Proc. Natl. Acad. Sci. USA Vol. 90, pp. 1642-1646, March 1993 Review Shared thematic elements in photochemical reaction centers (photosynthesis/Chlorbiaceae/Heliobacteriaceae/iron-sulfur clusters/evolution) John H. Golbeck Department of Biochemistry, Center for Biological Chemistry, University of Nebraska, Lincoln, NE 68583-0718

ABSTRACT The structural, func- electron is rapidly transferred to the qui- (e.g., Heliobacterium chlorum, Helio- tional, and evolutionary relationships be- none, which acts to stabilize against the bacterium gestii, and Heliobacillus mo- tween photosystem II and the purple non- rapid charge recombination between the bilis) has further stimulated interest in sulfur bacterial reaction center have been primary reactants. these organisms as evolutionary precur- recognized for several years. These can be As depicted in Table 1, the identities of sors of PSI (ref. 7; see also ref. 8). classified as "quinone type" (type I) pho- the components and the kinetics of the PSI is considered an "iron-sulfur- tosystems because the terminal electron initial forward electron transfer reactions type" RC in which the bound primary acceptor is a mobile quinone molecule. are remarkably similar in the purple bac- quinone, A1, donates its electron to an The analogous relationship between pho- terial RC, PSII, and PSI. A further shared iron-sulfur cluster (probably Fx) instead tosystem I and the green sulfur bacterial feature is that the photoactive compo- of to the secondary quinone (reviewed in (and heliobacterial) reaction centers has nents are located on a chlorophyll- ref. 9). Fx is a rare example of an inter- only recently become dear. These can be containing, homo- or heterodimer that is polypeptide [4Fe-4S] cluster that occu- dcassified as "iron-sulfur type" (type I) predicted to possess an overall C2 axis of pies the same relative position as the photosystems because the terminal elec- symmetry (this is only known for certain non-heme iron in the purple bacterial RC tron acceptor consists of one or more in the purple bacterial RC). The differ- and in PSII. However, it may serve a bound iron-sulfur clusters. At a funda- ences between the various RCs lie largely very different function. Unlike the non- mental level, the quinone type and iron- in the details of the protein scaffold that heme iron in quinone-type RCs, Fx is sulfur type reaction centers share a com- modulates the redox potentials of the thought to undergo redox chemistry un- mon photochemical motif in the early pro- photoactive components and in the iden- der physiologically relevant conditions. cess of charge separation, leading to the tities of the secondary electron donors Its role in PSI may be to divert the speculation that al photochemical reac- and acceptors that have evolved to pro- electron out of the membrane phase and tion centers have a common evolutionary vide the specialized functions of water into the stromal phase, leading to the origin. This review summarizes the cur- oxidation in PSII and nicotinamide ade- reduction of soluble ferredoxin through rent state of knowledge in comparative nine dinucleotide phosphate (NADP+) the involvement of the bound iron-sulfur reaction center biochemistry between pro- reduction in PSI. It is the purpose of this clusters FA and FB. The function of the karyotic bacteria, cyanobacteria, and article to review recent developments in secondary quinone in PSI is not known. green plants. the area of comparative RC biochemistry PSI is a heterodimer of 82- and 83-kDa and examine the evolutionary implica- polypeptides labeled PSI-A and PSI-B. tions offound similarities in structure and These proteins are highly hydrophobic, General Features of Photochemical function. each transversing the thylakoid mem- Charge Separation brane up to 11 times. The psaA genes PSI and the Chlorobium RC predict polypeptides of 739-751 amino All known classes of photochemical re- acids, and the presence of a short N-ter- action centers (RCs)-photosystem I The functional relationship between the minal extension is the only significant (PSI) and photosystem II (PSII) in green purple bacterial RC and PSII in green difference in length between the prokary- plants and cyanobacteria and the purple plants and cyanobacteria has been appar- otic and eukaryotic sequences. The psaB non-sulfur bacterial and green sulfur bac- ent for nearly a decade: both are consid- genes predict polypeptides of 733-736 terial RC-are now recognized to share a ered "quinone-type" photosystems in amino acids that are similar in length in strikingly similar motif. This shared which the bound primary quinone, QA, prokaryotes and eukaryotes. The higher theme consists of a dimeric protein core donates its electron across the het- plant sequences are about 95% identical, that functions as a scaffold for antenna erodimeric protein boundary to a mobile and when conservative replacements are chlorophylls and a series of bound elec- secondary quinone, QB (reviewed in refs. considered, the higher plant, green algal, tron donors and acceptors that serve to 1 and 2). Due to the overwhelming sim- and cyanobacterial sequences are 95% stabilize the initial charge separation be- ilarity in structure and function, it is well similar. The psaA and psaB sequence tween primary electron donor and accep- accepted that the purple bacterial RC and classes are 45% identical in sequence to tor. A "generic" photochemical RC can PSII share a common ancestor. In con- one another and another 10% when con- be depicted with the following notation: trast, there has been no definitive bacte- servative amino acid replacements are hi. rial analog for PSI. However, a series of considered. This high degree of homol- recent publications have provided excel- PIQ hP*IQP+I-QP+I Q, ogy strongly suggests that the sequences lent evidence that the RC in green sulfur arose by duplication and divergence from where P is a chlorophyll primary electron bacteria of the genus Chlorbiaceae (e.g., a single ancestral gene. The high degree donor, I is a chlorophyll primary electron Chlorobium limicola f. thiosulfato- acceptor, and Q is a quinone secondary philum, Chlorobium phaeobacteroides, In Abbreviations: PSI, photosystem I; PSII, pho- electron acceptor. this generalized Chlorobium vibriforme) contains bound tosystem II; RC, reaction center; ESR, elec- RC, the absorption of a photon results in iron-sulfur clusters (3-6). The discovery tron spin (paramagnetic) resonance; S/N, sig- charge separation between the chloro- of iron-sulfur clusters in Gram-positive nal-to-noise; NADP+, nicotinamide adenine phyll donor and acceptor molecules. The bacteria of the genus Heliobacteriaceae dinucleotide phosphate. 1642 Downloaded by guest on September 29, 2021 Review: Golbeck Proc. Natl. Acad. Sci. USA 90 (1993) 1643

Table 1. Shared components and kinetics of photochemical charge separation tical and the cyanobacterial sequences Purple non-sulfur bacteriat are >90% identical, and virtually all P860 BPh QA h P8* BPh QA -O P860+ BPh- QA C P860+ BPh QA- amino acid substitutions are conservative Photosystem Ilt replacements. The primary sequence Phye Ph QA -Vh p* Ph QA 2 P68o+ Ph- QA C P68o+ Ph QA contains nine cysteine residues and two Photosystem I§ I<. PS CxxCxxCxxxCP motifs characteristic of P7oo Ao Al 1-SPspoo* Ao Al 10~sP7oo+ Ao- Al S20P P700+ Ao Al- proteins that contain [4Fe-4S] clusters. A very similar folding pattern is exhibited tp8w is a bacteriochlorophyll dimer, BPh is a bacteriopheophytin monomer, and QA is a bound the ferredoxin from Pepto- molecule of menaquinone in Rhodobacter sphaeroides. by 2[4Fe-4S] tP680 is a chlorophyll a monomer or dimer, Ph is a pheophytin monomer, and QA is a bound coccus aerogenes (15) and the [3Fe- molecule of plastoquinone in cyanobacteria and green plants. 4S][4Fe-4S] ferredoxin fromAzotobacter §P7oo is a chlorophyll a dimer; Ao is a chlorophyll a monomer, and A1 is a tightly bound vinelandii (16), and there are significant molecule of phylloquinone (or 5'-monohydroxyphylloquinone) in cyanobacteria and green regions of homology with ferredoxins plants. from Desulfovibrio gigas (17) and Bacil- lus thermoproteolyticus (18) that contain of primary sequence similarity between erodimer. The sequence similarities indi- a single [4Fe-4S] cluster. Although the the subunit classes also leads to the ex- cate that the divergence of the green three-dimensional structure of PSI-C is pectation that the three-dimensional sulfur bacterium from PSI occurred be- not (yet) available, there is enough se- structures of the two subunits will be fore the gene duplication event that led to quence homology with the 54-amino acid similar. the heterodimeric protein core of the PSI P. aerogenes ferredoxin and the fist 58 The recent sequencing of the RC gene RC. The strong implication is that the residues of the 106-amino acid A. vine- from C. limicola f. thiosulfatophilum heliobacterial and green sulfur bacterial landiiferredoxin to make meaningful