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Clues from Nitrogenase and Chlorophyll Iron Proteins (Gene Duplication/Bacteriochlorophyll/Purple Bacteria/Protochlorophyilide Reductase/Chlorin Reductase) DONALD H

Clues from Nitrogenase and Chlorophyll Iron Proteins (Gene Duplication/Bacteriochlorophyll/Purple Bacteria/Protochlorophyilide Reductase/Chlorin Reductase) DONALD H

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 7134-7138, August 1993 Evolution Early evolution of : Clues from nitrogenase and iron proteins (gene duplication//purple /protochlorophyilide reductase/ reductase) DONALD H. BURKE*t, JOHN E. HEARST*, AND AREND SIDOWt *Department of Chemistry, University of California, Berkeley, CA 94720; and tDepartment of Molecular and Cell Biology, University of California, 401 Barker Hall, Berkeley, CA 94720 Communicated by Randy Schekman, March 24, 1993

ABSTRACT Chlorophyll (Chl) is often viewed as having to chlorophyll to bacteriochlorophyll preceded bacteriochlorophyll (BChl) as the primary photore- ceptor pigment in early photosynthetic systems because syn- bchL bchX A thesis of Chl requires one fewer enzymatic reduction than does bchN A, bchY / synthesis ofBChl. We have conducted statistical DNA sequence bchB bchZ _N,N \ ,Ma hN ,N- analyses of the two reductases involved in Chl and BChl N N- synthesis, reductase and chlorin reduc- tase. Both are three-subunit enzymes in which each subunit COOM° from one reductase shares significant amino acid identity with COOH OOH H M a subunit of the other, indicating that the two enzymes are derived from a common three-subunit ancestral reductase. The xmax = 628 nm Xmax = 663 nm Xmax = 716 nm "chlorophyll iron protein" subunits, encoded by the bchL and bchX genes in the purple bacterium Rhodobacter capsulatus, PChlide Chlorin Bacteriochlorin also share amino acid sequence identity with the nitrogenase FIG. 1. Reduction reactions of Chl and BChl synthesis. Above iron protein, encoded by niff. When nitrogenase iron proteins arrows, names of genes coding for the enzyme complexes that are used as outgroups, the chlorophyll iron protein tree is catalyze these steps in Rhodobacter capsulatus. The reductases' rooted on the chlorin reductase lineage. This rooting suggests substrates, rings B and D, are indicated by arrowheads. All pigments that the last common ancestor of all extant photosynthetic are shown in the monovinyl form. PChlide, monovinyl protochloro- eubacteria contained BChl, not Chl, in its reaction center, and phyllide a; chlorin, a; bacteriochlorin, 2-desacetyl-2- implies that Chl-containing reaction centers were a late inven- vinylbacteriochlorophyllide a. tion unique to the lineage. / iron proteins encoded by niIfH (8, 13). An analysis of the sequences of the PChlide and chlorin reductases shows (i) Chlorophyll (Chl) and bacteriochlorophyll (BChl) are the that they are probably derived from a common three-subunit photochemically active reaction center pigments for all ex- ancestral reductase and (ii) that the so-called "chlorophyll tant photosynthetic organisms except halobacteria, which iron protein" subunits encoded by bchX, bchL, and chlL use an unrelated, carotenoid-based photosystem. During the have been under similar structural constraints as the nitro- synthesis ofboth Chl and BChl, reduction ofthe genase iron proteins. ring system converts a pheoporphyrin, protochlorophyllide Given the strong similarities among chlL, bchL, and bchX (PChlide), into a chlorin (Fig. 1 Left). A second reduction that products and nitrogenase iron proteins, one can use the latter is unique to the synthesis of BChl converts the chlorin into a as an outgroup in establishing the phylogenetic relationships bacteriochlorin (Fig. 1 Right). Because compounds with among the chlorophyll iron proteins. Since Chl-based pho- shorter biosynthetic pathways are often presumed to be more tosynthesis is unique to eubacteria and , it prob- reflective of the ancestral