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

Open AccessPublished:February 09, 2001DOI:https://doi.org/10.1074/jbc.M008238200
      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.
      PSI/II
      photosystems I and II
      LHC
      light-harvesting complex
      CP1
      P700-chlorophylla-protein complex
      HPLC
      high-performance liquid chromatography
      PAGE
      polyacrylamide gel electrophoresis
      Photosynthetic organisms capture light energy by their light-harvesting systems that consist of core and peripheral antenna complexes (
      • Green B.R.
      • Durnford D.G.
      ). 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 (
      • Grossman A.R.
      • Bhaya D.
      • Apt K.E.
      • Kohoe D.M.
      ). 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 (
      • Cogdell R.J.
      • Fyfe P.K.
      • Barrett S.J.
      • Prince S.M.
      • Freer A.A.
      • Isaacs N.W.
      • McGlynn P.
      • Hunter C.N.
      ,
      • Pearlstein R.M.
      ), 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%) (
      • Anderson J.M.
      ). LHCII proteins of chlorophyll b-less mutants of higher plants are unstable in thylakoid membranes (
      • Thornber J.P.
      • Highkin H.R.
      ,
      • Tarao T.
      • Katoh S.
      ,
      • Murray D.L.
      • Kohorn B.D.
      ) 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 (
      • Paulsen H.
      • Finkenzeller B.
      • Kühlein N.
      ). 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 (
      • Sukenik A.
      • Wyman K.D.
      • Bennett J.
      • Falkowski P.G.
      ), 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 (
      • Polle J.E.W.
      • Benemann J.R.
      • Tanaka A.
      • Melis A.
      ). Moreover, the antenna size of PSI in the mutant is similar to that in the wild type (
      • Polle J.E.W.
      • Benemann J.R.
      • Tanaka A.
      • Melis A.
      ), 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 (
      • Green B.R.
      • Durnford D.G.
      ). PSI and PSII core complexes purified by nondenatured polyacrylamide gel electrophoresis (PAGE) from green plants had very little or no chlorophyll b (
      • Anderson J.M.
      • Waldron J.C.
      • Thorne S.W.
      ). 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) (
      • Tanaka A.
      • Ito H.
      • Tanaka R.
      • Tanaka N.K.
      • Yoshida K.
      • Okada K.
      ), 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.