pre- now places the green sulfur bacteria on RCs and PSI shared a common ancestor. dictions of tertiary structure. secure footing with PSI (10). The gene Gene duplication in the L and M subunits The P. aerogenes protein shows a re- encodes a protein of730 amino acids with ofbacteria and the Dl and D2 subunits of markable two-fold rotation symmetry a calculated mass of 82 kDa. The pre- PSII may have also taken place indepen- axis related to the two iron-sulfur binding dicted protein is highly hydrophobic, dently, which indicates that the purple sites (reviewed in refs. 19 and 20). When containing up to 11 transmembrane seg- bacterial and cyanobacterial precursors the protein is rotated 1800 around the ments. Although the similarity of amino also shared a single common ancestor symmetry axis, the main chain atoms acid residues is only 15% with PSI-A and (13). Accordingly, Dl did not descend approximately overlay, but the iron- 14% with PSI-B, this may not be the from L and D2 did not descend from M; sulfur clusters superimpose almost ex- problem it first appears. The overall pri- rather, divergence probably occurred af- actly. The protein can thus be divided mary sequence homology of the higher ter the existence of an ancestral ho- into two symmetrical domains, each plant Dl and D2 proteins with the L and modimer. However, no contemporary composed of the three N-terminal and M subunits of the purple bacterial RC is organism has yet been discovered with a three C-terminal segments. These three- only 15%, yet the three-dimensional fold- quinone-type homodimeric RC. The ex- segment domains have similar amino acid ing pattern of the five membrane- istence of a homodimer has conse- sequences and assume nearly the same spanning a-helices is predicted to be very quences for the pathway of electrons main chain conformation, and each con- similar (11). The similarity ofthe external within the RC. Ifthe analogy continues to tains a partial iron-sulfur binding site. loop regions between the green bacterial hold with the purple non-sulfur bacterial The first three cysteines of the CxxCxx- RC and PSI is especially low except for RC and (probably) with PSII, there will CxxxCP binding site in the segment are the loop between spans VIII and IX that be two pathways of nearly identical elec- used to ligate the N-terminal [4Fe-4S] contains two cysteines separated by 8 tron carriers in the C. limicola RC, the H. cluster and the fourth cysteine is used to amino acids, 5 of which are identical to mobilis RC, and PSI. The heterodimer in ligate the C-terminal [4Fe-4S] cluster. PSI-A and PSI-B. This sequence occurs the purple bacterial RC contains inherent The polypeptide chain wraps back to in the same relative position as in PSI and symmetry-breaking elements, ensuring allow the fourth cysteine of the C-termi- probably represents the binding site for that only one path is active. These ele- nal iron-sulfur binding motif to partici- iron-sulfur center Fx. A similar stretch of ments may not exist in a homodimer, pate in binding the N-terminal [4Fe-4S] amino acids has been found in a proteo- raising the issue of equal access to the cluster. Because of this geometry, the lytic fragment of the H. mobilis RC (12). two pathways for electron flow. The ex- two half-chains do not form separate In both C. limicola and H. mobilis it is istence oftwo active paths may represent globular iron-sulfur domains, but each striking that the highly conserved aspar- a new aspect in electron transfer in these half-chain cooperates in the same manner tic acid residues that flank three of the types of RCs. to form an approximate intramolecular cysteines in PSI-A and PSI-B are absent; diad axis. The protein can thus be con- the significance of this omission is not Evolutionary Aspects of PSI-C sidered a dimer of connected polypep- known. Unfortunately, the Fx cluster has tides containing two shared [4Fe-4S] not been positively identified in either C. PSI-C is an 8.8-kDa extrinsic protein clusters. The question then arises: How limicola or H. mobilis. A resonance rem- associated with green plant and cyano- are the cysteine ligands arranged in ferre- iniscent of the Fx iron-sulfur cluster has bacterial PSI that contains the FA and FB doxins that contain only one [4Fe-4S] been reported in C. limicola (4), but its terminal electron acceptors. This protein cluster? The polypeptide backbone is detailed electron spin (paramagnetic) res- is particularly interesting because it rep- roughly