biochemical state than compounds ably originated in eubacteria after the divergence of archaeal that require additional modifications, Chl is thought to have and eubacterial lineages. In contrast, nitrogen fixation occurs preceded BChl in ancient photosynthetic organisms (1, 2). in both archaea (methanogens) and eubacteria, and members The "Chl-first" application of the recapitulation theory is of both of these groups contain at least two types of nitro- part of the "Granick hypothesis," in honor of its first genase iron proteins. Two questions are therefore of central expositor (1). It has become incorporated into several (2-4), importance to our understanding of the evolution of photo- though not all (5), models of the origin and early evolution of synthetic pigment synthesis: (i) Which type of nitrogenase the photosynthetic reaction centers. iron protein gave rise to the chlorophyll iron proteins? (ii) The light-independent protochlorophyllide and chlorin re- Which chlorophyll iron protein(s) was present in the last ductases have recently been sequenced from the purple common ancestor of the Chl-containing cyanobacteria and nonsulfur bacterium Rhodobacter capsulatus. In R. capsu- the latus, the products of three genes are required for each BChl-containing ? reduction: bchL, bchN, and bchB for the PChlide reductase (6, 7) and bchX, bch Y, and bchZ for the chlorin reductase (8). METHODS The corresponding homologs in the PChlide reductase of Published nucleotide sequences were retrieved from the Chl-synthesizing organisms are chlL (frxC), chiN (gidA), and GenBank Release 71 Labo- chlB (Marchantia polymorpha chloroplast open reading or European Molecular Biology frame 513) (6, 9-12). The products of bchX, bchL, and chlL ratory Release 30 data base (Table 1). Alignment of the each share notable amino acid similarity with nitrogenase Abbreviations: Chl, chlorophyll; BChl, bacteriochlorophyll; PChlide, protochlorophyllide. The publication costs of this article were defrayed in part by page charge TTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Molecular, Cell, and Developmental Biology, University of Colo- in accordance with 18 U.S.C. §1734 solely to indicate this fact. rado, Boulder, CO 80309-0347. 7134 Downloaded by guest on September 26, 2021 Evolution: Burke et al. Proc. Natl. Acad. Sci. USA 90 (1993) 7135

Table 1. Sequences used in this study Abbreviation Species Classification* Sourcet Chlorophyll iron proteins bchX Rhodobacter capsulatus a proteobacteria Z11165 bchL Rhodobacter capsulatus a proteobacteria Z11165 PchlL Plectonema boryanum Cyanobacteria MchlL Marchantia polymorpha chloroplast chloroplasts X04465 Nitrogenase iron proteins CpI Clostridium pasteurianum Low-G+C Gram-positive X07472 CpIII Clostridium pasteurianum Low-G+C Gram-positive X07474 MtI (nifH2)* Methanococcus thermolithotrophicus Methanococcales (Archaea) X07500 MtIII (nifHl)* Methanococcus thermolithotrophicus Methanococcales (Archaea) X13830 AzI§ Azotobacter vinelandii y proteobacteria M11579 AzIII Azotobacter vinelandii y proteobacteria M23528 Rc§ Rhodobacter capsulatus a proteobacteria X07866 Rm§ Rhizobium meliloti a proteobacteria J01781 TfV Thiobacillus ferrooxidans 13 proteobacteria M15238 Kp§ Klebsiella pneumoniae y proteobacteria J01740 An§ Anabaena oscillarioides Cyanobacteria VOOOO1 *According to ref. 14. tEuropean Molecular Biology Laboratory or GenBank accession number of the gene encoding the protein. PchlL is from ref. 12. tOld nomenclature from ref. 15. §Used for estimating the transition-to-transversion ratio. translated amino acid sequences was done with ALIGN (16) transition-to-transversion ratio was estimated from six and refined by hand. Nucleotide sequences were then aligned closely related eubacterial type I nifH sequences that were according to the amino acid alignment and output as files in not included in the phylogenetic analysis (Table 1). DNAML PHYLIP format (17) with program MAKEINF (available with was repeatedly run on one tree that is consistent with PHYLIP v3.5). Positions of uncertain homology-i.e., most previously published analyses (21), while the transition-to- gaps and surrounding areas-were excluded from all analyses transversion ratio was changed in increments of 0.05. For (Fig. 2). C or T in first positions of leucine codons and A or first positions, the best ratio was 0.70; for second positions it was 0.95. These ratios were then used in all phylogenetic C in first positions ofarginine codons were converted to their analyses, except the first positions of nitrogenase iron pro- degenerate bases (Y and M, respectively) for all analyses. As teins, for which DNAML had to apply a ratio of 0.80 because a result, only base substitutions that cause amino acid of the skewed base composition. For paired sites tests (22), replacements are used. Such elimination of noise generated option U (user-specified trees) was used. Otherwise, options by silent substitutions is essential for comparisons over large G (global rearrangements) and J (random order of sequence evolutionary distances. addition to tree search) were in effect. Results from paired First and second positions of codons were analyzed sep- sites tests were combined by adding the likelihoods of the arately because their base compositions differ significantly same trees from the separate analyses of first and second (first: 42% G, 29% A, 16% T, 13% C; second: 20%o G, 29% A, positions of codons; standard deviations of the differences 32% T, 19% C). Phylogenetic analyses using maximum between the tree with the highest combined log likelihood and likelihood were done with DNAML version 3.4 (PHYLIP pack- the other trees were computed by taking the square root of age; ref. 18), which implements a six-parameter stochastic the added variances (sum of squares of standard deviations). model of DNA sequence evolution (19) appropriate for se- This is possible because the differences are normally distrib- quence analyses of this kind (20). For analyses of first uted (22). The best tree is considered significantly better at positions of codons, base frequencies as given by MAKEINF 95% confidence (P < 0.05) if the difference in log likelihood were used, with Ys and Ms counted as Y3 T or A, respec- between it and the one it is tested against exceeds 1.96 tively, and Y3 C, consistent with the genetic code. The standard deviations.

****T***** *********TT********* sn **********TTT******10fn** *********TV*** AzI MAM------RQCAIYGUZXTTTQNLVAAL-AEMGKKVMIVGCDPKADSTRLILHSKAQNTIMEMAAEAGTV------EDLELEDVLKAGYGGVKCVESGGPEPGVGCAGRGVITAINFLEEEGAYED-DLDFVFYDVLGDW CpI ------V...... TSG.-HA ... TI .V. ... L.GGL ..KSVLDTLR.E.------V..DSI..E ...... I.IR.S..MQL . T.-. .Y.. MtI L------K.I.F...... VC.IA ...-.DQ....V.H.H.C.SNLRGGQEIP.VLDILR.K.LDKLGLETIIEK.MI.IN.IIYEN..NIY . ..A ..5.K.Y. W.DL.KKMNL.IK.LK.I .... CpIII .T------KI...... Q.TA. .MAHFYD. ..F.H.... GGMP.K.L.D.LRDE.E------.KITT.NIVRV . EDIR ...... DLM.KN . TE-. F. MtIII .SFDEIAPDAKKV .TA.. .AYFFD. .H .. HG.P.D.V.DVLR.E.E------.AVT ..K.R.I.FKDIL ...... VDMMR.LEG.P.-. ..NL.F. MchlL .------KI ...... SC.ISI ..-.RR.. LQI. H.. .FTLTGF-LIP .. IDTLQSKDYHY------.VWP . IYK ...RCD . .A.. PA.A ..G.YV.GETVKL.K.LN.FY--EY.IILF. PchlL ------KL.V...... SC.ISV ..-.KR... LQI. .H.. .FTLTGF-LIP ..IDTLQ.KDYHY------... . IYK...... A..APA.A ..G.YV.GETVKL.K.LN.FD--EY.VILF. bchL .---insert--FSV ...... SS .SF-SLL.. .R.LQI . H...FTLTGR-L.E.VIDILKQVNFHP------.E.RP ..YVTE.FN. .M .. A...PA.T ..G.YV.GQTVKL.KQHHLL.--.T.V.VF . bchX .---insert-T s F LA SH- - R LIT.T.T _ S-TI S -LFC.NCP--T-T.TKKKTG------EUrV CFKs- rAM-L V W- T H -.T._

150 200 250 AzI OGGFAMPIRENKAQEIYIVCSGEMMAMYAANNISKGIV-KYANSGSVRLGGLICNSRNTDREDELIIALANKLGTQMIHFVPRDNVVQR------AEIRRMTVIEYDPKAKQADEYRALARKVVDNKLLVIPNPITMDELEELLMEF ....CpI A...... L -...... QA...K.. ..G.. I . KVAN.Y ..LD.F.KE..S.L...... SPM.TK------. NKQ.. TCE .E...E.E. DA.E.F ... K.M.QER. ..I..QY MtI ...... L.MLGL.EQ ..V.T.SDY...... CR. .S-EFVKR.GSK.....Y.V.GSMDAYDI.NEF.D . .ANIVGK ..NSHLIPE------EGK...... NDEISQV. .E .K.IYE.NEGT. .K.LEHI.IMTIGKKI CpIII ...... G....V...A...V.. C. .L.