Evolutionary relationships between “Q-type” photosynthetic reaction centres: Hypothesis-testing using parsimony

J. theor. Biol. (1990) 145, 535-545

Evolutionary Relationships between "Q-type" Photosynthetic Reaction Centres: Hypothesis-testing using Parsimony TIMOTHY J. BEANLAND

Department of Biochemistry, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QW, U.K. (Received on 10 February 1990, Accepted on 17 March 1990) Hypotheses concerning the evolutionary relationships between "Q-type" photosynthetic reaction centres are tested using amino acid parsimony analysis of subunit sequences and an alignment based on dot matrix comparisons. Strong evidence is found for independent gene duplications having produced the L and M subunits of the photosynthetic purple bacterial reaction centre and D1 and D2 of PhotosystemII. Much support is also found for the L and M subunits of the green filamentous bacterium Chloroflexus aurantiacus arising from the same gene duplication as the purple bacterial subunits, suggesting there was an ancestral bacterial heterodimeric reaction centre. These conclusions caution against over-extrapolation from the purple bacterial reaction centre to Photosystem-II, and suggest that the latter is more ancient than previously supposed.

Introduction Photosynthetic reaction centres (RCs) are pigment-protein complexes located in membranes that use the energy from incident photons to effect a transmembrane charge-separation and reduce high-potential acceptor species. Chlorophyll-dependent photosynthesis is found in four groups of Gram-negative and one group of Gram-positive bacteria. The former are the green bacteria (two groups, "sulphur" and "filamentous"), the photosynthetic purple bacteria, and the species of the oxygenic lineage (comprising cyanobacteria and their evolutionary descendants, the chloroplasts of plants and algae). The sole photosynthetic group of Gram-positive bacteria are the Heliobacteria. Analysis of the pigment groups in all these RCs suggests they fall into two distinct families. The RCs of green sulphur bacteria, Heliobacterium, and Photosystem-I of cyanobacteria and chloroplasts form a group characterized by possession of ironsulphur ( " F e - S " ) centres as the secondary acceptor, hence they have been called "F-type" RCs (Amesz & Duysens, 1986). The reaction centres of photosynthetic purple bacteria, green filamentous bacteria and Photosystem-II (PS-II) of the oxygenic groups form a family (the "Q-type" RCs) typified by the possession of phaeophytin as the primary acceptor and quinone as the secondary acceptor. In this paper I concentrate on the relationships between the different "Q-type" RCs.

"Q-type" Reaction Centres The purple bacterial RC (Deisenhofer & Michel, 1989) carries out anoxygenic photosynthesis and comprises four polypeptide species; H, L and M subunits and 535 0022-5193/90/016535 + 12 $03.00/0

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a cytochrome. Green filamentous bacteria also undergo anoxygenic photosynthesis, and possess a RC with L and M subunits and a cytochrome, but apparently lacking the H subunit (Pierson et al., 1983). The third "Q-type" RC, Photosystem-II, is the oxygenic water-splitting component of the "Z-scheme". Minimal core complexes of PS-II showing light-driven charge separation (Nanba & Satoh, 1987) contain D1, D2 and cytochrome b-559 as the major protein subunits. Comparisons between the primary sequences of the core subunits of the "Q-type" RCs suggest they have common ancestry and form a phylogenetic group to the exclusion of the "F-type" RCs. One may therefore ask what are the evolutionary relationships between the subunit proteins of "Q-type" RCs. The primary sequence homologies between the L and M subunits of the purple photosynthetic bacterial RC (e.g. from Rhodopseudomonas viridis) and D1 and D2 of PS-II (e.g. from spinach chloroplasts) are well documented (Michel et al., 1986; Williams et al., 1983). They have led to a proposed structure for PS-II (Trebst, 1986; Hearst, 1986) based to a large extent on the purple bacterial RC, for which a combination of crystallography, spectroscopy and molecular biology has produced a detailed structural model. The L and M subunits form a heterodimer that binds the pigment groups and comprises the core of the purple bacterial RC. The model for PS-II proposes that DI and D2 similarly form a heterodimer at the core of this photosystem. One scheme for the evolutionary origin of heterodimeric RCs (Deisenhofer & Michel, 1989) proposes that the ancestral RC core was a homodimeric structure encoded by a single gene that evolved into a heterodimeric structure by gene duplication and divergence, resulting in modern RC cores encoded by two related but separate genes. The question of whether there was a single gene duplication for the purple bacterial RC and PS-II taking place before the lineages diverged, or whether there were two independent duplications occurring since this divergence has not been adequately addressed. There now exist sufficient data (in the form of subunit primary sequences) and the necessary tools (methods of phylogenetic inference) formally to test such alternatives. I report here that parsimony analysis of the sequence data strongly indicates that a scheme in which independent gene duplications gave rise to the purple bacterial L-M pair and to the PS-II D1-D2 pair is correct. I also test the relationship of the Chloroflexus RC to the other two "Q-type" RCs, and find strong evidence that the L and M subunits of Chloroflexus arose from the same gene duplication as the purple bacterial subunits. These findings suggest that PS-II may be more ancient than previously supposed.

