Proc. Natl. Acad. Sci. USAVol. 90, pp. 7134-7138, August 1993Evolution

Early evolution of photosynthesis: Clues from nitrogenase andchlorophyll iron proteins

(gene duplication/bacteriochlorophyll/purple bacteria/protochlorophyilide reductase/chlorin 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, 401Barker Hall, Berkeley, CA 94720

Communicated by Randy Schekman, March 24, 1993

ABSTRACT Chlorophyll (Chl) is often viewed as havingpreceded bacteriochlorophyll (BChl) as the primary photore-ceptor pigment in early photosynthetic systems because syn-thesis of Chl requires one fewer enzymatic reduction than doessynthesis of BChl. We have conducted statistical DNA sequenceanalyses of the two reductases involved in Chl and BChlsynthesis, protochlorophyllide reductase and chlorin reduc-tase. Both are three-subunit enzymes in which each subunitfrom one reductase shares significant amino acid identity witha subunit of the other, indicating that the two enzymes arederived from a common three-subunit ancestral reductase. The"chlorophyll iron protein" subunits, encoded by the bchL andbchX genes in the purple bacterium Rhodobacter capsulatus,also share amino acid sequence identity with the nitrogenaseiron protein, encoded by niff. When nitrogenase iron proteinsare used as outgroups, the chlorophyll iron protein tree isrooted on the chlorin reductase lineage. This rooting suggeststhat the last common ancestor of all extant photosyntheticeubacteria contained BChl, not Chl, in its reaction center, andimplies that Chl-containing reaction centers were a late inven-tion unique to the cyanobacteria/chloroplast lineage.

Chlorophyll (Chl) and bacteriochlorophyll (BChl) are thephotochemically active reaction center pigments for all ex-tant photosynthetic organisms except halobacteria, whichuse an unrelated, carotenoid-based photosystem. During thesynthesis of both Chl and BChl, reduction of the tetrapyrrolering system converts a pheoporphyrin, protochlorophyllide(PChlide), into a chlorin (Fig. 1 Left). A second reduction thatis unique to the synthesis of BChl converts the chlorin into abacteriochlorin (Fig. 1 Right). Because compounds withshorter biosynthetic pathways are often presumed to be morereflective of the ancestral biochemical state than compoundsthat require additional modifications, Chl is thought to havepreceded BChl in ancient photosynthetic organisms (1, 2).The "Chl-first" application of the recapitulation theory ispart of the "Granick hypothesis," in honor of its firstexpositor (1). It has become incorporated into several (2-4),though not all (5), models of the origin and early evolution ofthe photosynthetic reaction centers.The light-independent protochlorophyllide and chlorin re-

ductases have recently been sequenced from the purplenonsulfur bacterium Rhodobacter capsulatus. In R. capsu-latus, the products of three genes are required for eachreduction: bchL, bchN, and bchB for the PChlide reductase(6, 7) and bchX, bch Y, and bchZ for the chlorin reductase (8).The corresponding homologs in the PChlide reductase ofChl-synthesizing organisms are chlL (frxC), chiN (gidA), andchlB (Marchantia polymorpha chloroplast open readingframe 513) (6, 9-12). The products of bchX, bchL, and chlLeach share notable amino acid similarity with nitrogenase

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\ ,MaN N-

COOH

xmax = 628 nm

PChlide

bchLbchNbchB

to chlorophyll

A,hN ,N-

OOHCOOM°

Xmax = 663 nm

Chlorin

to bacteriochlorophyll

bchX AbchY /bchZ _N,N

H M

Xmax = 716 nm

Bacteriochlorin

FIG. 1. Reduction reactions of Chl and BChl synthesis. Abovearrows, names of genes coding for the enzyme complexes thatcatalyze these steps in Rhodobacter capsulatus. The reductases'substrates, rings B and D, are indicated by arrowheads. All pigmentsare shown in the monovinyl form. PChlide, monovinyl protochloro-phyllide a; chlorin, chlorophyllide a; bacteriochlorin, 2-desacetyl-2-vinylbacteriochlorophyllide a.