-...... I. MV.L.R.F.EEF.ASI . ....I..K-- FNKQ... .F.DTCN ..K..GE.. . IIE.EMF ...T.LK .D..AMVVKY MtIII .L.DGL...... T. . A.. AL-.. .EQSG.I.. A. .V.G.K ..MDEFCD.... KL.Y.. I. .K------.FNK.F. .ECN .K.. .T. .KNIDE.DE ..K.T.M... VVKY MchlL .....A.L--.Y.DYCI.ITDNGFD.LF.. .R.AASVR-EK.RTHPL ..A..VG.R---TSKRD ..DKYVEACPMPVLEVL.LIEDIRV------SRVKGK.LF. PchlL .....A.L--.Y.DYCM ..TDNGFD.LF.. .R.AASVR-EK.RTHPL ..A ... .CR---TAKRD. .EKYVDAVPMPILEVL.LIEDIRV------SRVKGK.LF. C-termini not bchL .....A.L--QH.DRAL ..TANDFDSI. .M.R.IAAVQ-AKSVNYK ... AA.CVA.R---S ..TNEVDRYCEAANFKR.AHM.DLDSIR.------SRLKKR.LF. alignable bchX ..XL..ADM KV-V-- G VTNAVEYFUKFTV N GTT--SCTOFAA F AF.VTPILAAT A FFR KSAYQIVGSHATPWGLLE FIG. 2. Alignment of nitrogenase and chlorophyll iron protein sequences. The reference sequence at the top and the numbering are those of Azotobacter vinelandii nifH, for comparison with the recently published crystal structure. "Insert" in the Rhodobacter bchL and bchX sequences indicates charged (13 of 36 positions in each) amino-terminal extensions that are absent from the other sequences (8). Dots indicate identity, hyphens are gaps. Sequences involved in binding MgATP and a [4Fe-4S] cluster are in boldface. Asterisk-delineated regions I-IV mark highly conserved motifs discussed in the text. Codons used for the analyses of nitrogenase iron proteins alone are overlined and those used for analyses of chlorophyll iron proteins alone are underlined. Positions that are both over- and underlined were used in analyses involving all sequences. Abbreviations are as in Table 1. Downloaded by guest on September 26, 2021 7136 Evolution: Burke et al. Proc. Natl. Acad. Sci. USA 90 (1993) Table 2. Percent amino acid identity among homologous side chains of Tyr-159, Ala-160, and Asn-162) of the bound reductase subunits ATP (28), suggesting similar ATP-binding between the two PChlide, Chlorin, subunits. Second, there are many charged residues between chloroplast proteobacterium sites III and IV (Fig. 2). Since this region has been shown to (Marchantia) (Rhodobacter) be involved in ionic interactions of nifH with the ,3 subunit of nitrogenase, nifK (32-35), similar ionic interactions may bind Reductase Subunit chlL chlN chlB bchX bchY bchZ the chlorophyll iron proteins to one or both oftheir respective PChlide, chIL 32 ancillary proteins bchYZ, bchNB, or chlNB. Third, in all chloroplast chlN <20* nitrogenase iron proteins and in bchX, position 100 is occu- (Marchantia) chlB 20 pied by an arginine residue. In both bchL and chlL, it is PChlide, bchL 50 34 tyrosine. In site-directed mutations ofAzotobacter vinelandii proteobacterium bchN 36 22* nifH, tyrosine is the only other amino acid at this position that (Rhodobacter) bchB 34 24 still allows a significant amount of electron transfer activity *These two alignments contain a large number of gaps, so that exact (35). Finally, the structural compatibility method of Bowie et scores cannot be obtained with certainty. al. (36), which assesses the likelihood of burying and expos- ing residues in a given sequence, predicts all chlorophyll iron RESULTS AND DISCUSSION proteins to be compatible with adopting a nitrogenase iron protein-like structure (D. Eisenberg, personal communica- Chlorin Reductase and PChlide Reductase Share Common tion). Structural Features with Each Other and with Nitrogenase More than half (54%) of the matches in two-way compar- Reductase. Each of the three subunits from the chlorin isons among bchZ, bchB, and chlB are shared by all three reductase can be aligned with one from the PChlide reduc- proteins. Conservation between bchN and chlN is also strik- tase. All three PChlide reductase genes from Rhodobacter ing (36%; Table 2), and there is a cluster ofthree-way matches capsulatus are more similar to those in cyanobacteria and when bchY is added to the alignment (6). All of these chloroplasts than they are to the respective subunits of the three-way matches are also conserved in chlN from March- chlorin reductase (Table 2). The strongest conservation be- antia polymorpha (37) and from the cyanobacterium Syn- tween PChlide and chlorin reductases is among bchX, bchL, echocystis sp. 6803 (31). chlN shares 19% identity with nifK and chlL (Fig. 2; ref. 8). These also share notable sequence of nitrogenase (31), but a similar alignment between chlB and identity with the nitrogenase iron proteins. The greatest nifD is elusive. It is intriguing to speculate that all three conservation is in sites known to be important for binding the subunits of the PChlide and chlorin reductases are derived y-phosphate ofMgATP (23, 24), for binding a [4Fe-4S] cluster from homologous nitrogenase genes, though three-dimen- (25-28), and in those postulated to have a role in catalyzing sional structural information from the other two subunits will ATP hydrolysis (Asp-39 and Asp-43; ref. 28). All of these probably be required to prove this hypothesis. imply mechanistic similarities between the chlorophyll and Ancient Duplication of Genes for Nitrogenase Iron Proteins. nitrogenase iron proteins (8, 11, 29-31). In eubacteria, there are three types of nitrogenase iron There is also evidence for structural similarities. First, proteins, whose catalytic subunits contain both molybdenum when conserved regions I-IV (Fig. 2) are mapped onto the and iron (type I, the main nitrogenase of eubacteria), both Azotobacter vinelandii nifH crystal structure (28), a number vanadium and iron (type II), or iron only (type III) (38). Type of the matches are located within the subunit interiors and at II sequences have been shown to be a recent derivation from the subunit-subunit interface of the homodimer (Fig. 3). type I sequences within the proteobacteria (39). We therefore Some of these residues form salt bridge (Lys-15 Asp-125 and focus our attention on type I and III sequences. Lys-41-Asp-129) and van der Waals contacts across the Two homologs of nifH have been cloned and sequenced subunit-subunit interface (28), suggesting similar interho- from the nitrogen-fixing archaebacterium Methanococcus modimeric interactions. Others contact the ribose (Lys-41 thermolithotrophicus (15, 40). In phylogenetic comparisons and Asp-129) or adenine (main chain ofresidues 128-130 and of amino acid sequences, Methanococcus nifHl groups with eubacterial type III sequences to the exclusion of eubacterial type I-associated nifHs and Methanococcus nifH2 (39). To test whether this association is confirmed by maximum likelihood analysis of DNA sequences (18), we conducted four-taxon paired sites tests (22) of the two sequences from Methanococcus thermolithotrophicus with sequences of nifH type I and III from Clostridium pasteurianum. For both first and second positions of codons, the same grouping as that found in the previous analysis (39) is supported. When the likelihoods ofthe trees from first and second positions are combined, the best tree is more than one standard deviation better than the two alternative trees (P < 0.25 and P < 0.10). Similar results were obtained when the type I and III se- quences from Clostridium pasteurianum were exchanged for those from Azotobacter vinelandii. In the absence of statis- tically significant resolution, the bias in the data is expected to go toward grouping together the Methanococcus se- quences on the one hand and the eubacterial sequences on the FIG. 3. Ribbon diagram derived from the crystal structure of other, because they have resided in the same genomes for Azotobacter vinelandii nitrogenase iron protein, nifH (28). Residues three billion years or more. As this is not the case, we prefer identical in all of the proteins in Fig. 2 are highlighted in light blue, the that the of iron positions with conservative replacements are in red, and