Ferrochelatase (FeCH) catalyzes the insertion of Fe(2+) into protoporphyrin, forming protoheme. In photosynthetic organisms, FeCH and magnesium chelatase lie at a biosynthetic branch point where partitioning down the heme and chlorophyll (Chl) pathways occurs. Unlike their mammalian, yeast, and other bacterial counterparts, cyanobacterial and algal FeCHs as well as FeCH2 isoform from plants possess a carboxyl-terminal Chl a/b-binding (CAB) domain with a conserved Chl-binding motif. The CAB domain is connected to the FeCH catalytic core by a proline-rich linker sequence (region II). In order to dissect the regulatory, catalytic, and structural roles of the region II and CAB domains, we analyzed a FeCH ΔH347 mutant that retains region II but lacks the CAB domain and compared it with the ΔH324-FeCH mutant that lacks both these domains. We found that the CAB domain is not required for catalytic activity but is essential for dimerization of FeCH; its absence causes aberrant accumulation of Chl-protein complexes under high light accompanied by high levels of the Chl precursor chlorophyllide. Thus, the CAB domain appears to serve mainly a regulatory function, possibly in balancing Chl biosynthesis with the synthesis of cognate apoproteins. Region II is essential for the catalytic function of the plastid-type FeCH enzyme, although the low residual activity of the ΔH324-FeCH is more than sufficient to furnish the cellular demand for heme. We propose that the apparent surplus of FeCH activity in the wild type is critical for cell viability under high light due to a regulatory role of FeCH in the distribution of Chl into apoproteins.

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

See more in PubMed

Cornah JE, Terry MJ, Smith AG. (2003) Green or red: what stops the traffic in the tetrapyrrole pathway?. Trends Plant Sci 8: 224–230 PubMed

Davidson RE, Chesters CJ, Reid JD. (2009) Metal ion selectivity and substrate inhibition in the metal ion chelation catalyzed by human ferrochelatase. J Biol Chem 284: 33795–33799 PubMed PMC

Dolganov NA, Bhaya D, Grossman AR. (1995) Cyanobacterial protein with similarity to the chlorophyll a/b binding proteins of higher plants: evolution and regulation. Proc Natl Acad Sci USA 92: 636–640 PubMed PMC

Goslings D, Meskauskiene R, Kim C, Lee KP, Nater M, Apel K. (2004) Concurrent interactions of heme and FLU with Glu tRNA reductase (HEMA1), the target of metabolic feedback inhibition of tetrapyrrole biosynthesis, in dark- and light-grown Arabidopsis plants. Plant J 40: 957–967 PubMed

Grzybowska E, Góra M, Plochocka D, Rytka J. (2002) Saccharomyces cerevisiae ferrochelatase forms a homodimer. Arch Biochem Biophys 398: 170–178 PubMed

Hihara Y, Sonoike K, Ikeuchi M. (1998) A novel gene, pmgA, specifically regulates photosystem stoichiometry in the cyanobacterium Synechocystis species PCC 6803 in response to high light. Plant Physiol 117: 1205–1216 PubMed PMC

Jensen PE, Gibson LCD, Henningsen KW, Hunter CN. (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 271: 16662–16667 PubMed

Kilian O, Steunou AS, Grossman AR, Bhaya D. (2008) A novel two domain-fusion protein in cyanobacteria with similarity to the CAB/ELIP/HLIP superfamily: evolutionary implications and regulation. Mol Plant 1: 155–166 PubMed

Kumar AM, Csankovszki G, Söll D. (1996) A second and differentially expressed glutamyl-tRNA reductase gene from Arabidopsis thaliana. Plant Mol Biol 30: 419–426 PubMed

Lagarde D, Beuf L, Vermaas W. (2000) Increased production of zeaxanthin and other pigments by application of genetic engineering techniques to Synechocystis sp. strain PCC 6803. Appl Environ Microbiol 66: 64–72 PubMed PMC

Larkin RM, Alonso JM, Ecker JR, Chory J. (2003) GUN4, a regulator of chlorophyll synthesis and intracellular signaling. Science 299: 902–906 PubMed

