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

Zobrazit více v PubMed

Beck, C. F., and B. Grimm. 2006. Involvement of tetrapyrroles in cellular regulation, p. 223-235. In B. Grimm, R. J. Porra, W. Rüdiger, and H. Scheer (ed.), Chlorophylls and bacteriochlorophylls: biochemistry, biophysics, functions and applications. Advances in photosynthesis and respiration, vol. 25. Springer, Dordrecht, The Netherlands.

Boudreau, E., Y. Takahashi, C. Lemieux, M. Turmel, and J. D. Rochaix. 1997. The chloroplast ycf3 and ycf4 open reading frames of Chlamydomonas reinhardtii are required for the accumulation of the photosystem I complex. EMBO J. 166095-6104. PubMed PMC

Camadro, J. M., and P. Labbe. 1988. Purification and properties of ferrochelatase from the yeast Saccharomyces cerevisiae. Evidence for a precursor form of the protein. J. Biol. Chem. 26311675-11682. 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. In K. M. Kadish, K. M. Smith, and R. Guillard (ed.), The porphyrin handbook. Elsevier Science, St. Louis, MO.

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 Saccharomyces cerevisiae of chimeric mouse/yeast and Bacillus subtilis/yeast ferrochelatases. Arch. Biochem. Biophys. 361231-240. PubMed

Grzybowska, E., M. Gora, D. Plochocka, and J. Rytka. 2002. Saccharomyces cerevisiae ferrochelatase forms a homodimer. Arch. Biochem. Biophys. 398170-178. 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 Rhodopseudomonas sphaeroides and its effect in promoting biosynthesis of magnesium tetrapyrroles. Biochem. J. 208479-486. PubMed PMC

Jansson, S. 1999. A guide to the Lhc genes and their relatives in Arabidopsis. Trends Plant Sci. 4236-240. PubMed

Jensen, P. E., L. C. D. Gibson, K. W. Henningsen, and C. N. Hunter. 1996. Expression of the chlI, chlD and chlH genes from the cyanobacterium Synechocystis PCC6803 in Escherichia coli and demonstration that the three cognate proteins are required for magnesium protoporphyrin chelatase activity. J. Biol. Chem. 27116662-16667. 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 Synechocystis PCC 6803. J. Biol. Chem. 27948620-48629. PubMed

Kwon, S. J., A. L. de Boer, R. Petri, and C. Schmidt-Dannert. 2003. High-level production of porphyrins in metabolically engineered Escherichia coli: systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis. Appl. Environ. Microbiol. 694875-4883. 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 Arabidopsis with a mini-array system. Plant Physiol. 1352379-2391. 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 Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 9812826-12831. PubMed PMC

Müller, B., and L. A. Eichacker. 1999. Assembly of the D1 precursor in monomeric photosystem II reaction center precomplexes precedes chlorophyll a-triggered accumulation of reaction center II in barley etioplasts. Plant Cell 112365-2377. 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 Bacillus subtilis ferrochelatase mutants suggest substrate channeling in the heme biosynthesis pathway. J. Bacteriol. 1844018-4024. 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 a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975384-394.

Rieble, S., and S. I. Beale. 1991. Purification of glutamyl-transfer RNA reductase from Synechocystis sp. PCC 6803. J. Biol. Chem. 2669740-9745. 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 Synechocystis sp. PCC 6803 is dependent on the level of chlorophyll precursors regulated by ferrochelatase. J. Biol. Chem. 28031595-31602. PubMed

Srivastava, A., and S. I. Beale. 2005. Glutamyl-tRNA reductase of Chlorobium vibrioforme is a dissociable homodimer that contains one tightly bound heme per subunit. J. Bacteriol. 1874444-4450. 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 Synechocystis sp. PCC 6803. Eur. J. Biochem. 2676296-6301. 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 Chlamydomonas reinhardtii. Plant Physiol. 1391946-1958. 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 Synechocystis sp. PCC 6803. Plant Mol. Biol. 49149-160. PubMed

Yang, H., H. Inokuchi, and J. Adler. 1995. Phototaxis away from blue light by an Escherichia coli mutant accumulating protoporphyrin IX. Proc. Natl. Acad. Sci. USA 927332-7336. 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 Albostrians. Plant J. 35512-522. 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

Epigenetic control of tetrapyrrole biosynthesis by m4C DNA methylation in a cyanobacterium

Enigmatic Evolutionary History of Porphobilinogen Deaminase in Eukaryotic Phototrophs

A Photosynthesis-Specific Rubredoxin-Like Protein Is Required for Efficient Association of the D1 and D2 Proteins during the Initial Steps of Photosystem II Assembly

The antenna-like domain of the cyanobacterial ferrochelatase can bind chlorophyll and carotenoids in an energy-dissipative configuration

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

Lack of Phosphatidylglycerol Inhibits Chlorophyll Biosynthesis at Multiple Sites and Limits Chlorophyllide Reutilization in Synechocystis sp. Strain PCC 6803

Making proteins green; biosynthesis of chlorophyll-binding proteins in cyanobacteria

Functional assignments for the carboxyl-terminal domains of the ferrochelatase from Synechocystis PCC 6803: the CAB domain plays a regulatory role, and region II is essential for catalysis

Importance of the cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes