Characterization of a Synechococcus sp. strain PCC 7002 mutant lacking Photosystem I. Protein assembly and energy distribution in the absence of the Photosystem I reaction center core complex

A Synechococcus sp. strain PCC 7002 psaAB::cat mutant has been constructed by deletional interposon mutagenesis of the psaA and psaB genes through selection and segregation under low-light conditions. This strain can grow photoheterotrophically with glycerol as carbon source with a doubling time of 25 h at low light intensity (10 E m^–2 s^–1). No Photosystem I (PS I)-associated chlorophyll fluorescence emission peak was detected in the psaAB::cat mutant. The chlorophyll content of the psaAB::cat mutant was approximately 20% that of the wild-type strain on a per cell basis. In the absence of the PsaA and PsaB proteins, several other PS I proteins do not accumulate to normal levels. Assembly of the peripheral PS I proteins PsaC,PsaD, PsaE, and PsaL is dependent on the presence of the PsaA and PsaB heterodimer core. The precursor form of PsaF may be inserted into the thylakoid membrane but is not processed to its mature form in the absence of PsaA and PsaB. The absence of PS I reaction centers has no apparent effect on Photosystem II (PS II) assembly and activity. Although the mutant exhibited somewhat greater fluorescence emission from phycocyanin, most of the light energy absorbed by phycobilisomes was efficiently transferred to the PS II reaction centers in the absence of the PS I. No light state transition could be detected in the psaAB::cat strain; in the absence of PS I, cells remain in state 1. Development of this relatively light-tolerant strain lacking PS I provides an important new tool for the genetic manipulation of PS I and further demonstrates the utility of Synechococcus sp. PCC 7002 for structural and functional analyses of the PS I reaction center.Abbreviations ATCC American type culture collection - Chl chlorophyll - DCMU 3-(3,4-dichlorophyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] - PCC Pasteur culture collection - PS I Photosystem I - PS II Photosystem II - SDS sodium dodecyl sulfate     

The single chlorophyll a molecule in the cytochrome b6f complex: unusual optical properties protect the complex against singlet oxygen

The cytochrome b(6)f complex of oxygenic photosynthesis mediates electron transfer between the reaction centers of photosystems I and II and facilitates coupled proton translocation across the membrane. High-resolution x-ray crystallographic structures (Kurisu et al., 2003; Stroebel et al., 2003) of the cytochrome b(6)f complex unambiguously show that a Chl a molecule is an intrinsic component of the cytochrome b(6)f complex. Although the functional role of this Chl a is presently unclear (Kuhlbrandt, 2003), an excited Chl a molecule is known to produce toxic singlet oxygen as the result of energy transfer from the excited triplet state of the Chl a to oxygen molecules. To prevent singlet oxygen formation in light-harvesting complexes, a carotenoid is typically positioned within approximately 4 A of the Chl a molecule, effectively quenching the triplet excited state of the Chl a. However, in the cytochrome b(6)f complex, the beta-carotene is too far (> or =14 Angstroms) from the Chl a for effective quenching of the Chl a triplet excited state. In this study, we propose that in this complex, the protection is at least partly realized through special arrangement of the local protein structure, which shortens the singlet excited state lifetime of the Chl a by a factor of 20-25 and thus significantly reduces the formation of the Chl a triplet state. Based on optical ultrafast absorption difference experiments and structure-based calculations, it is proposed that the Chl a singlet excited state lifetime is shortened due to electron exchange transfer with the nearby tyrosine residue. To our knowledge, this kind of protection mechanism against singlet oxygen has not yet been reported for any other chlorophyll-containing protein complex. It is also reported that the Chl a molecule in the cytochrome b(6)f complex does not change orientation in its excited state.     

Structure of the cytochrome b6f complex: quinone analogue inhibitors as ligands of heme cn

A native structure of the cytochrome b(6)f complex with improved resolution was obtained from crystals of the complex grown in the presence of divalent cadmium. Two Cd(2+) binding sites with different occupancy were determined: (i) a higher affinity site, Cd1, which bridges His143 of cytochrome f and the acidic residue, Glu75, of cyt b(6); in addition, Cd1 is coordinated by 1-2 H(2)O or 1-2 Cl(-); (ii) a second site, Cd2, of lower affinity for which three identified ligands are Asp58 (subunit IV), Glu3 (PetG subunit) and Glu4 (PetM subunit). Binding sites of quinone analogue inhibitors were sought to map the pathway of transfer of the lipophilic quinone across the b(6)f complex and to define the function of the novel heme c(n). Two sites were found for the chromone ring of the tridecyl-stigmatellin (TDS) quinone analogue inhibitor, one near the p-side [2Fe-2S] cluster. A second TDS site was found on the n-side of the complex facing the quinone exchange cavity as an axial ligand of heme c(n). A similar binding site proximal to heme c(n) was found for the n-side inhibitor, NQNO. Binding of these inhibitors required their addition to the complex before lipid used to facilitate crystallization. The similar binding of NQNO and TDS as axial ligands to heme c(n) implies that this heme utilizes plastoquinone as a natural ligand, thus defining an electron transfer complex consisting of hemes b(n), c(n), and PQ, and the pathway of n-side reduction of the PQ pool. The NQNO binding site explains several effects associated with its inhibitory action: the negative shift in heme c(n) midpoint potential, the increased amplitude of light-induced heme b(n) reduction, and an altered EPR spectrum attributed to interaction between hemes c(n) and b(n). A decreased extent of heme c(n) reduction by reduced ferredoxin in the presence of NQNO allows observation of the heme c(n) Soret band in a chemical difference spectrum.     

Molecular cloning and nucleotide sequence of the psaA and psaB genes of the cyanobacterium Synechococcus sp. PCC 7002

The psaA and psaB genes, which encode the P700 chlorophyll a apoproteins of the Photosystem I complex, have been cloned from the unicellular, transformable cyanobacterium Synechococcus sp. PCC 7002. The nucleotide sequence of these genes and of their flanking sequences have been determined by the chain termination method. As found in the chloroplast genomes of higher plants, the psaA gene lies 5 to the psaB gene; however, the cyanobacterial genes are separated by a greater distance (173 vs. 25–26 bp). The psaA gene is predicted to encode a polypeptide of 739 amino acid residues (81.7 kDa), and the psaB gene is predicted to encode a polypeptide of 733 residues (81.4 kDa). The cyanobacterial psa gene products are 76% to 81% identical to their higher plant homologues; moreover, because of conservative amino acid replacements, the cyanobacterial sequences are more than 95% homologous to those determined for higher plants. These results provide the basis for a genetic analysis of Photosystem I, and are discussed in relationship to structural and functional aspects of the Photosystem I complexes of both cyanobacteria and higher plants.     

Cytochrome bc 1 and b 6 f complexes of photosynthetic membranes

All photosynthetic membranes contain a cytochrome bc ₁ or b ₆ f complex that catalyzes the oxidation of quinols and the reduction of a high-potential electron carrier, such as cytochrome c ₂ or plastocyanin. The cytochrome complex also functions in the translocation of protons across the membrane and as a consequence, establishes the proton motive force that is used for the synthesis of ATP. The structure and function of the cytochrome complexes are first reviewed in this chapter. Amino acid sequence information for almost all of the protein subunits of these complexes is now available, and these allow for a detailed consideration of functional domains in the protein subunits and for a further discussion of the evolution of the cytochrome complex in photosynthetic organisms.     

Structural and compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002. Analyses of the wild-type strain and a phycocyanin-less mutant constructed by interposon mutagenesis

The phycobilisomes and phycobiliproteins of Synechococcus sp. PCC 7002 wild-type strain PR6000 have been isolated and characterized. The hemidiscoidal phycobilisomes of strain PR6000 are composed of eleven different polypeptides: phycocyanin and subunits; allophycocyanin and subunits; subunit of allophycocyanin B; the allophycocyanin -subunit-like polypeptide of M_r 18 000; the linker phycobiliprotein of M_r 99 000; and non-chromophore-carrying linker polypeptides of M_r 33 000, 29 000, 9000, and 8000. Several of these polypeptides were purified to homogeneity and their amino acid compositions and amino-terminal amino acid sequences were determined. Analyses of the phycobiliproteins of Synechococcus sp. PCC 7002 were greatly facilitated by comparative studies performed with a mutant strain, PR6008, constructed to be devoid of the phycocyanin and subunits by recombinant DNA techniques and transformation of strain PR6000. The absence of phycocyanin did not greatly affect the allophycocyanin content of the mutant strain but caused the doubling time to increase 2–7-fold depending upon the light intensity at which the cells were grown. Although intact phycobilisome cores could not be isolated from this mutant, it is probable that functionally intact cores do exist in vivo.Abbreviations used SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate - 2D-PAGE two-dimensional gel electrophoresis in which the first dimension consisted of isoelectric focusing in the presence of 8.0 M urea in the pH range 4–6 and the second dimension consisted of electrophoresis in the presence of sodium dodecylsulfate. The nomenclature employed for the phycobiliprotein subunits and linker polypeptides is that defined by Glazer (1985)     

The cloning and sequencing of Synechococcus sp. PCC 7002 petCA operon: Implications for the cytochrome c-553 binding domain of cytochrome f

The genes encoding the Rieske iron-sulfur protein and cytochrome f from a unicellular, naturally transformable, photoheterotrophic cyanobacterium, Synechococcus sp. PCC 7002, formerly Agmenellum quadruplicatum, have been isolated and sequenced. The two genes were found to be on a single operon, petCA.The Synechococcus sp. PCC 7002 iron-sulfur protein contains 181 amino acids, the conserved putative iron-binding domains CTHLGCV, residues 108–114, and CPCHGS, residues 128–133, no presequence and has a 73% sequence identity to the Nostoc PCC 7906 iron-sulfur protein. The 325 amino acid apocytochrome f sequence contains a 42 amino acid presequence, a CANCH heme binding domain, residues 20–24 from the presumed start of the mature protein, and a predicted hydrophobic membrane-spanning domain, residues 250–269. The mature cytochrome f sequence has a 71.5% sequence identity with Nostoc PCC 7906 cytochrome f and possesses a large (-14) negative charge and low calculated pI of 4.47 compared to higher plant chloroplast sequences. Nine separate domains showing differences in charged residues among cyanobacteria and plants have been identified and the possibility that these domains are involved in the ionic interactions with plastocyanin or cytochrome c-553 is discussed.The sequences reported in this paper have been deposited in the EMBL/Genbank data base (IntelliGenetics, Mountain View, CA, and Eur. Mol. Biol. Lab., Heidelberg) (accession no. M74514).     

ApcD is necessary for efficient energy transfer from phycobilisomes to photosystem I and helps to prevent photoinhibition in the cyanobacterium Synechococcus sp. PCC 7002

Phycobilisomes (PBS) are the major light-harvesting, protein-pigment complexes in cyanobacteria and red algae. PBS absorb and transfer light energy to photosystem (PS) II as well as PS I, and the distribution of light energy from PBS to the two photosystems is regulated by light conditions through a mechanism known as state transitions. In this study the quantum efficiency of excitation energy transfer from PBS to PS I in the cyanobacterium Synechococcus sp. PCC 7002 was determined, and the results showed that energy transfer from PBS to PS I is extremely efficient. The results further demonstrated that energy transfer from PBS to PS I occurred directly and that efficient energy transfer was dependent upon the allophycocyanin-B alpha subunit, ApcD. In the absence of ApcD, cells were unable to perform state transitions and were trapped in state 1. Action spectra showed that light energy transfer from PBS to PS I was severely impaired in the absence of ApcD. An apcD mutant grew more slowly than the wild type in light preferentially absorbed by phycobiliproteins and was more sensitive to high light intensity. On the other hand, a mutant lacking ApcF, which is required for efficient energy transfer from PBS to PS II, showed greater resistance to high light treatment. Therefore, state transitions in cyanobacteria have two roles: (1) they regulate light energy distribution between the two photosystems; and (2) they help to protect cells from the effects of light energy excess at high light intensities.     

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome b(L) to b(H)

The key step of the "protonmotive Q-cycle" mechanism for cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. ISP is reduced when its head domain is at the b-position and subsequent move to the c1 position, to reduce cytochrome c1, upon protein conformational changes caused by the electron transfer from cytochrome b(L) to b(H). Results of analyses of the inhibitory efficacy and the binding affinity, determined by isothermal titration calorimetry, of Pm and Pf, on different redox states of cytochrome bc1 complexes, confirm this speculation. Pm inhibitor has a higher affinity and better efficacy with the cytochrome b(H) reduced complex and Pf binds better and has a higher efficacy with the ISP reduced complex.     