iron proteins encoded by niIfH (8, 13). An analysis of thesequences of the PChlide and chlorin reductases shows (i)that they are probably derived from a common three-subunitancestral reductase and (ii) that the so-called "chlorophylliron protein" subunits encoded by bchX, bchL, and chlLhave been under similar structural constraints as the nitro-genase iron proteins.Given the strong similarities among chlL, bchL, and bchX

products and nitrogenase iron proteins, one can use the latteras an outgroup in establishing the phylogenetic relationshipsamong the chlorophyll iron proteins. Since Chl-based pho-tosynthesis is unique to eubacteria and chloroplasts, it prob-ably originated in eubacteria after the divergence of archaealand eubacterial lineages. In contrast, nitrogen fixation occursin both archaea (methanogens) and eubacteria, and membersof both of these groups contain at least two types of nitro-genase iron proteins. Two questions are therefore of centralimportance to our understanding of the evolution of photo-synthetic pigment synthesis: (i) Which type of nitrogenaseiron protein gave rise to the chlorophyll iron proteins? (ii)Which chlorophyll iron protein(s) was present in the lastcommon ancestor of the Chl-containing cyanobacteria andthe BChl-containing purple bacteria?

METHODSPublished nucleotide sequences were retrieved from theGenBank Release 71 or European Molecular Biology Labo-ratory Release 30 data base (Table 1). Alignment of the

Abbreviations: Chl, chlorophyll; BChl, bacteriochlorophyll; PChlide,protochlorophyllide.TTo whom reprint requests should be addressed at: Department ofMolecular, Cell, and Developmental Biology, University of Colo-rado, Boulder, CO 80309-0347.

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Table 1. Sequences used in this studyAbbreviation Species Classification* Sourcet

Chlorophyll iron proteinsbchX Rhodobacter capsulatus a proteobacteria Z11165bchL Rhodobacter capsulatus a proteobacteria Z11165PchlL Plectonema boryanum CyanobacteriaMchlL Marchantia polymorpha chloroplast Plant chloroplasts X04465

Nitrogenase iron proteinsCpI Clostridium pasteurianum Low-G+C Gram-positive X07472CpIII Clostridium pasteurianum Low-G+C Gram-positive X07474MtI (nifH2)* Methanococcus thermolithotrophicus Methanococcales (Archaea) X07500MtIII (nifHl)* Methanococcus thermolithotrophicus Methanococcales (Archaea) X13830AzI§ Azotobacter vinelandii y proteobacteria M11579AzIII Azotobacter vinelandii y proteobacteria M23528Rc§ Rhodobacter capsulatus a proteobacteria X07866Rm§ Rhizobium meliloti a proteobacteria J01781TfV Thiobacillus ferrooxidans 13 proteobacteria M15238Kp§ Klebsiella pneumoniae y proteobacteria J01740An§ Anabaena oscillarioides Cyanobacteria VOOOO1

*According to ref. 14.tEuropean Molecular Biology Laboratory or GenBank accession number of the gene encoding the protein. PchlL is fromref. 12.tOld nomenclature from ref. 15.§Used for estimating the transition-to-transversion ratio.

translated amino acid sequences was done with ALIGN (16)and refined by hand. Nucleotide sequences were then alignedaccording to the amino acid alignment and output as files inPHYLIP format (17) with program MAKEINF (available withPHYLIP v3.5). Positions of uncertain homology-i.e., mostgaps and surrounding areas-were excluded from all analyses(Fig. 2). C or T in first positions of leucine codons and A orC in first positions of arginine codons were converted to theirdegenerate bases (Y and M, respectively) for all analyses. Asa result, only base substitutions that cause amino acidreplacements are used. Such elimination of noise generatedby silent substitutions is essential for comparisons over largeevolutionary distances.