noncon- hypothesis duplication nitrogenase pro- served positions are in purple. Roman numerals designate conserved teins into types I and III preceded the divergence of eubac- regions I-IV of Fig. 2. The bound cofactors ([4Fe-4S] cluster, top, teria and methanogenic archaea. We therefore refer to Meth- and ATP, bottom) are shown as ball and stick models in the center anococcus nifHl sequences as type III, and to nifH2 as type of the homodimer. (Photo courtesy of Douglas Rees.) I. In a similar analysis of the four available chlorophyll iron Downloaded by guest on September 26, 2021 Evolution: Burke et al. Proc. Natl. Acad. Sci. USA 90 (1993) 7137 rooting of the chlorophyll iron protein tree is always on the bchX lineage, no matter where the root inserts on the nitrogenase iron protein tree (Fig. 4). All alternative rootings are ruled out at statistical significance in the four-by-four tests (P < 0.05). This result is notable because it indicates that the last common ancestor of cyanobacteria and purple bac- teria contained both a PChlide reductase and a chlorin reductase, and was thus capable of BChl synthesis. Because BChls absorb at longer wavelengths than do Chls (Fig. 1), the Cp Mt Cp Mt bchX bchL MchlL PchlL BChl made by this organism must have been in the photo- synthetic reaction center to permit downhill energy transfer Type III Type I from the antennae pigments. Nitrogenase iron proteins * Chlorophyll iron proteins Enzymatic Activity of the Ancestral Reductase. The central theme of the "recapitulation theory" and of the Granick FIG. 4. Results of the phylogenetic analyses. Names of se- hypothesis is that biosynthetic pathways are extended one quences are the same as in Table 1 and Fig. 2. *, Gene duplication evolutionary step at a time. As soon as a plausible mechanism oftype I and III sequences, assumed to have occurred before the last is proposed by which a particular pathway could leap ahead common ancestor of eubacteria and archaea lived (o). *, Gene two or more biochemical steps in a single evolutionary step, duplication giving rise to chlorophyll iron proteins. Uncertainty in then both the recapitulation theory and the Granick hypoth- this region of the tree is represented by the trifurcation. O, Gene esis can be called into question for that specific case. Such a duplication ofthe ancestral chlorophyll iron protein. e, Last common ancestor of mechanism is apparent in the common ancestry of the cyanobacteria and proteobacteria. PChlide and chlorin reductases: The first pheoporphyrin- proteins (Table 1), the chloroplast and cyanobacterial se- reducing chlorophyll iron protein system may have reduced quences PchlL and MchlL group together to the exclusion of both rings B and D (Fig. 1, arrowheads), thereby reducing the bchX and bchL at statistical significance (P < 0.05). pheoporphyrin directly to a bacteriochlorin with a single The Four-by-Four Test. Given the four-taxon trees for enzyme system. The chemistries of the two ring reductions nitrogenase iron proteins on one hand and for chlorophyll are nearly identical: the outer double bond of a five- iron proteins on the other, we adopted a four-by-four strategy membered ring flanked by two other conjugated rings is of conducting paired sites tests. Keeping the two reduced to a single bond (Fig. 1). Invention of this reductase four-taxon would then have advanced the synthesis of photosynthetic topologies constant, we tested every possible rooting of the reaction center pigments chlorophyll tree on from a pheoporphyrin such as iron protein the nitrogenase iron protein PChlide (2, 42) to a bacteriochlorin in a single evolutionary tree. Since there are five branches in each tree, 25 specific step. Only later, when a predecessor to modern cyanobac- trees were tested. This approach maximizes the amount of teria acquired the ability to synthesize singly reduced pig- available data by allowing construction of subtrees of closely ments, did Chl