Photosynthetic Reaction Center Genes in Green Sulfur Bacteria and in Photosystem 1 Are Related
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Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8135-8139, September 1992 Biochemistry Photosynthetic reaction center genes in green sulfur bacteria and in photosystem 1 are related (photosynthesis/Chlorobium) MICHAEL BUTTNER*, DIAN-LIN XIEt, HANNAH NELSONt, WILFRIED PINTHER*, GUNTER HAUSKA*, AND NATHAN NELSONt *Lehrstuhl fir Zelibiologie und Pflanzenphysiologie, Universitit Regensburg, 8400 Regensburg, Federal Republic of Germany; and tRoche Institute of Molecular Biology, Nutley, NJ 07110 Communicated by George Feher, May 6, 1992 (receivedfor review January 20, 1992) ABSTRACT Oxygenic photosynthesis of chloroplasts and components known for PS1 (7, 8), the "special pair" of cyanobacteria involves two photosystems, which originate from chlorophylls as electron donor, A. and A1 as early electron different prokaryotic ancestors. The reaction center of photo- acceptors, and the subsequent FeS centers X, A, and B, have system 2 (PS2) is related to the well-characterized reaction also been detected in the green sulfur bacterium Chlorobium center of purple bacteria, while the reaction center of photo- limicola (11, 13), and they seem to exist as well in the system 1 (PS1) is related to the green sulfur bacteria, as is distantly related Heliobacteria (14, 16). Moreover, the iso- convincingly documented here. An operon encoding the P840 lated reaction center from Chlorobium contains large sub- reaction center of Chlorobium limicola f.sp. thiosulfatophilum units (10), very similar to PS1 preparations (17). The primary has been cloned and sequenced. It contains two structural donor and the acceptors AO, A1, and X are thought to be genes, coding for proteins of730 and 232 amino acids. The first located on these large subunits of PS1, while the two 4Fe4S protein resembles the large subunits ofthe PS1 reaction center. centers A and B are contained in an extra subunit (7, 8). Putative binding elements for the primary donor, P840 in Numerous genes from chloroplasts and cyanobacteria for Chlorobium and P700 in PS1, and for the acceptors A., Al, and the two large subunits, called psaA and psaB (8, 18), and for FeS center X are conserved. The second protein is related to the the polypeptide carrying the FeS centers A and B, called PS1 subunit carrying the FeS centers A and B. An adjacent psaC (8, 19), have been sequenced. Here we report on the third gene, not belonging to the reaction center, encodes a genes from the green sulfur bacterium Chlorobium limicola protein related to dolichyl-phosphate-D-maunose synthase corresponding to psaA/B and psaC.t from yeast. The different origins ofPS1 and PS2 are discussed. METHODS Conversion of light into chemical free energy is fundamental for life, and photosynthetic reaction centers play a major role Chlorobium limicola f.sp. thiosulfatophilum (Deutsche in this process. These are special protein-chlorophyll com- Sammlung von Mikroorganismen, Gottingen, F.R.G.) was plexes in the core of light-harvesting photosystems, which grown, and its reaction center with the primary electron use excitation energy to drive charge separation across the donor P840 was prepared as described (10), with minor photosynthetic membranes. Two prototypes of photosyn- modifications (11). From the major polypeptide at 65 kDa thetic reaction centers are known. One functions in the (see Fig. 1) proteolytic fragments were obtained either by oxygen-evolving photosystem 2 (PS2) of chloroplasts and soaking lyophilized gel pieces with staphylococcal protease cyanobacteria, as well as in the nonoxygenic photosystem of V8 overnight, electroeluting the digest from the gel pieces on purple bacteria. The other functions in ferredoxin reduction a second SDS gel according to Schagger and von Jagow (20), by photosystem 1 (PS1) ofchloroplasts and cyanobacteria. A and electroblotting on Immobilon (Millipore) or by electro- current view of the structure, common to both types, is a blotting first, digesting the 65-kDa band cut from the Immo- transmembrane heterodimeric arrangement of two proteins bilon filter with trypsin, and fractionating the digest by