First and second positions of codons were analyzed sep-arately because their base compositions differ significantly(first: 42% G, 29% A, 16% T, 13% C; second: 20%o G, 29% A,32% T, 19% C). Phylogenetic analyses using maximumlikelihood were done with DNAML version 3.4 (PHYLIP pack-age; ref. 18), which implements a six-parameter stochasticmodel of DNA sequence evolution (19) appropriate for se-quence analyses of this kind (20). For analyses of firstpositions of codons, base frequencies as given by MAKEINFwere used, with Ys and Ms counted as Y3 T or A, respec-tively, and Y3 C, consistent with the genetic code. The

AzICpIMtICpIIIMtIIIMchlLPchlLbchLbchX

AzICpIMtICpIIIMtIIIMchlLPchlLbchLbchX

transition-to-transversion ratio was estimated from sixclosely related eubacterial type I nifH sequences that werenot included in the phylogenetic analysis (Table 1). DNAMLwas repeatedly run on one tree that is consistent withpreviously published analyses (21), while the transition-to-transversion ratio was changed in increments of 0.05. Forfirst positions, the best ratio was 0.70; for second positions itwas 0.95. These ratios were then used in all phylogeneticanalyses, except the first positions of nitrogenase iron pro-teins, for which DNAML had to apply a ratio of 0.80 becauseof the skewed base composition. For paired sites tests (22),option U (user-specified trees) was used. Otherwise, optionsG (global rearrangements) and J (random order of sequenceaddition to tree search) were in effect. Results from pairedsites tests were combined by adding the likelihoods of thesame trees from the separate analyses of first and secondpositions of codons; standard deviations of the differencesbetween the tree with the highest combined log likelihood andthe other trees were computed by taking the square root ofthe added variances (sum of squares of standard deviations).This is possible because the differences are normally distrib-uted (22). The best tree is considered significantly better at95% confidence (P < 0.05) if the difference in log likelihoodbetween it and the one it is tested against exceeds 1.96standard deviations.

*****T**** *********TT********* sn **********TTT******10fn** *********TV***MAM-------RQCAIYGUZXTTTQNLVAAL-AEMGKKVMIVGCDPKADSTRLILHSKAQNTIMEMAAEAGTV----------EDLELEDVLKAGYGGVKCVESGGPEPGVGCAGRGVITAINFLEEEGAYED-DLDFVFYDVLGDW--------- V. .......TSG.-HA ...TI.V. ...L.GGL ..KSVLDTLR.E.------------ V..DSI..E .... ................I.IR.S..MQL . T.-. ..Y.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.....T--------KI........... Q.TA. .MAHFYD. ..F.H.... GGMP.K.L.D.LRDE.E-----------.KITT.NIVRV . EDIR ...... DLM.KN . TE-. F..SFDEIAPDAKKV .TA.. .AYFFD. .H .. HG.P.D.V.DVLR.E.E-----------.AVT ..K.R.I.FKDIL .......... VDMMR.LEG.P.-. ..NL.F.

.----------KI ........... SC.ISI ..-.RR.. LQI. H.. .FTLTGF-LIP .. IDTLQSKDYHY---------- .VWP . IYK ...RCD . .A.. PA.A ..G.YV.GETVKL.K.LN.FY--EY.IILF.----------KL.V...........SC.ISV ..-.KR... LQI. .H.. .FTLTGF-LIP ..IDTLQ.KDYHY--------- ... . IYK..... ...A..APA.A ..G.YV.GETVKL.K.LN.FD--EY.VILF..---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..---insert-T s F LA H-S - R LIT.T.T _ S-TI S -LFC.NCP--T-T.TKKKTG-------- -EUrV CFKs- rAM-L V W- T H -.T._

150 200 250OGGFAMPIRENKAQEIYIVCSGEMMAMYAANNISKGIV-KYANSGSVRLGGLICNSRNTDREDELIIALANKLGTQMIHFVPRDNVVQR----------AEIRRMTVIEYDPKAKQADEYRALARKVVDNKLLVIPNPITMDELEELLMEF

.... 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.... ..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

........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......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

.....A.L--.Y.DYCI.ITDNGFD.LF.. .R.AASVR-EK.RTHPL ..A..VG.R---TSKRD ..DKYVEACPMPVLEVL.LIEDIRV----------SRVKGK.LF.