Sequence Alignment and Programs of Phylogenetic Inference The sequence data to test the RC hypotheses are all available in the EMBL or GenBank sequence databases. Six subunit protein sequences were obtained from DNA sequences using the program TRANSLATE within the University of Wisconsin G C G package (Devereux et al., 1984). The nucleotide sequences are database entries

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Embl:Rvprclm (Michel et al., 1986), Embl:Caprcm (Ovchinnikov et al., 1988a), GenBank:Spicppsba (Zurawski et al., 1982) and GenBank:Spicpd2cb (Alt et al., 1984; Holschuh et al., 1984). The Chloroflexus L subunit sequence (Ovchinnikov et al., 1988b) is not present in any database and was entered manually. For all subsequent analyses, the sequences were used as primary translation products, the bacterial subunits showing post-translational removal of the N-terminal methionine residue, and D1 limited processing in vivo. A prerequisite for phylogenetic inference from primary sequences is that strictly homologous sites across all the sequences are aligned. The basis for the published alignments of these six sequences is often not stated, most seeming to be a blend of maximal global homology (by application of a Needleman-Wunsch type algorithm) and manual alignment of functionally similar key residues such as the ligands to the non-haem iron atom. Not surprisingly therefore, published alignments for the L, M, D1 and D2 sequences (Deisenhofer & Michel, 1989; Hearst, 1986) agree only for a central highly-conserved block of about 120 amino acids. I have used dot matrix comparisons (Maizel & Lenk, 1981) between the sequences (Fig. 1) as the basis for generating a multiple alignment, with regions of extended similarity in Fig. 1 lying on or near the diagonal being taken as regions of homology. All 15 pairwise comparisons in Fig. 1 contain extensive regions of similarity, suggesting the six subunits are ultimately all derived from one ancestral polypeptide. The alignment generated (Fig. 2) agrees with published alignments over the central region but not elsewhere. Several of the cofactor ligands (e.g. the histidine residues to the accessory bacteriochlorophylls, leucine to the accessory bacteriophaeophytin of the Chloroflexus M subunit) are conserved across the four bacterial sequences to the exclusion of D1 and D2. The aligned sequences were subjected to phylogenetic analysis (Felsenstein, 1988) using the program PROTPARS from the PHYLIP 3.2 package (Felsenstein, 1985a). PROTPARS carries out parsimony ("minimal mutation" or "ancestral sequence") analysis on aligned amino acid sequence data. For a set of sequences one can either look for the tree topology that requires the fewest total number of amino acid substitutions (i.e. is the most parsimonious), or one can supply specific topologies, determine the number of substitutions each would require, and test whether the differences between the topologies tested are statistically significant. In calculating the number of mutational steps a tree requires, PROTPARS uses a matrix of distances between pairs of amino acids that is intermediate between a simple unitary one (mutation between any two different amino acid residues scored as 1.00) and a purely genetic one (Fitch, 1971). In the latter scheme, distances are based on the minimum number of DNA mutations implied in converting between the two codons. The frequency with which an amino acid substitutes for another in a biological situation (Schwartz & Dayhoff, 1979) is a combination of this distance and selective constraints on which residues are acceptable at that site, although since functionally similar amino acids are often encoded by closely related codons these effects are not independent. The statistical testing used within PROTPARS determines whether the differences between the steps required for

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FIG. 1. (Left) Dot matrix comparisons between RC subunit sequences produced using COMPARE and DOTPLOT (Devereux et al., 1984). The axes represent the complete protein sequences N-terminus to C-terminus left-to-right and bottom-to-top and are all to the same scale. Rps, Rhodopseudomonas oiridis; Cfx, Chloroflexus aurantiacus. Each point represents the centre of a window within which the two sequences have an aggregate similarity greater than or equal to the stringency value. The window size in all cases was 30 residues (default 30). The stringency (default value 9.0) was set to 15-0 for (a), and 17.0 for the intraspecific comparisons in (b). The similarity matrix used by COMPARE for pairs of amino acids (Gribskov & Burgess, 1986) is an empirically-based one derived from the Dayhoff log-odds matrix (Schwartz & Dayhoff, 1979).

