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Chlorophyll b Expressed in Cyanobacteria Functions as a Light-harvesting Antenna in Photosystem I through Flexibility of the Proteins

2001; Elsevier BV; Volume: 276; Issue: 6 Linguagem: Inglês

10.1074/jbc.m008238200

1083-351X

Soichirou Satoh, Masahiko Ikeuchi, Mamoru Mimuro, Ayumi Tanaka,

Mitochondrial Function and Pathology

Photosynthetic pigments bind to their specific proteins to form pigment-protein complexes. To investigate the pigment-binding activities of the proteins, chlorophyll b was for introduced the first time to a cyanobacterium that did not synthesize that pigment, and expression of its function in the native pigment-protein complex of cyanobacterium was confirmed by energy transfer. Arabidopsis CAO (chlorophylla oxygenase) cDNA was introduced into the genome ofSynechocystis sp. PCC6803. The transformant cells accumulated chlorophyll b, with the chlorophyllb content being in the range of 1.4 to 10.6% of the total chlorophyll depending on the growth phase. Polyacrylamide gel electrophoresis analysis of the chlorophyll-protein complexes of transformant cells showed that chlorophyll b was incorporated preferentially into the P700-chlorophylla-protein complex (CP1). Furthermore, chlorophyllb in CP1 transferred light energy to chlorophylla, indicating a functional transformation. We also found that CP1 of Chlamydomonas reinhardtii, believed to be a chlorophyll a protein, bound chlorophyll b with a chlorophyll b content of ∼4.4%. On the basis of these results, the evolution of pigment systems in an early stage of cyanobacterial development is discussed in this paper. Photosynthetic pigments bind to their specific proteins to form pigment-protein complexes. To investigate the pigment-binding activities of the proteins, chlorophyll b was for introduced the first time to a cyanobacterium that did not synthesize that pigment, and expression of its function in the native pigment-protein complex of cyanobacterium was confirmed by energy transfer. Arabidopsis CAO (chlorophylla oxygenase) cDNA was introduced into the genome ofSynechocystis sp. PCC6803. The transformant cells accumulated chlorophyll b, with the chlorophyllb content being in the range of 1.4 to 10.6% of the total chlorophyll depending on the growth phase. Polyacrylamide gel electrophoresis analysis of the chlorophyll-protein complexes of transformant cells showed that chlorophyll b was incorporated preferentially into the P700-chlorophylla-protein complex (CP1). Furthermore, chlorophyllb in CP1 transferred light energy to chlorophylla, indicating a functional transformation. We also found that CP1 of Chlamydomonas reinhardtii, believed to be a chlorophyll a protein, bound chlorophyll b with a chlorophyll b content of ∼4.4%. On the basis of these results, the evolution of pigment systems in an early stage of cyanobacterial development is discussed in this paper. photosystems I and II light-harvesting complex P700-chlorophylla-protein complex high-performance liquid chromatography polyacrylamide gel electrophoresis Photosynthetic organisms capture light energy by their light-harvesting systems that consist of core and peripheral antenna complexes (1Green B.R. Durnford D.G. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 685-714Crossref PubMed Scopus (475) Google Scholar). Core antenna complexes of oxygen-evolving photosynthetic organisms are highly conserved and have chlorophyll a as a pigment, whereas peripheral antenna complexes, especially for photosystem II (PSII),1 have various pigments such as chlorophyll b, chlorophyllc, phycobilins, fucoxanthin, and peridinin depending on the group of photosynthetic organisms (2Grossman A.R. Bhaya D. Apt K.E. Kohoe D.M. Annu. Rev. Genet. 1995; 29: 231-288Crossref PubMed Scopus (223) Google Scholar). Cyanobacteria and red algae have phycobilins that harvest light energy in a wavelength region between 500 and 650 nm. Chlorophytes and prochlorophytes contain chlorophyllb, which captures light energy at around 470 and 650 nm. These antenna pigments are acquired during the evolution of photosystems. These new antenna systems are thought to play an important role in the adaptation to various light conditions or in competition with other organisms because they capture light energy that had not been harvested by pre-existing pigments. Thus, the new pigments have been a driving force in the divergence of photosynthetic organisms. However a new pigment(s) would not have its specific binding sites in pre-existing pigmented proteins. All pigments bind to their specific proteins to form pigment-protein complexes. Studies of the crystal structure of pigment-protein