.....A.L--.Y.DYCM ..TDNGFD.LF.. .R.AASVR-EK.RTHPL ..A ... .CR---TAKRD. .EKYVDAVPMPILEVL.LIEDIRV----------SRVKGK.LF. C-termini not

.....A.L--QH.DRAL ..TANDFDSI. .M.R.IAAVQ-AKSVNYK ... AA.CVA.R---S ..TNEVDRYCEAANFKR.AHM.DLDSIR.-----------SRLKKR.LF. alignable..XL..ADM KV-V-- G FTVVTNAVEYFUK 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 thoseof Azotobacter vinelandii nifH, for comparison with the recently published crystal structure. "Insert" in the Rhodobacter bchL and bchXsequences indicates charged (13 of 36 positions in each) amino-terminal extensions that are absent from the other sequences (8). Dots indicateidentity, hyphens are gaps. Sequences involved in binding MgATP and a [4Fe-4S] cluster are in boldface. Asterisk-delineated regions I-IV markhighly conserved motifs discussed in the text. Codons used for the analyses of nitrogenase iron proteins alone are overlined and those used foranalyses of chlorophyll iron proteins alone are underlined. Positions that are both over- and underlined were used in analyses involving allsequences. Abbreviations are as in Table 1.

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Table 2. Percent amino acid identity among homologousreductase subunits

PChlide, Chlorin,chloroplast proteobacterium(Marchantia) (Rhodobacter)

Reductase Subunit chlL chlN chlB bchX bchY bchZPChlide, chIL 32

chloroplast chlN <20*(Marchantia) chlB 20

PChlide, bchL 50 34proteobacterium bchN 36 22*(Rhodobacter) bchB 34 24

*These two alignments contain a large number of gaps, so that exactscores cannot be obtained with certainty.

RESULTS AND DISCUSSIONChlorin Reductase and PChlide Reductase Share Common

Structural Features with Each Other and with NitrogenaseReductase. Each of the three subunits from the chlorinreductase can be aligned with one from the PChlide reduc-tase. All three PChlide reductase genes from Rhodobactercapsulatus are more similar to those in cyanobacteria andchloroplasts than they are to the respective subunits of thechlorin reductase (Table 2). The strongest conservation be-tween PChlide and chlorin reductases is among bchX, bchL,and chlL (Fig. 2; ref. 8). These also share notable sequenceidentity with the nitrogenase iron proteins. The greatestconservation is in sites known to be important for binding they-phosphate ofMgATP (23, 24), for binding a [4Fe-4S] cluster(25-28), and in those postulated to have a role in catalyzingATP hydrolysis (Asp-39 and Asp-43; ref. 28). All of theseimply mechanistic similarities between the chlorophyll andnitrogenase iron proteins (8, 11, 29-31).There is also evidence for structural similarities. First,

when conserved regions I-IV (Fig. 2) are mapped onto theAzotobacter vinelandii nifH crystal structure (28), a numberof the matches are located within the subunit interiors and atthe subunit-subunit interface of the homodimer (Fig. 3).Some of these residues form salt bridge (Lys-15 Asp-125 andLys-41-Asp-129) and van der Waals contacts across thesubunit-subunit interface (28), suggesting similar interho-modimeric interactions. Others contact the ribose (Lys-41and Asp-129) or adenine (main chain of residues 128-130 and

FIG. 3. Ribbon diagram derived from the crystal structure ofAzotobacter vinelandii nitrogenase iron protein, nifH (28). Residuesidentical in all of the proteins in Fig. 2 are highlighted in light blue,positions with conservative replacements are in red, and noncon-served positions are in purple. Roman numerals designate conservedregions I-IV of Fig. 2. The bound cofactors ([4Fe-4S] cluster, top,and ATP, bottom) are shown as ball and stick models in the centerof the homodimer. (Photo courtesy of Douglas Rees.)