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FIG. 2. Alignment of RC subunit sequences based on homology. This alignment is consistent with the regions of extended similarity in Fig. 1. For regions not covered by COMPARE/DOTPLOT, the alignment is derived using the pairwise alignment program GAP. Default values for gap weight (5-0) and gap length weight (0.3) were used. GAP uses the same similarity table as COMPARE (Gribskov & Burgess, 1986). The numbering is arbitrary. Cfxaur, Chloroflexus aurantiacus; Rpsvir, Rhodopseudomonas viridis; Spin, spinach chloroplast. Asterisks, sites at which four or more residues are identical; boxes I-V, regions used for parsimony analysis; Sp.pr, ligands to the special pair (bacterio)chlorophylls; Fe, ligands to the non-haem iron atom; Q, quinone-binding residues.

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specified trees are significant (the null hypothesis being that they are not) and is a version of a test proposed by Templeton (Templeton, 1983; Felsenstein, 1985b). To avoid artificially forcing the bacterial subunits to group together, regions where extensive gaps have been introduced into the bacterial sequences were omitted from the analysis, leaving the five boxed regions shown (Fig. 2). Analysis was carried out with two data sets, either on all five boxes together (a total of 247 sites) or on the more highly conserved central boxIII alone (123 sites).

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Hypothesis Testing Figure 3 shows the hypotheses tested represented as unrooted gene tree topologies. The hypotheses in trees 1-3 test Michel's scheme (Deisenhofer & Michel, 1989) for purple bacterial R C - P S - I I relationships, and cover all possible relationships between the four sequences. Tree 1 tests whether independent duplications produced the L-M and D 1 - D 2 pairs, with the two RC types having separate heterodimeric ancestors, the last RC ancestral to the bacterial RC and PS-II being a homodimer. Tree 2 tests whether a single gene duplication produced L-D1 and M-D2 pairs (tree 3 : L - D 2 and M-D1), consistent with a single heterodimeric RC ancestral to the bacterial RC and PS-II. RpsL

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TABLE 1 Parsimony analysis using PROTPARS, testing Michel's hypotheses of Fig. 3 using as data sets the boxed regions indicated in Fig. 2. The "number of steps" for each tree is related to, but not the same as, the number of total amino acid substitutions. S.D., the standard deviation of the step difference between each tree and the best topology (details are in the main text) Boxes I-V

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S.D.

No. of steps

Differences from best topology

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637 678 680

(Best) 41 43

9.56 9.24

287 307 309

(Best) 20 22

7.10 6.81

Table 1 shows the results from PROTPARS with boxes I-V or box III alone as the data set. In both cases parsimony analysis favours tree 1, the alternative topologies being significantly worse at below the 1% level with either data set. This is therefore strong evidence for independent gene duplications having produced the purple bacterial L - M and PS-II D 1 - D 2 pairs. The same conclusion results, although I have not shown the data, from taking the L and M subunit sequences of another purple bacterium, Rhodobacter sphaeroides (Williams et al., 1983, 1984) with D1 (psbAt) and D2 from the cyanobacterium Anacystis nidulans R2 (Synechococcus PCC 7942) (Golden et al., 1986; Golden & Stearns, 1988) using an equivalent alignment to Fig. 2, or even from taking the Rhodopseudomonas viridis and spinach sequences with a commonly published alignment (Deisenhofer & Michel, 1989), extensive gaps again being omitted. The conclusion is therefore not an artefact of either the sequences selected or the alignment used. Trees 4-7 (Fig. 3) allow testing of four hypotheses about the relationship of the Chloroflexus RC to the other two RCs, and are a subset of the 105 possible unrooted tree topologies with six sequences, selected to test the most feasible biological relationships. Tree 4 tests whether three separate gene duplications independently produced the three subunit pairs, all RCs sharing an ancestral homodimer. Tree 5 tests whether the Chloroflexus and Rhodopseudomonas subunits arose from the same duplication and diverged from an ancestral bacterial heterodimer independently of the D 1 - D 2 duplication, the two bacterial L subunits forming a natural pair and the M subunits likewise. Tree 6 tests the same basic scheme as tree 5 but with the Chioroflexus L and M subunits interchanged. Tree 7 tests whether one duplication produced the Chloroflexus proteins and D1-D2, independently of the purple bacterial subunits. Table 2 shows that parsimony analysis of trees 4-7 favours tree 5, the other topologies being significantly worse (at below the 1% level) for either data set. This indicates a common gene duplication for the ChloroJlexus-Rhodopseudomonas L-M pair, independent of the D 1 - D 2 duplication, and thus suggests a heterodimeric RC ancestral to the two bacterial RCs. Again, by taking the Rhodobacter and Anacystis