similar (for example, in D. gi- onance (ESR) characteristics have not resents a 2[4Fe-4S] ferredoxin that may gas), but an a-helix replaces the segment been described. have been borrowed from a preexisting that extends from the (missing) C-termi- One surprising finding is that there is bacterial protein, modified slightly, and nal iron-sulfur site to place the fourth no evidence for a second RC gene in C. then added to a PSI core after the mem- cysteine in ligand position to the N-ter- limicola that would code for a second brane-bound components were already in minal [4Fe-4S] cluster. It is possible that homologous RC protein (10) (the data will place and functional. The 80-amino acid the a-helix replaces one of the antiparal- be available shortly for H. mobilis). This PSI-C is very highly conserved in all 13 lel /3-segments primarily to restrict the implies that the RC in C. limicola is species sequenced thus far (14). The number of degrees of freedom of the probably a homodimer instead of a het- higher plant sequences are virtually iden- polypeptide chain so as to place the Downloaded by guest on September 29, 2021 1644 Review: Golbeck Proc. Natl. Acad. Sci. USA 90 (1993) fourth cysteine with the correct geometry [3Fe-4S] or a [4Fe-4S] cluster is reconsti- nearly doubles in size, and the reso- to ligate the iron. It is therefore probable tuted depending on whether 3 Fe or 4 Fe nances occur at gx = 2.05, gy = 1.95, and that a 2[4Fe-4S] ferredoxin rather than a is added per molecule of apoprotein (30). gz = 1.90. These centers have been des- single [4Fe-4S] ferredoxin is the ancestral There are instances where a [3Fe-4S] clus- ignated FB and FA by comparison of the prototype. The former has probably ter can incorporate a fourth iron to reform gy lines with those of green plants and arisen by gene duplication, the latter by a [4Fe-4S] cluster. The best example is cyanobacteria (the g., lines are problem- the deletion of the second iron-sulfur aconitase, which has a [3Fe-4S] cluster atical). Unlike PSI in green plants and cluster and substitution of one polypep- due to the presence of only-three appro- cyanobacteria, the H. chlorum FA has a tide segment by the a-helix later in evo- priately located cysteine ligands (31). The lower midpoint potential than FB. lution. fourth iron in the [4Fe-4S] cluster proba- ESR spectra characteristic of bound What was the nature ofthe polypeptide bly has a water or hydroxyl as ligand iron-sulfur clusters have also been found and iron-sulfur cluster that preceded the (discussed in ref. 32), and the cluster has in membrane preparations (4) and lauryl early gene duplication event? At issue is high propensity for losing iron when in the maltoside particles (6) from C. limicola, the inability of a half-protein, containing oxidized state to reform the [3Fe-4S] clus- but with a lower S/N. When the former is a complete CxxCxxCxxxCP binding mo- ter. The accumulation of oxygen in the illuminated at 4 K, an iron-sulfur center is tif, to accommodate an intact iron-sulfur atmosphere could have been the driving reduced with reported resonances at gz = cluster. There are two obvious possibil- force that led to the formation of the 2.07, gy = 1.91, and g. = 1.86. When ities. The first is that the ancestral protein four-cysteine structure in order to confer illuminated at 200 K, a second iron-sulfur was, indeed, a dimer, where two separate stability to the cluster. center is found at gz = 2.05, gy = 1.94, but identical polypeptide chains each The present structure of a 2[4Fe-4S] and gx = 1.88. Based on analogy with contributed cysteine ligands to produce ferredoxin probably represents the most PSI, the former is labeled FB, and the an interpolypeptide iron-sulfur cluster frugal manner in which the polypeptide latter FA. Although this assignment is (21). In this rudimentary, but effective, can fold to create the internal iron-sulfur reasonable, it suffers the same difficulty method of cluster ligation, the cysteine binding pockets. Indeed, the extremely that only two ofthe three resonances (the ligands would occur on the surface rather small size of these 2[4Fe-4S] ferredoxins gy and gz lines) match those of FA (gz = than in an interior pocket of the protein. has led several groups to suggest that 2.05, gy = 1.94, and g. = 1.85) and FB (gz We shall presuppose that the very small PSI-C will possess a very similar struc- = 2.07, gy = 1.92, and gx = 1.89) in PSI amount of oxygen in the early biotic ture (33-35). The