side chains of Tyr-159, Ala-160, and Asn-162) of the boundATP (28), suggesting similar ATP-binding between the twosubunits. Second, there are many charged residues betweensites III and IV (Fig. 2). Since this region has been shown tobe involved in ionic interactions of nifH with the ,3 subunit ofnitrogenase, nifK (32-35), similar ionic interactions may bindthe chlorophyll iron proteins to one or both oftheir respectiveancillary proteins bchYZ, bchNB, or chlNB. Third, in allnitrogenase iron proteins and in bchX, position 100 is occu-pied by an arginine residue. In both bchL and chlL, it istyrosine. In site-directed mutations ofAzotobacter vinelandiinifH, tyrosine is the only other amino acid at this position thatstill allows a significant amount of electron transfer activity(35). Finally, the structural compatibility method of Bowie etal. (36), which assesses the likelihood of burying and expos-ing residues in a given sequence, predicts all chlorophyll ironproteins to be compatible with adopting a nitrogenase ironprotein-like structure (D. Eisenberg, personal communica-tion).More than half (54%) of the matches in two-way compar-

isons among bchZ, bchB, and chlB are shared by all threeproteins. Conservation between bchN and chlN is also strik-ing (36%; Table 2), and there is a cluster ofthree-way matcheswhen bchY is added to the alignment (6). All of thesethree-way matches are also conserved in chlN from March-antia polymorpha (37) and from the cyanobacterium Syn-echocystis sp. 6803 (31). chlN shares 19% identity with nifKof nitrogenase (31), but a similar alignment between chlB andnifD is elusive. It is intriguing to speculate that all threesubunits of the PChlide and chlorin reductases are derivedfrom homologous nitrogenase genes, though three-dimen-sional structural information from the other two subunits willprobably be required to prove this hypothesis.

Ancient Duplication of Genes for Nitrogenase Iron Proteins.In eubacteria, there are three types of nitrogenase ironproteins, whose catalytic subunits contain both molybdenumand iron (type I, the main nitrogenase of eubacteria), bothvanadium and iron (type II), or iron only (type III) (38). TypeII sequences have been shown to be a recent derivation fromtype I sequences within the proteobacteria (39). We thereforefocus our attention on type I and III sequences.Two homologs of nifH have been cloned and sequenced

from the nitrogen-fixing archaebacterium Methanococcusthermolithotrophicus (15, 40). In phylogenetic comparisonsof amino acid sequences, Methanococcus nifHl groups witheubacterial type III sequences to the exclusion of eubacterialtype I-associated nifHs and Methanococcus nifH2 (39). Totest whether this association is confirmed by maximumlikelihood analysis of DNA sequences (18), we conductedfour-taxon paired sites tests (22) of the two sequences fromMethanococcus thermolithotrophicus with sequences ofnifH type I and III from Clostridium pasteurianum. For bothfirst and second positions of codons, the same grouping asthat found in the previous analysis (39) is supported. Whenthe likelihoods of the trees from first and second positions arecombined, the best tree is more than one standard deviationbetter 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 forthose from Azotobacter vinelandii. In the absence of statis-tically significant resolution, the bias in the data is expectedto go toward grouping together the Methanococcus se-quences on the one hand and the eubacterial sequences on theother, because they have resided in the same genomes forthree billion years or more. As this is not the case, we preferthe hypothesis that the duplication of nitrogenase iron pro-teins into types I and III preceded the divergence of eubac-teria and methanogenic archaea. We therefore refer to Meth-anococcus nifHl sequences as type III, and to nifH2 as typeI. In a similar analysis of the four available chlorophyll iron

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Cp Mt Cp Mt bchX bchL MchlL PchlL

Type III Type I

Nitrogenase iron proteins * Chlorophyll iron proteins

FIG. 4. Results of the phylogenetic analyses. Names of se-quences are the same as in Table 1 and Fig. 2. *, Gene duplicationof type I and III sequences, assumed to have occurred before the lastcommon ancestor of eubacteria and archaea lived (o). *, Geneduplication giving rise to chlorophyll iron proteins. Uncertainty inthis region of the tree is represented by the trifurcation. O, Geneduplication ofthe ancestral chlorophyll iron protein. e, Last commonancestor of cyanobacteria and proteobacteria.