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TABLE 2

Parsimony analysis using PROTPARS, testing the Chloroflexus hypotheses of Fig. 3 using as data sets the boxed regions from Fig. 2. Details in the legend to Table 1 and in the main text Boxes I-V

Tree Tree Tree Tree

4 5 6 7

No. of steps

Differences from best topology

958 919 963 1002

39 (Best) 44 83

Box III only

S.D. 10-07 9.50 13.55

No. of steps

Differences from best topology

433 412 433 455

21 (Best) 21 43

S.D. 7.31 7.17 10-28

subunit sequences, this result was shown not to be an artefact of either the sequences or the alignment used. Analysis of both the Rhodopseudomonas-Spinach-Chioroflexus and the Rhodobacter-Anacystis-Chloroflexus data sets using PROTPARS simply to find the minimal mutation tree shows tree 5 is indeed the most parsimonious of all the 105 topologies. Discussion

Hypothesis-testing suggests the scheme shown in Fig. 4 in which PS-II emerges as the most deeply-rooting o f the three " Q - t y p e " RCs. This is in agreement with recent findings (Dekker et al., 1989) that suggest the similarities between the purple Purple bacteria

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bacterial RC and PS-II have been exaggerated, and cautions against over-extrapolation from the former to the latter. The scheme in Fig. 4 however contradicts phylogenies based on 16S r R N A sequences (Woese, 1987) which place Chloroflexus as the most anciently-diverging species. Lateral gene transfer is one possible explanation for this conflict. One can also criticise the 16S rRNA phylogeny because no attempt was made at testing its statistical robustness, this being particularly important here because most o f the taxa radiate from within one area o f the tree, lending it a fan-like topology. A greater antiquity for PS-II than previously supposed is not necessarily to be taken as suggesting that oxygenic photosynthesis per se predates the purple-green filamentous bacterial divergence, likely to have been around 3 billion years ago (Olson & Pierson, 1987), although the evidence from microfossils (Schopf & Packer, 1987) suggests the cyanobacteria are an extremely ancient group (possibly 3.5billion-years-old) and therefore such a possibility is not excluded. It is perhaps more likely that a primitive " P S - I I " acted cyclically (possibly with H2S as electron d o n o r for non-cyclic flow) as seen in purple and green bacterial RCs, but distinct from the facultative anoxygenic photosynthesis seen in uncoupled PS-I in some extant cyanobacteria. At later stages this early PS-II became linked to PS-I in a Z-scheme and began using water as an electron donor. Such a scheme is consistent with the evolution of oxygenic photosynthesis at a date more in agreement with the geochemical record o f around 2-5 billion years ago (Olson & Pierson, 1987). I would like to thank Derek Bendall, Tony Larkum, Peter Lockhart and Christopher Howe for useful discussions, and David Judge for help with computing. I am supported by the Science and Engineering Research Council, the Cambridge Philosophical Society and Corpus Christi College. REFERENCES ALT, J., MORRIS, J., WESTHOFF, P. & H ERRMANN, R. G. (1984). Nucleotide sequence of the clustered genes for the 44 kd chlorophyll a apoprotein and the "32kd"-like protein of the photosystem I1 reaction center in the spinach plastid chromosome. Curt. Genet. 8, 597-606. AMESZ, J. & DUYSENS, L. N. M. (1986). Electron donors and acceptors in photosynthetic reaction centers. Photosynth. Res. 10, 337-346. DEISENHOFER, J. & MICHEL, H. (1989). The photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis. EMBO J. 8, 2149-2170. DEKKER, J. P., BOWLBY, N. R. & YOCUM, C. F. (1989). Chlorophyll and cytochrome b-559 content of the photochemical reaction center of photosystem II. FEBS Lett. 254, 150-154. DEVEREUX, J., HAEBERLI, P. & SMITHIES, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucl. Acids Res. 12, 387-395. FELSENSTEI N, J. (1985a). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783-791. FELSENSTEIN, J. (1985b). Confidence limits on phylogenies with a molecular clock. Syst. Zool. 34, 152-161. FELSENSTEIN, J. (1988). Phylogenies from molecular sequences: inference and reliability. Ann. Rev. Genet. 22, 521-565. FITet-t, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst. Zool. 20, 406-416. GOLDEN, S. S., BRUSSLANs J. & HASELKORN, R. (1986). Expression of a family of psbA genes encoding a photosystem II polypeptide in the cyanobacterium Anacystis nidulans R2. EMBO £ 5, 2789-2798.

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