only significant differ- of cyanobacteria and higher plants. Sim- atmosphere would not require elaborate ence between P. aerogenes ferredoxin ilar to H. chlorum, the FA cluster in C. shielding of the iron-sulfur clusters. An and PSI-C is an additional 5 amino acids limicola has a lower midpoint potential interpolypeptide [4Fe-4S] cluster exists between the two iron-sulfur binding sites, than FB, but, unlike H. chlorum, the in at least two ancient electron transport which should have the effect of enlarging clusters are not reducible with dithionite proteins: the Fx iron-sulfur cluster in PSI the internal loop as the polypeptide chain at high pH values. Miller et al. (5) also and the iron-protein of nitrogenase (22). folds back on itself twice between the reported resonances at gx = 1.87 and gy = The former is an example of a [4Fe-4S] iron-sulfur clusters, and an additional 8 1.93 in C. limicola membranes after illu- cluster shared by two homologous pro- amino acids on the C terminus. This mination at 13 K. The loss ofthese signals teins, and the latter is a [4Fe-4S] cluster addition does not necessarily translate to after treatment with chaotropic agents shared by the two identical proteins (23). a greater distance between the iron-sulfur and their recovery after reconstitution The nitrogenase iron-protein retains ox- clusters. One can consider the additional with FeCl3, Na2S, and 2-mercaptoetha- ygen sensitivity, primarily due to the amino acids on the C terminus as partial nol supports the assertion that they are exposed nature of the iron-sulfur cluster, compensation for the extra length cre- derived from iron-sulfur clusters similar whereas the Fx cluster is inherently ox- ated in the intercluster loop in partial to FA and FB. Unlike PSI, the cluster ygen-stable, probably due to its seques- preservation of the two-fold symmetry insertion and restoration protocol did not tered location. The advantage of this axis. These additions to the primordial require the addition ofprotein, indicating structure is that the constraints on the PSI-C protein could have provided bind- that the protein containing the FA/FB protein may be fewer if the folding pat- ing sites to the PSI-A/PSI-B heterodimer clusters was not removed from the RC tern need not define both the cluster and/or to the PSI-D and PSI-E proteins core by chaotrope treatment. Based on cavity and those features needed for in- that most likely overlie PSI-C on the PSI the difference between the amount ofP840 teraction with other proteins. Ultimately, core (36). determined chemically and that deter- gene duplication would have produced a mined by time-resolved optical spectros- fused protein that would utilize a single PSI-C in C. lmicola f. thiosulfatophilum copy, only 5% enters into a paired reac- polypeptide to ligate the 2[4Fe-4S] clus- and in H. chlorum tion with FA/FB in these membranes. The ters (see refs. 24 and 25 for further dis- inference is that the bound iron-sulfur cussion). If C. limicola and H. chlorum have iron- clusters are extremely susceptible to ox- The second possibility is that the an- sulfur-type RCs, then there should also idative denaturation during sample prep- cestral half-chain might have formed a exist a low molecular mass polypeptide aration. single globular cluster using an amino acid that contains FA and FB-like iron-sulfur A further difference with PSI is that in near the C-terminal end of the protein. clusters. A major technical difficulty in both H. chlorum and C. limicola FB The fourth ligand to the [4Fe-4S] cluster in this work is that the ESR spectra are rather than FA is preferentially photore- the half-chain protein may, or may not, extremely difficult to measure because of duced at 15 K. In PSI, the only instances have been a cysteine. In the [4Fe-4S] the low yield of active membranes and where the FB cluster undergoes signifi- ferredoxin from Pyrococcus furiosus (26) the reported oxygen sensitivity of the cant photoreduction at 15 K are in Phor- and in the 2[4Fe-4S] ferredoxin from Des- clusters. The data with the best signal- midium laminosum RCs (37), in a spinach ulfovibrio africanus (27), an aspartic acid to-noise (S/N) are from H. chlorum (7), PSI RC treated with high concentrations replaces the second cysteine in the where illumination of isolated plasma of glycerol (38), when PSI-C is rebound CxxDxxCxxxC motif. A [4Fe-4S] cluster membranes at 4 K leads to the photore- on a P700-Fx core in the absence ofPSI-D forms in both proteins, but it is easily duction of an iron-sulfur cluster with (39), and in a PSI-D-less mutant of Syn- converted to a [3Fe-4S] cluster under ox- broad resonances at g, = 2.07, gy = 1.93, echocystis sp. PCC 6803 (P. Warren, idizing conditions (28, 29). In Des- and gx = 1.89. When illuminated at 200 K Y.