      DISCUSSION

      In a previous paper, we described a possible reaction mechanism ofCAO based on the results of in vitro experiments with CAO gene products (
      • Oster U.
      • Tanaka R.
      • Tanaka A.
      • Rüdiger W.
      ). CAO catalyzed two-step oxygenation reactions and converted chlorophyllidea to chlorophyllide b without any other enzymes. The results of those in vitro experiments are supported by the results of present experiments showing that cyanobacteria accumulated chlorophyll b by acquiring only theCAO gene. An in vitro study (
      • Oster U.
      • Tanaka R.
      • Tanaka A.
      • Rüdiger W.
      ) also showed that reduced ferredoxin was required for the oxygenation reactions byCAO. The cb1–3 transformant cells probably utilized endogenous ferredoxin for chlorophyll b synthesis.
      Chlorophyll b synthesized in the cb1–3 transformant cells was incorporated preferentially into CP1 apoproteins, and chlorophyllb transferred light energy to chlorophyll a in PSI. Fluorescence from free chlorophyll b was not observed on the transformant cells. These results indicate that chlorophyllb was incorporated into the chlorophyll a binding sites of CP1 instead of chlorophyll a, because it is generally observed that nonspecifically bound chlorophyll fluoresces even in a low yield (
      • Uehara K.
      • Mimuro M.
      • Fujita Y.
      • Tanaka M.
      ). The notion of specific binding of chlorophyllb was also supported by the observation that chlorophyllb was found only in CP1. CP43/47 and free chlorophyll bands on the green gels and that of other colorless proteins never bound chlorophyll b. The chlorophyll b content of 8% in CP1 indicated that at least eight chlorophyll binding sites could be replaced with chlorophyll b. These results are consistent with recent reports that some chlorophyll binding sites in LHC are replaceable by either chlorophyll a or chlorophyllb and that the chlorophyll b content of LHCII could change (
      • Sukenik A.
      • Wyman K.D.
      • Bennett J.
      • Falkowski P.G.
      ,
      • Nakayama K.
      • Mimuro M.
      ).
      Native SDS-PAGE is a powerful tool for isolating chlorophyll-protein complexes without contamination. This method has been used in many studies to determine the chlorophyll contents of chlorophyll-protein complexes. Most of these studies showed that CP1 has no chlorophyllb (
      • Anderson J.M.
      • Waldron J.C.
      • Thorne S.W.
      ), although some studies suggested the presence of chlorophyll b in CP1 (
      • Tanaka A.
      • Tanaka Y.
      • Tsuji H.
      ). Ikegami and Ke (
      • Ikegami I.
      • Ke B.
      ), using the ether extraction method, reported the existence of chlorophyllb in PSI reaction center particles in which the chlorophyll/P700 ratio was 13. These discrepant results concerning the chlorophyll a/b ratio of CP1 may be due in part to the method used for chlorophyll determination, because high chlorophyll a/b ratios cannot be determined spectroscopically. Although CP1 is believed to be a chlorophylla-protein complex, there have been no results providing clear evidence of this idea. In the present study, we found by HPLC that CP1, which was completely free from LHCI, bound a considerable amount of chlorophyll b. Our results suggested that CP1 is not a chlorophyll-a protein complex but a chlorophyll a/b-protein complex in C. reinhardtii. Our observation was also supported by the results of experiments showing that cyanobacterial CP1 apoproteins, which have amino acid sequence homology to that of green plants, bound chlorophyllb. Further studies are needed to determine whether core antenna complexes of oxygen-evolving photosynthetic organisms bind exclusively chlorophyll a.
      On the basis of the above results, we propose the progression of an antenna system in which a new pigment is acquired without the presence of a corresponding new protein. Our results demonstrated that the PSI core complex in a prokaryote has the capacity to incorporate chlorophyll b flexibly to its functional sites and that the complex in green algae indeed binds chlorophyll b. These findings led us to hypothesize as to how chlorophyll b, a new pigment, is incorporated into an antenna system of a prototype of cyanobacteria. When photosynthetic organisms acquired a CAO gene during an early evolutionary phase (
      • Tomitani A.
      • Okada K.
      • Miyashita H.
      • Matthijs H.C.
      • Ohno T.
      • Tanaka A.
      ), chlorophyll b began to be synthesized and bound to the core antenna of PSI through its flexibility. Chlorophyll b in CP1 immediately began to function as a photosynthetic pigment, and the organisms became able to use light energy at around 470 and 650 nm, which would be favorable for competition for light energy capturing. It would also have been important for the organisms that a new pigment did not induce photodamage. Our experiments reproduced this process. Cyanobacteria do not contain chlorophyll b, and it is therefore probable that they lost the CAO gene. Prochlorophytes bind chlorophyll b to prochlorophyte chlorophyll b-binding protein (
      • La Roche J.
      • van der Staay G.W.M.
      • Partensky F.
      • Ducret A.
      • Aebersold R.
      • Li R.
      • Golden S.S.
      • Hiller R.G.
      • Wrench P.M.
      • Larkum A.W.D.
      • Green B.R.
      ) by acquiring a new protein. In the evolutionary progression to eukaryotes, CP1 in green plants has retained an ancestral character to bind chlorophyllb by keeping a CAO gene in the common ancestor. On the other hand, chlorophyll b in peripheral antenna systems has changed its locations from prochlorophyte chlorophyllb-binding protein to LHC, which appears after an endosymbiotic event by the duplication of high light-inducible protein (
      • Green B.R.
      • Pichersky E.
      ).

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