Construction of insertion mutants of Synechocystis sp. PCC 6803: evidence for an essential function of subunit IV of the cytochrome b6/f complex

The gene encoding subunit IV of the cytochrome b₆/f complex (petD) has been isolated from a genomic library of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 480 nucleotides and can code for a polypeptide with a molecular weight of 17.5 kDa. The deduced amino acid sequence shows high identity with the corresponding sequences of both the photoautotrophic prokaryote Nostos sp. PCC 7906 as well as of lower and higher photoautotrophic eukaryotes (e.g. Chlorella protothecoides, Nicotiana tabacum). Transformation of Synechocystis sp. PCC 6803 with a plasmid containing the cloned petD gene in which the coding sequence is interrupted by the aminoglycoside 3-phosphotransferase gene (aph) from Tn903 resulted in the formation of km resistant transformants. The molecular analysis of independent transformants revealed that all clones were merodiploid containing both uninterrupted wild-type as well as interrupted mutant petD copies. Approaches to segregate these two genomes were unsuccessful implying an essential function of the petD gene product in Synechocystis sp. PCC 6803.Abbreviations aph aminoglycoside 3-phosphotransferase - cpDNA chloroplast DNA - km kanamycin - PSI photosystem I - PSII photosystem II     

Genetic analysis of two new mutations resulting in herbicide resistance in the cyanobacterium Synechcoccus sp. PCC 7002

Two herbicide-resistant strains of the cyanobacterium Synechococcus sp. PCC 7002 are compared to the wild-type with respect to the DNA changes which result in herbicide resstance. The mutations have previously been mapped to a region of the cyanobacterial genome which encodes oneof three copies of psbA, the gene which encodes the 32 kDa Q_b-binding protein also known as D1 (Buzby et al. 1987). The DNA sequence of the wild-type gene was first determined and used as a comparison to that of the mutant alleles. A point mutation at codon 211 in the psbA1 coding locus (TTC) to TCC) results in an amino acid change from phenylalanine to serine in the D1 protein. This mutation confers resistance to atrazine and diuron at seven times and at two times the minimal inhibitory concentration (MIC) for the wild-type, respectively. A mutation at codon 211 resulting in herbicide resistance has not previously been described in the literature. A second point mutation at codon 219 in the psbA1 coding locus (GTA to ATA) results in an amino acid change from valine to isoleucine in the D1 protein. This mutation confers resistance to diuron and atrazine at ten times and at two times the MIC for the wild-type, respectively. An identical codon change conferring similar herbicide resistance patterns has previously been described in Chlamydomonas reinhardtii. The atrazine-resistance phenotype in Synechococcus sp. PCC 7002 was shown to be dominant by plasmid segregation analysis.Abbreviations At ^r atrazine resistance - Du ^r diuron resistance - Km ^r kanamycin resistance - Ap ^r ampicillin resistance - MIC Minimum inhibitory concentration     

Multiple step assembly of the transmembrane cytochrome b6

We have analyzed the role of individual heme-ligating histidine residues for assembly of holo-cytochrome b₆, and we show that the two hemes b_L and b_H bind in two subsequent steps to the apo-protein. Binding of the low-potential heme b_L is a prerequisite for binding the high-potential heme b_H. After substitution of His86, which serves as an axial ligand for heme b_L, the apo-protein did not bind heme, while substitution of the heme b_L-ligating residue His187 still allowed binding of both hemes. Similarly, after replacement of His202, one axial ligand to heme b_H, binding of only heme b_L was observed, whereas replacement of His100, the other heme b_H ligand, resulted in binding of both hemes. These data indicate sequential heme binding during formation of the holo-cytochrome, and the two histidine residues, which serve as axial ligands to the same heme molecule (heme b_L or heme b_H), have different importance during heme binding and cytochrome assembly. Furthermore, determination of the heme midpoint potentials of the various cytochrome b₆ variants indicates a cooperative adjustment of the heme midpoint potentials in cytochrome b₆.     

Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b(6) in Synechococcus sp. PCC 7002

The cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b(6) subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b(6) at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc(1) complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc(1) complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc(1) complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure-function relationships in the cytochrome bf complex.     

Study on energy transfer between carotenoid and chlorophyll a in cytochrome b 6 f complex from Bryopsis corticulans

The excitation energy transfer between carotenoid and chlorophyll (Chl) in the cytochrome b ₆ f complex from Bryopsis corticulans (B. corticulans), in which the carotenoid is 9-cis-α-carotene, was investigated by means of fluorescence excitation and sub-microsecond time-resolved absorption spectroscopies. The presence of efficient singlet excitation transfer from α-carotene to Chl a was found with an overall efficiency as high as ∼ ∼24%, meanwhile the Chl a-to-α-carotene triplet excitation transfer was also evidenced. Circular dichroism spectroscopy showed that α-carotene molecule existed in an asymmetric environment and Chl a molecule had a certain orientation in this complex.Bin-Xing Li and Ping Zuo contributed equally to this work.     