-S. Jung, V. P. Chitnis, P. R. Chitnis, ulfovibrio gigas ferredoxin, FdII, either a in the presence of dithionite, the signal J. D. Zhao, D. A. Bryant, and J.H.G., Downloaded by guest on September 29, 2021 Review: Golbeck Proc. Natl. Acad. Sci. USA 90 (1993) 1645 unpublished data). A further distinction amino acids and a smaller C-terminal erodimer, and the pseudo-C2 axis ofsym- is that the resonances in H. chlorum are extension. Further, only 20% of the metry strongly indicate that both types of broader, and the magnetic interaction amino acids are conserved in the relevant RCs may share a common ancestor. between the two iron-sulfur clusters ap- 80-amino acid stretch, and 13% of these There should remain regions of similarity pears to be weaker than FA and FB in PSI. are represented by the two flanking pro- in the primary amino acid sequences of Broad resonances and weak levels of lines and the eight cysteines that com- the RC polypeptides, but attempts to magnetic interaction have been observed prise the iron-sulfur ligands. correlate similar regions of PSI with the in cyanobacterial FA and FB when PSI-D Another difficulty arises because the g purple bacterial RC or with PSII have not is absent (39). The data supporting mag- tensor that gives rise to the ESR spectrum been overwhelmingly successful. There netic interaction between the two clus- of an iron-sulfur cluster is sensitive to the is a similarity between helix VIII of PSI- ters in C. limicola are also limited, and it protein conformation (41) and ionic envi- A/PSI-B and helix D of the bacterial RC is questionable whether the absence of ronment (42). One consequence is that if and between helix VI of PSI-A/PSI-B the g, resonance of FB at g = 2.08 upon the line shapes of a set of resonances are and helix B of the bacterial RC (44). The illumination at 200 K is sufficient evi- nearly identical to those of FA and FB, as difficulty is that the C. limicola RC pro- dence to assert a high level ofinteraction found for C. limicola, then the protein tein is different in these regions. A sim- between the clusters (4). A much stron- should be similar to PSI-C. Also, if the ilarly weak homology was noted between ger case can be made for magnetic inter- clusters undergo significant magnetic in- helix X ofPSI-B and helix D ofthe purple action in RCs that have been oriented on teraction and the resonances are suffi- bacterial RC (45); the difficulty here is Mylar film. A comparison of the g values ciently distinct and sharp, then the protein that helix X in PSI-A is quite different. and g-tensor orientations of the Chloro- environment surrounding PSI-C should Helix D is important in bacteria because bium RC with PSI strengthens the argu- be similar. This implies the existence of a it contains the ligands to the bacterio- ment that there is significant interaction PSI-D-like protein. Yet, the putative chlorophyll dimer, the bacteriopheophy- between FA and FB (4). Nitschke and "PSI-C" in Chlorobium would be highly tin acceptor, and the iron-quinone com- Rutherford (40) conclude that the marked dissimilar in primary amino acid sequence plex. A greater degree of similarity has similarities between these data sets and from the FA/FB protein in PSI. There also been found between the low molecular PSI provide strong evidence for a very is no evidence for the existence of a PSI- mass polypeptides PSI-I/PSI-J and helix similar structure in the two RCs. D-like protein. One might argue that the E ofD2/D1 in PSII (and by analogy, L/M The presence of FA and FB-like iron- tertiary structure around the iron-sulfur in purple bacteria) (46). It may be signif- sulfur clusters implies the existence of a clusters is all that matters; yet, ifthis were icant that these proteins are encoded in PSI-C-like polypeptide on the H. chlo- strictly true, the highly homologous [4Fe- the chloroplast genome of higher plants, rum and C. limicola RCs. However, even 4S] ferredoxins from C. pasteuranium and which is where most of the catalytic at relatively low stringencies, a search of P. aerogenes should show ESR spectra components of the RCs are encoded. the Chlorobium genomic library has identical to those ofFA and FB. Except