Lee J, Lee HJ, Shin MK, Ryu WS. (2004) Versatile PCR-mediated insertion or deletion mutagenesis. Biotechniques 36: 398–400 PubMed

Levicán G, Katz A, de Armas M, Núñez H, Orellana O. (2007) Regulation of a glutamyl-tRNA synthetase by the heme status. Proc Natl Acad Sci USA 104: 3135–3140 PubMed PMC

Liu Z, Yan H, Wang K, Kuang T, Zhang J, Gui L, An X, Chang W. (2004) Crystal structure of spinach major light-harvesting complex at 2.72 A resolution. Nature 428: 287–292 PubMed

Masoumi A, Heinemann IU, Rohde M, Koch M, Jahn M, Jahn D. (2008) Complex formation between protoporphyrinogen IX oxidase and ferrochelatase during haem biosynthesis in Thermosynechococcus elongatus. Microbiology 154: 3707–3714 PubMed

Masuda T, Fujita Y. (2008) Regulation and evolution of chlorophyll metabolism. Photochem Photobiol Sci 7: 1131–1149 PubMed

Mauzerall D, Granick S. (1956) The occurrence and determination of δ-amino-levulinic acid and porphobilinogen in urine. J Biol Chem 219: 435–446 PubMed

McCormac AC, Fischer A, Kumar AM, Söll D, Terry MJ. (2001) Regulation of HEMA1 expression by phytochrome and a plastid signal during de-etiolation in Arabidopsis thaliana. Plant J 25: 549–561 PubMed

Müller B, Eichacker LA. (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 11: 2365–2377 PubMed PMC

Muramatsu M, Sonoike K, Hihara Y. (2009) Mechanism of downregulation of photosystem I content under high-light conditions in the cyanobacterium Synechocystis sp. PCC 6803. Microbiology 155: 989–996 PubMed

Nowaczyk MM, Hebeler R, Schlodder E, Meyer HE, Warscheid B, Rögner M. (2006) Psb27, a cyanobacterial lipoprotein, is involved in the repair cycle of photosystem II. Plant Cell 18: 3121–3131 PubMed PMC

Ohgari Y, Sawamoto M, Yamamoto M, Kohno H, Taketani S. (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 14: 327–334 PubMed

Papenbrock J, Mishra S, Mock HP, Kruse E, Schmidt EK, Petersmann A, Braun HP, Grimm B. (2001) Impaired expression of the plastidic ferrochelatase by antisense RNA synthesis leads to a necrotic phenotype of transformed tobacco plants. Plant J 28: 41–50 PubMed

Papenbrock J, Mock HP, Tanaka R, Kruse E, Grimm B. (2000) Role of magnesium chelatase activity in the early steps of the tetrapyrrole biosynthetic pathway. Plant Physiol 122: 1161–1169 PubMed PMC

Porra RJ, Thompson WA, Kriedemann PE. (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 975: 384–394

Promnares K, Komenda J, Bumba L, Nebesarova J, Vacha F, Tichy M. (2006) Cyanobacterial small chlorophyll-binding protein ScpD (HliB) is located on the periphery of photosystem II in the vicinity of PsbH and CP47 subunits. J Biol Chem 281: 32705–32713 PubMed

Rippka R, Deruelles J, Waterbury JB, Herman M, Stanier RY. (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111: 1–61

Sobotka R, Dühring U, Komenda J, Peter E, Gardian Z, Tichy M, Grimm B, Wilde A. (2008a) Importance of the cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes. J Biol Chem 283: 25794–25802 PubMed PMC

Sobotka R, Komenda J, Bumba L, Tichy M. (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 280: 31595–31602 PubMed

Sobotka R, McLean S, Zuberova M, Hunter CN, Tichy M. (2008b) The C-terminal extension of ferrochelatase is critical for enzyme activity and for functioning of the tetrapyrrole pathway in Synechocystis strain PCC 6803. J Bacteriol 190: 2086–2095 PubMed PMC