The proteins involved in sucrose synthesis in the marine cyanobacterium Synechococcus sp. PCC 7002 are encoded by two genes transcribed from a gene cluster

It has been reported that higher plants and cyanobacteria synthesize sucrose (Suc) by a similar sequential action of sucrose-phosphate synthase (SPS) and sucrose-phosphate phosphatase (SPP). In the genome of the marine unicellular cyanobacterium Synechococcus sp. PCC 7002 there is a sequence that was not annotated as a putative SPP encoding gene (sppA), although the sequence was available. In this study, we functionally characterize the sppA gene of that strain and demonstrate that it is cotranscribed with spsA, the SPS encoding gene. This is the first report on the coordination of Suc synthesis gene expression in an oxygenic-photosynthetic organism.     

Hydrogen peroxide-induced chlorophyll a bleaching in the cytochrome b 6 f complex: a simple and effective assay for stability of the complex in detergent solutions

The instability of cytochrome b ₆ f complex in detergent solutions is a well-known problem that has been studied extensively, but without finding a satisfactory solution. One of the important reasons can be short of the useful method to verify whether the complex suspended in different detergent is in an intact state or not. In this article, a simple and effective assay for stability of the complex was proposed based on the investigation on the different effects of the two detergents, n-octyl-β-d-glucopyranoside (OG) and dodecyl-β-d-maltoside (DDM), on the properties of the complex. DDM stabilizes the complex preparation more effectively whereas OG denatures the interactions of the heme groups and pigment molecules with the protein environment, leading to the bleaching of chlorophyll a induced by addition of hydrogen peroxide. The assay of the use of hydrogen peroxide to characterize the complex by studying the bleaching of chlorophyll induced by hydrogen peroxide and the peroxidase activity of the complex was discussed. This simple method will probably be useful to study the stability of the complex. Xiao-Bo Chen and Xiao-Hui Zhao contributed equally to this work.     

Functional activities of monomeric and dimeric forms of the chloroplast cytochrome b6f complex

A monomeric form of the isolated cytochrome b6f complex from spinach chloroplast membranes has been isolated after treatment of the dimeric complex with varying concentrations of Triton X-100. The two forms of the complex are similar as regards electron transfer components and subunit composition. In contrast to a previous report (Huang et al. (1994) Biochemistry 33: 4401–4409) both the monomer and dimer are enzymatically active. However, after incorporation of the respective complexes into phospholipid vesicles, only the dimeric form of the cytochrome complex shows uncoupler sensitive electron transport, an indication of coupling of electron transport to proton translocation. The absence of this activity with the monomeric form of the cytochrome complex may be related to an inhibition by added lipids.Abbreviations CCCP- carbonyl cyanide m-chlorophenylhydrazone - mega-9- nonanoyl-N-methylglucamide     

Fractional control of photosynthesis by the QB protein,the cytochrome f/b 6 complex and other components of the photosynthesic apparatus

In order to obtain information on fractional control of photosynthesis by individual catalysts, catalytic activities in photosynthetic electron transport and carbon metabolism were modified by the addition of inhibitors, and the effect on photosynthetic flux was measured using chloroplasts of Spinacia oleracea L. In thylakoids with coupled electron transport, light-limited electron flow to ferricyanide was largely controlled by the Q_B protein of the electron-transport chain. Fractional control by the cytochrome f/b ₆ complex was insignificant under these conditions. Control by the cytochrome f/b ₆ complex dominated at high energy fluence rates where the contribution to control of the Q_B protein was very small. Uncoupling shifted control from the cytochrome f/b ₆ complex to the Q_B protein. Control of electron flow was more complex in assimilating chloroplasts than in thylakoids. The contributions of the cytochrome f/b ₆ complex and of the Q_B protein to control were smaller in intact chloroplasts than in thylakoids. Thus, even though the transit time for an electron through the electron-transport chain may be below 5 ms in leaves, oxidation of plastohydroquinone was only partially responsible for limiting photosynthesis under conditions of light and CO₂ saturation. The energy fluence rate influenced control coefficients. Fractional control of photosynthesis by the ATP synthetase, the cytochrome f/b ₆ complex and by ribulose-1,5-bisphosphate carboxylase increased with increasing fluence rates, whereas the contributions of the Q_B protein and of enzymes sensitive to SH-blocking agents decreased. The results show that the burdens of control are borne by several components of the photosynthetic apparatus, and that burdens are shifted as conditions for photosynthesis change.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2,4-dinitro phenylether of 2-iodo-4-nitrothymol - pCMBS p-chloromercuribenzosulfonate