for Helix E is important because it partici- failed to find a psaC-like gene. This is the presence of rhombic symmetry, the pates in binding the quinone and iron in rather surprising in light of the very few line shapes ofthese spectra are broad and D2. Accordingly, there has been a suspi- changes that have occurred in PSI-C over quite different in appearance from FA/FB. cion that PSI-I may contain a partial a the course of the 3.5 thousand million Finally, a 24-kDa protein containing quinone-binding site in PSI (46). Ifthis is years that separate cyanobacteria and 2[4Fe-4S] clusters remains to be identified borne out experimentally, a similar low green plants. If the Chlorbiaceae and in green bacterial RCs. There is a 22-kDa molecular mass polypeptide should exist Heliobacteriaceae diverged early from protein present in a lauryl maltoside prep- in the C. limicola and H. mobilis RCs. the line that led ultimately to cyanobac- aration from C. limicola, but a highly Since the overall sequence identity be- teria, the primary and tertiary structure resolved RC has been isolated from the tween the RC proteins ofChlorobium and of the 2[4Fe-4S] protein may be quite related species C. vibriforme showing PSI is only slightly higher than the level different from that ofmodem PSI-C, par- only an 18- and a 40-kDa band in this of identity between completely unrelated ticularly if the clusters did not require region (43). It should be kept in mind that proteins, there appears to be little point in protection from molecular oxygen. An iron-sulfur proteins frequently run anom- further cross-comparing the primary open reading frame has been found alously on SDS/PAGE and that the iden- amino acid sequences. A more satisfac- downstream from the C. limicola RC tification ofPsaC in PSI was long overdue tory approach may lie in comparing their gene that codes for a 24-kDa iron-sulfur because it migrated as a highly diffuse secondary and tertiary structures. The protein (10). The 232-amino acid se- band between 8 and 10 kDa. Neverthe- relative positions ofthe photoactive chlo- quence contains a traditional CxxCxx- less, a firm correspondence between the rophylls, quinone, and iron-sulfur cluster CxxxCP [4Fe-4S] binding motif and an 24-kDa downstream gene product and of PSI should be available shortly from unusual CxxCxxCxxxxxCP sequence "PSI-C" will require the identification of the 6-A x-ray structure of the cyanobac- that could represent a second iron-sulfur aprotein in RC preparations ofC. limicola terial RC (47). If the correspondence be- binding region. The 2 additional amino or C. vibriforme with the appropriate tween the two types ofRCs is as strong as acids occur between the third and fourth N-terminal amino acid sequence or with I suspect, P700, AO, A1, and Fx will su- cysteines, which would not affect the cross-reactivity using antibody prepared perimpose over P890, the bacteriopheo- normal pattern of iron-sulfur ligation. It against the expressed gene product. phytin acceptor, the bound quinone, and may, instead, increase the distance be- the non-heme iron of the purple non- tween the two iron-sulfur clusters, Are the Quinone-Type and sulfur bacterial RC (48). Ifthis prediction thereby reducing the likelihood of mag- Iron-Sulfur-Type RCs Related? holds, the challenge will be to determine netic interaction. The identification of how a minimal protein scaffold could the gene product with "PSI-C" of PSI is A major advance in photosynthesis re- accommodate the photoactive compo- based entirely on the fact that it is search has been the recognition that there nents and how the scaffold has expanded cotranscribed along with the RC gene as is a unifying theme for photochemical to provide binding sites for carotenoids a bicistronic mRNA. This is probably the charge separation among the quinone- and antenna chlorophyll molecules. correct identification of the FA/Fe- type and iron-sulfur-type RCs. We have It is more difficult to determine which containing protein. However, a compli- come to realize that the presence of very is the most ancestral: the quinone-type or cation arises because there is a large similar inorganic and organic cofactors, iron-sulfur-type RC. Blankenship (49) N-terminal extension of highly positive the existence of a protein homo- or het- has argued that no simple linear branch- Downloaded by guest on September 29, 2021 1646 Review: Golbeck Proc. Natl. Acad. Sci. USA 90 (1993)

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