Sonoike K, Hihara Y, Ikeuchi M. (2001) Physiological significance of the regulation of photosystem stoichiometry upon high light acclimation of Synechocystis sp. PCC 6803. Plant Cell Physiol 42: 379–384 PubMed

Srivastava A, Lake V, Nogaj LA, Mayer SM, Willows RD, Beale SI. (2005) The Chlamydomonas reinhardtii gtr gene encoding the tetrapyrrole biosynthetic enzyme glutamyl-trna reductase: structure of the gene and properties of the expressed enzyme. Plant Mol Biol 58: 643–658 PubMed

Tanaka R, Tanaka A. (2007) Tetrapyrrole biosynthesis in higher plants. Annu Rev Plant Biol 58: 321–346 PubMed

Tanaka R, Yoshida K, Nakayashiki T, Masuda T, Tsuji H, Inokuchi H, Tanaka A. (1996) Differential expression of two hemA mRNAs encoding glutamyl-tRNA reductase proteins in greening cucumber seedlings. Plant Physiol 110: 1223–1230 PubMed PMC

Tous C, Vega-Palas MA, Vioque A. (2001) Conditional expression of RNase P in the cyanobacterium Synechocystis sp. PCC6803 allows detection of precursor RNAs: insight in the in vivo maturation pathway of transfer and other stable RNAs. J Biol Chem 276: 29059–29066 PubMed

Tzvetkova-Chevolleau T, Franck F, Alawady AE, Dall’Osto L, Carrière F, Bassi R, Grimm B, Nussaume L, Havaux M. (2007) The light stress-induced protein ELIP2 is a regulator of chlorophyll synthesis in Arabidopsis thaliana. Plant J 50: 795–809 PubMed

Vasileuskaya Z, Oster U, Beck CF. (2005) Mg-protoporphyrin IX and heme control HEMA, the gene encoding the first specific step of tetrapyrrole biosynthesis, in Chlamydomonas reinhardtii. Eukaryot Cell 4: 1620–1628 PubMed PMC

Vavilin D, Vermaas W. (2007) Continuous chlorophyll degradation accompanied by chlorophyllide and phytol reutilization for chlorophyll synthesis in Synechocystis sp. PCC 6803. Biochim Biophys Acta 1767: 920–929 PubMed

Vothknecht UC, Kannangara CG, von Wettstein D. (1998) Barley glutamyl tRNAGlu reductase: mutations affecting haem inhibition and enzyme activity. Phytochemistry 47: 513–519 PubMed

Wang L, Elliott M, Elliott T. (1999) Conditional stability of the HemA protein (glutamyl-tRNA reductase) regulates heme biosynthesis in Salmonella typhimurium. J Bacteriol 181: 1211–1219 PubMed PMC

Weinstein JD, Howell RW, Leverette RD, Grooms SY, Brignola PS, Mayer SM, Beale SI. (1993) Heme inhibition of δ-aminolevulinic acid synthesis is enhanced by glutathione in cell-free extracts of Chlorella. Plant Physiol 101: 657–665 PubMed PMC

Xu H, Vavilin D, Funk C, Vermaas W. (2002) Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol 49: 149–160 PubMed

Zouni A, Kern J, Frank J, Hellweg T, Behlke J, Saenger W, Irrgang KD. (2005) Size determination of cyanobacterial and higher plant photosystem II by gel permeation chromatography, light scattering, and ultracentrifugation. Biochemistry 44: 4572–4581 PubMed

Using Diatom and Apicomplexan Models to Study the Heme Pathway of Chromera velia

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

Synthesis of Chlorophyll-Binding Proteins in a Fully Segregated Δycf54 Strain of the Cyanobacterium Synechocystis PCC 6803

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

Long-term acclimation of the cyanobacterium Synechocystis sp. PCC 6803 to high light is accompanied by an enhanced production of chlorophyll that is preferentially channeled to trimeric photosystem I

Sequence evidence for the presence of two tetrapyrrole pathways in Euglena gracilis