proteins (Table 1), the chloroplast and cyanobacterial se-quences PchlL and MchlL group together to the exclusion ofbchX and bchL at statistical significance (P < 0.05).The Four-by-Four Test. Given the four-taxon trees for

nitrogenase iron proteins on one hand and for chlorophylliron proteins on the other, we adopted a four-by-four strategyof conducting paired sites tests. Keeping the two four-taxontopologies constant, we tested every possible rooting of thechlorophyll iron protein tree on the nitrogenase iron proteintree. Since there are five branches in each tree, 25 specifictrees were tested. This approach maximizes the amount ofavailable data by allowing construction of subtrees of closelyrelated sequences that utilizes more homologous sites be-cause alignments are less problematic. (There were 262unambiguously homologous codons for the nitrogenase ironproteins, 207 for the chlorophyll iron proteins, and 200 whenboth were combined; Fig. 2.) It also bypasses computation-ally intensive maximum likelihood analyses of bootstrappeddata sets and instead relies on the paired sites test (22) forassessing statistical significance. Note that there is no legit-imate way of combining bootstrap values from two analyses,whereas results from paired sites tests are easily combined(see Methods). As a control, the best trees were also searchedfor in separate analyses with all eight sequences, by bothmaximum likelihood and parsimony. For both first andsecond positions, the best trees found by these searches werethe same as those obtained by the four-by-four tests.

Duplication ofType I, not Type III, Nitrogenase Iron ProteinGave Rise to the Ancestral Chlorophyll Iron Protein. Associ-ation of the chlorophyll iron proteins with either of the twotype III nitrogenase iron proteins is ruled out at statisticalsignificance in the four-by-four tests (P < 0.05). In the besttree for first positions of codons, the root inserts on theMethanococcus type I lineage, whereas in the best tree forsecond positions, it inserts on the Clostridium type I lineage.When the likelihoods from both analyses are combined, theroot falls on the Methanococcus type I lineage, as in the treefor first positions. Note that this implies that there might beundiscovered iron proteins in extant archaebacteria. On theother hand, second positions are known to be more reliableover long evolutionary time periods (41). We therefore preferthe tree in which the chlorophyll iron proteins originatewithin eubacteria on the lineage to the Clostridium type Isequence, but we emphasize that statistically significantdistinction between the alternatives is elusive (Fig. 4).

Duplication of Chlorophyll Iron Proteins Before the Diver-sification of Extant Photosynthesizing Eubacteria. The best

rooting of the chlorophyll iron protein tree is always on thebchX lineage, no matter where the root inserts on thenitrogenase iron protein tree (Fig. 4). All alternative rootingsare ruled out at statistical significance in the four-by-fourtests (P < 0.05). This result is notable because it indicates thatthe last common ancestor of cyanobacteria and purple bac-teria contained both a PChlide reductase and a chlorinreductase, and was thus capable of BChl synthesis. BecauseBChls absorb at longer wavelengths than do Chls (Fig. 1), theBChl made by this organism must have been in the photo-synthetic reaction center to permit downhill energy transferfrom the antennae pigments.Enzymatic Activity of the Ancestral Reductase. The central

theme of the "recapitulation theory" and of the Granickhypothesis is that biosynthetic pathways are extended oneevolutionary step at a time. As soon as a plausible mechanismis proposed by which a particular pathway could leap aheadtwo or more biochemical steps in a single evolutionary step,then both the recapitulation theory and the Granick hypoth-esis can be called into question for that specific case. Such amechanism is apparent in the common ancestry of thePChlide and chlorin reductases: The first pheoporphyrin-reducing chlorophyll iron protein system may have reducedboth rings B and D (Fig. 1, arrowheads), thereby reducing thepheoporphyrin directly to a bacteriochlorin with a singleenzyme system. The chemistries of the two ring reductionsare nearly identical: the outer double bond of a five-membered ring flanked by two other conjugated rings isreduced to a single bond (Fig. 1). Invention of this reductasewould then have advanced the synthesis of photosyntheticreaction center pigments from a pheoporphyrin such asPChlide (2, 42) to a bacteriochlorin in a single evolutionarystep. Only later, when a predecessor to modern cyanobac-teria acquired the ability to synthesize singly reduced pig-ments, did Chl appear, perhaps first serving as an antennaepigment [such as the chlorins (BChlc, d, and e) in theantennae of modern Chlorobium] and later as a component ofthe reaction center (as in modern cyanobacteria and chloro-plasts).