appear, perhaps first serving as an antennae related sequences that utilizes more homologous sites be- pigment [such as the (BChlc, d, and e) in the cause alignments are less problematic. (There were 262 antennae ofmodern ] and later as a component of unambiguously homologous codons for the nitrogenase iron the reaction center (as in modern cyanobacteria and chloro- proteins, 207 for the chlorophyll iron proteins, and 200 when plasts). both were combined; Fig. 2.) It also bypasses computation- This model resolves the apparent conflict between the ally intensive maximum likelihood analyses of bootstrapped Granick hypothesis and the observed phylogenetic distribu- data sets and instead relies on the paired sites test (22) for tion of pigments used in photosynthetic reaction centers. assessing statistical significance. Note that there is no legit- There are four commonly recognized tetrapyrrole reaction imate way of combining bootstrap values from two analyses, center pigments: BChla, BChlb, and BChlg, and Chla (43). whereas results from paired sites tests are easily combined BChla is in the reaction center and antennae of the green (see Methods). As a control, the best trees were also searched gliding bacteria (Chloroflexaceae), most purple bacteria (pro- for in separate analyses with all eight sequences, by both teobacteria), including Rhodobacter capsulatus, and the maximum likelihood and parsimony. For both first and (Chlorobiaceae). BChlg (found in He- second positions, the best trees found by these searches were liobacteriaceae) and BChlb (in the proteobacterium Rhodo- the same as those obtained by the four-by-four tests. pseudomonas viridis) are in a redox state intermediate be- Duplication ofType I, not Type III, Nitrogenase Iron Protein tween chlorins and bacteriochlorins, in that the 4-ethyl group Gave Rise to the Ancestral Chlorophyll Iron Protein. Associ- of BChla is replaced by an ethylidene. Their rings are three ation of the chlorophyll iron proteins with either of the two electrons reduced from PChlide, as opposed to two electrons type III nitrogenase iron proteins is ruled out at statistical in chlorins and four electrons in bacteriochlorins. The only significance in the four-by-four tests (P < 0.05). In the best group that uses a Chl in place of a BChl in its photosynthetic tree for first positions of codons, the root inserts on the reaction centers is the cyanobacteria/chloroplasts, which is Methanococcus type I lineage, whereas in the best tree for also the only group whose members couple two distinct second positions, it inserts on the Clostridium type I lineage. photosystems to oxidize water and produce . A pos- When the likelihoods from both analyses are combined, the sible exception to this rule is 8-hydroxychlorophyll, which root falls on the Methanococcus type I lineage, as in the tree has recently been reported to be in the reaction centers of for first positions. Note that this implies that there might be (44). The broad phylogenetic distribution ofthe undiscovered iron proteins in extant archaebacteria. On the more reduced photopigments among four of the five photo- other hand, second positions are known to be more reliable synthetic bacterial phyla is consistent with the ancestral over long evolutionary time periods (41). We therefore prefer pigment's having been more reduced than a chlorin, in the tree in which the chlorophyll iron proteins originate agreement with the phylogenetic analysis above. The pres- within eubacteria on the lineage to the Clostridium type I ence of reduced pigments, particularly BChla, in both pho- sequence, but we emphasize that statistically significant tosystem I (PSI)-like (heliobacteria and Chlorobiaceae) and distinction between the alternatives is elusive (Fig. 4). photosystem II (PSII)-like (proteobacteria and Chloroflex- Duplication of Chlorophyll Iron Proteins Before the Diver- aceae) reaction centers (2, 45-48) may even suggest that sification of Extant Photosynthesizing Eubacteria. The best there was an ancestral bacterium with both PSI and PSII, Downloaded by guest on September 26, 2021 7138 Evolution: Burke et al. Proc. Natl. Acad. Sci. USA 90 (1993) similar to that postulated by Pierson and Olson (2, 3) but with 12. Fujita, Y., Takahashi, Y., Chuganji, M. & Matsubara, H. (1992) BChl instead of Chl in its reaction centers. Subsequent Plant Cell Physiol. 