complexes at the atomic level indicate that the arrangement and molecular species of pigments are strictly determined in the complexes (3Cogdell R.J. Fyfe P.K. Barrett S.J. Prince S.M. Freer A.A. Isaacs N.W. McGlynn P. Hunter C.N. Photosynth. Res. 1996; 48: 55-63Crossref PubMed Scopus (86) Google Scholar, 4Pearlstein R.M. Photosynth. Res. 1996; 48: 75-82Crossref PubMed Scopus (14) Google Scholar), and this strict determination is thought to be important for efficient energy transfer among pigments. This idea is supported by the results of studies using the light-harvesting complex II (LHCII) of higher plants. The chlorophyll b content of LHCII in higher plants is highly conserved (between 45 and 50%) (5Anderson J.M. Annu. Rev. Plant Physiol. 1986; 37: 93-136Crossref Google Scholar). LHCII proteins of chlorophyll b-less mutants of higher plants are unstable in thylakoid membranes (6Thornber J.P. Highkin H.R. Eur. J. Biochem. 1974; 41: 109-116Crossref PubMed Scopus (189) Google Scholar, 7Tarao T. Katoh S. Plant Cell Physiol. 1989; 30: 571-580Crossref Scopus (39) Google Scholar, 8Murray D.L. Kohorn B.D. Plant. Mol. Biol. 1991; 16: 71-79Crossref PubMed Scopus (58) Google Scholar) and do not accumulate without chlorophyllb, probably because of the breakdown of apoproteins.In vitro reconstitution experiments have shown that folding of LHCII required both chlorophyll a and chlorophyllb (9Paulsen H. Finkenzeller B. Kühlein N. Eur. J. Biochem. 1993; 215: 809-816Crossref PubMed Scopus (164) Google Scholar). These are thought to be the mechanisms by which chlorophyll a/b ratios were conserved in LHCII. In contrast to LHCII of higher plants, some studies suggest that the chlorophyll b content of LHCII of green algae are variable. The chlorophyll a/b ratio of LHCII ofDunaliella tertiolecta varies according to the light intensity, and the content of chlorophyll b in LHCII regulates the effective absorption cross-section of PSII (10Sukenik A. Wyman K.D. Bennett J. Falkowski P.G. Nature. 1987; 327: 704-707Crossref Scopus (58) Google Scholar), indicating that the flexibility of proteins for pigments plays an important role in adaptation to light environments. It has also been reported that LHCII proteins are stable in thylakoid membranes of a chlorophyll b-less mutant of Chlamydomonas reinhardtii (11Polle J.E.W. Benemann J.R. Tanaka A. Melis A. Planta. 2000; 211: 335-344Crossref PubMed Scopus (102) Google Scholar). Moreover, the antenna size of PSI in the mutant is similar to that in the wild type (11Polle J.E.W. Benemann J.R. Tanaka A. Melis A. Planta. 2000; 211: 335-344Crossref PubMed Scopus (102) Google Scholar), suggesting that chlorophylla molecules bind to LHC at chlorophyll b binding sites. Many studies have indicated that the protein and pigment compositions of core antenna complexes are highly conserved among oxygen-evolving photosynthetic organisms (1Green B.R. Durnford D.G. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 685-714Crossref PubMed Scopus (475) Google Scholar). PSI and PSII core complexes purified by nondenatured polyacrylamide gel electrophoresis (PAGE) from green plants had very little or no chlorophyll b (12Anderson J.M. Waldron J.C. Thorne S.W. FEBS Lett. 1978; 92: 227-233Crossref Scopus (266) Google Scholar). These observations led to the conclusion that core antenna complexes of chlorophytes have chlorophyll a and do not bind chlorophyllb despite the presence of chlorophyll b. The ability of the proteins to bind pigments has been studied by biochemical, physiological, and biophysical methods. However, we considered that the introduction of a new pigment into cells by a molecular genetics method would be a useful means of investigating the distribution of a new pigment among light-harvesting complexes to understand their pigment-binding activity. We therefore introduced the chlorophyll b synthesis gene,i.e. chlorophyll a oxygenase (CAO) (13Tanaka A. Ito H. Tanaka R. Tanaka N.K. Yoshida K. Okada K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12719-12723Crossref PubMed Scopus (324) Google Scholar), into a cyanobacterium that does not synthesize chlorophyllb. This is the first report on the introduction of a new pigment into a photosynthetic organism. Chlorophyll b was synthesized in transformant cyanobacteria cells and incorporated into the P700-chlorophyll a-protein complex (CP1). The chlorophyll a-protein was then functionally transformed to the chlorophyll a/b protein. It was found that CP1 of C. reinhardtii, believed to be a chlorophylla protein, bound chlorophyll b. We propose herein a hypothesis for the evolution of light-harvesting systems on the basis of flexibility of antenna proteins.