This model resolves the apparent conflict between theGranick hypothesis and the observed phylogenetic distribu-tion of pigments used in photosynthetic reaction centers.There are four commonly recognized tetrapyrrole reactioncenter pigments: BChla, BChlb, and BChlg, and Chla (43).BChla is in the reaction center and antennae of the greengliding bacteria (Chloroflexaceae), most purple bacteria (pro-teobacteria), including Rhodobacter capsulatus, and thegreen sulfur bacteria (Chlorobiaceae). BChlg (found in He-liobacteriaceae) and BChlb (in the proteobacterium Rhodo-pseudomonas viridis) are in a redox state intermediate be-tween chlorins and bacteriochlorins, in that the 4-ethyl groupof BChla is replaced by an ethylidene. Their rings are threeelectrons reduced from PChlide, as opposed to two electronsin chlorins and four electrons in bacteriochlorins. The onlygroup that uses a Chl in place of a BChl in its photosyntheticreaction centers is the cyanobacteria/chloroplasts, which isalso the only group whose members couple two distinctphotosystems to oxidize water and produce oxygen. A pos-sible exception to this rule is 8-hydroxychlorophyll, whichhas recently been reported to be in the reaction centers ofheliobacteria (44). The broad phylogenetic distribution of themore reduced photopigments among four of the five photo-synthetic bacterial phyla is consistent with the ancestralpigment's having been more reduced than a chlorin, inagreement with the phylogenetic analysis above. The pres-ence of reduced pigments, particularly BChla, in both pho-tosystem I (PSI)-like (heliobacteria and Chlorobiaceae) andphotosystem II (PSII)-like (proteobacteria and Chloroflex-aceae) reaction centers (2, 45-48) may even suggest thatthere was an ancestral bacterium with both PSI and PSII,

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similar to that postulated by Pierson and Olson (2, 3) but withBChl instead of Chl in its reaction centers. Subsequentradiation and loss of one type of reaction center or anothercould then account for all extant forms with fewer changes inpigment types than are needed in the scheme proposedpreviously (2, 3).

In an alternative scenario, Chl was used in reaction centersbefore BChl. Since the Chl-containing cyanobacteria arederived from organisms that made BChl, a Chl-first scenariorequires that BChl-containing organisms displaced all of theorganisms with Chl in their reaction centers prior to theradiation of all extant photosynthesizers. This is less parsi-monious than a BChl-first scenario, in that it invokes a switchin reaction center pigments in addition to the switch fromBChl to Chl in an ancestor of the cyanobacteria.Proposed History of the Iron Protein Family. The ancestral

iron protein duplicated and diverged into nitrogenase type Iand type III prior to the speciation event that separated theeubacteria from the methanogenic archaebacteria. Subse-quent to this speciation, there was a duplication of thenitrogenase iron protein gene in the eubacterial line, possiblyaccompanied by a duplication of nifK and nipD. One of thecopies diverged sufficiently to enable it to interact with aPChlide-binding protein (or even directly with the PChlide),thereby forming the first PChlide reductase. This enzymereduced its substrate twice to form a bacteriochlorin. Sub-sequent duplication of each of the three subunits of thisreductase allowed the two copies to specialize toward reduc-tion of PChlides on one hand and chlorins on the other. Chlappeared later during the early evolution of cyanobacteria.Reduced reaction center pigments and the modern oxygen-rich atmosphere are therefore partly consequences of ancientduplications of the type I nitrogenase iron protein.

We thank Robert Blankenship, three anonymous reviewers, andespecially Ellen Prager for useful comments on the manuscript.D.H.B. and J.E.H. were supported in part by National Institutes ofHealth Grant GM 30786, by the Office of Basic Energy Science in theBiological Energy Division of the Department of Energy undercontract DE-ACO30-76F000978, and by a graduate research fellow-ship from Bristol-Meyers Squibb to D.H.B.; A.S. was supported bya National Institutes of Health grant to the late Allan C. Wilson.

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