33, 81-92. 13. Hearst, J. E., Alberti, M. & Doolittle, R. F. (1985) Cell 40, radiation and loss of one type of reaction center or another 219-220. could then account for all extant forms with fewer changes in 14. Woese, C. R. (1987) Microbiol. Rev. 51, 221-271. pigment types than are needed in the scheme proposed 15. Souillard, N. & Sibold, L. (1989) Mol. Microbiol. 3, 541-551. previously (2, 3). 16. Hein, J. (1989) Mol. Biol. Evol. 6, 649-668. In an alternative scenario, Chl was used in reaction centers 17. Felsenstein, J. (1989) Cladistics 5, 164-166. before BChl. Since the Chl-containing cyanobacteria are 18. Felsenstein, J. (1981) J. Mol. Evol. 17, 368-376. derived from organisms that made BChl, a Chl-first scenario 19. Hasegawa, M., Kishino, H. & Yano, T. (1985) J. Mol. Evol. 22, the 160-174. requires that BChl-containing organisms displaced all of 20. Nguyen, T. (1991) Ph.D. thesis (Univ. ofCalifornia, Berkeley). organisms with Chl in their reaction centers prior to the 21. Normand, P., Gouy, M., Cournoyer, B. & Simonet, P. (1992) radiation of all extant photosynthesizers. This is less parsi- Mol. Biol. Evol. 9, 495-506. monious than a BChl-first scenario, in that it invokes a switch 22. Kishino, H. & Hasegawa, M. (1989) J. Mol. Evol. 29, 170-179. in reaction center pigments in addition to the switch from 23. Robson, R. L. (1984) FEBS Lett. 173, 394-398. BChl to Chl in an ancestor of the cyanobacteria. 24. Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. Proposed History of the Iron Protein Family. The ancestral (1982) EMBO J. 1, 945-951. duplicated and diverged into nitrogenase type I 25. Howard, J. B., Davis, R., Moldenhauer, B., Cash, V. L. & iron protein Dean, D. (1989) J. Biol. Chem. 264, 11270-11274. and type III prior to the speciation event that separated the 26. Orme-Johnson, W. H. (1985) Annu. Rev. Biophys. Biophys. eubacteria from the methanogenic archaebacteria. Subse- Chem. 14, 419-459. quent to this speciation, there was a duplication of the 27. Mortenson, L. E. & Thornley, R. N. F. (1979) Annu. Rev. nitrogenase iron protein gene in the eubacterial line, possibly Biochem. 48, 387-418. accompanied by a duplication of nifK and nipD. One of the 28. Georgiadis, M. M., Komiya, H., Chakrabarti, P., Woo, D., copies diverged sufficiently to enable it to interact with a Kornuc, J. J. & Rees, D. C. (1992) Science 257, 1653-1659. PChlide-binding protein (or even directly with the PChlide), 29. Burke, D. H., Alberti, M. & Hearst, J. (1991) Photochem. Photobiol. 53, Suppl., 85S-86S. thereby forming the first PChlide reductase. This enzyme 30. Fujita, Y., Takahashi, Y., Kohchi, T., Ozeki, H., Ohyama, K. reduced its substrate twice to form a bacteriochlorin. Sub- & Matsubara, H. (1989) Plant Mol. Biol. 13, 551-561. sequent duplication of each of the three subunits of this 31. Ogura, Y., Takemura, M., Oda, K., Yamato, K., Ohta, E., reductase allowed the two copies to specialize toward reduc- Fukuzawa, H. & Ohyama, K. (1992) Biosci. Biotech. Biochem. tion of PChlides on one hand and chlorins on the other. Chl 56, 788-793. appeared later during the early evolution of cyanobacteria. 32. Deits, T. L. & Howard, J. B. (1990) J. Biol. Chem. 265, Reduced reaction center pigments and the modern oxygen- 3859-3867. 33. Willing, A. H. & Howard, J. B. (1990) J. Biol. Chem. 265, rich atmosphere are therefore partly consequences ofancient 6596-6599. duplications of the type I nitrogenase iron protein. 34. Willing, A. H., Georgiadis, M. M., Rees, D. 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