. Vavilin, C. Funk, and W. Vermaas. 2002. Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium PubMed

Yang, H., H. Inokuchi, and J. Adler. 1995. Phototaxis away from blue light by an PubMed PMC

Yaronskaya, E., V. Ziemann, G. Walter, N. Averina, T. Börner, and B. Grimm. 2003. Metabolic control of the tetrapyrrole biosynthetic pathway for porphyrin distribution in the barley mutant PubMed

Zouni, A., J. Kern, J. Frank, T. Hellweg, J. Behlke, W. Saenger, and K. D. Irrgang. 2005. Size determination of cyanobacterial and higher plant photosystem II by gel permeation chromatography, light scattering, and ultracentrifugation. Biochemistry 444572-4581. PubMed

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