HPLC that hold the primary electron donor and the early electron on a Serva RP8 column with a gradient from 0 to 100% acceptors. Two major differences between PS1-type and acetonitrile in 0.1% trifluoroacetic acid. The peptides cut PS2-type reaction centers are that the former involves from the blot or in the HPLC fractions were sequenced in a strongly reductive FeS centers as electron acceptors and gas-phase sequenator (Applied Biosystems). Four peptide contains many more chlorophyll molecules. sequences were obtained (see Fig. 3), and corresponding Research on PS2 was highly stimulated by the identities oligonucleotides were prepared to screen a gene bank of discovered in the genes for its heterodimer subunits, D1 and Chlorobium DNA, which we got by partial digestion with D2, and for the subunits L and M of a corresponding Sau3Al, isolation of 1- to 5-kilobase (kb) fragments, ligation heterodimer in purple bacteria (1-4). Detailed modeling of into pBluescript SK (Stratagene), and transformation of the PS2 reaction center was possible because the reaction Escherichia coli DH5a cells. Positive clones yielded over- center of purple bacteria had been crystallized and its three- lapping sequences of 1629 base pairs (bp) (position 190-1819 dimensional structure had been resolved by x-ray crystallog- in Fig. 3) and included the sequence coding for FPCLG- raphy (4-6). Our picture of the reaction center of PS1 is PVYGGTC (position 1570-1618 in Fig. 3). This dodecapep- lagging behind. Its structure has not been resolved yet, tide is related to the conserved peptides in PS1 that bind the although crystallization has been achieved (7, 8). Interest- ingly, however, a similarity to the reaction center of green Abbreviations: PS1 and PS2, photosystem 1 and 2; P700 and P840, sulfur bacteria has been recognized (9-15). All the redox primary electron donors of the reaction centers in PS1 and ChMoro- bium; A., A1, and X, electron acceptors in the reaction center ofPS1; L and M and D1 and D2, subunits of the reaction center of purple The publication costs of this article were defrayed in part by page charge bacteria and of PS2, respectively. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M94675). 8135 Downloaded by guest on September 28, 2021 8136 Biochemistry: Buttner et al. Proc. Nad. Acad Sci. USA 89 (1992) FeS center X, suggesting that we had found the desired gene. The sequenced region also contained a HindIII site (position 424 in Fig. 3). Digestion of Chlorobium DNA with Eag I -68 FIG. 1. Polypeptides of the reaction yielded a fragment of about 5 kb in Southern blots that 65-W center preparation from Chlorobium. hybridized with the oligonucleotides above. To obtain the -45 Lane 1, P840 reaction center. The top complete sequence ofthe region shown in Fig. 2, Chlorobium band is an incompletely unfolded, still DNA was digested with Eag I and HindIII, and the two 32 - -30 green form of the 65-kDa band, as is fragments (about 1 and 4 kb) reacting in the Southern blot observed also in preparations of the PS1 24- - reaction center. Lane 2, stan- Sau3AI , protein with the 0.7-kb insert of a positive clone from the !dard. Numbers indicate molecular mass library were ligated into pBluescript SK and cloned in E. coli - 4 values in kDa. The reaction center was DH5a. DNA sequencing with oligonucleotide primers and prepared, electrophoresed on SDS/15% dideoxynucleotides was carried out by the exonuclease III polyacrylamide gels, and stained with deletion technique (21). The complementary nature of the Coomassie blue as described (10, 11). two Eag I-HindIII clones was proven by the sequences of several overlapping Sau3AI clones. All molecular biology sequences may constitute part ofthe promoter system for the procedures used can be found in Sambrook et al. (22). operon. Comparison ofthe P840 Protein with Other Reaction Center Proteins. From the hydropathy plot ofthe P840 protein up to RESULTS AND DISCUSSION 11 hydrophobic segments with a potential for forming trans- Cloning and Sequencing the Operon Encoding Chlorobium membrane helices can be identified. Fig. 3 shows a fit of the Reaction Center Proteins. The reaction center from the green P840 sequence into a consensus alignment for the two large sulfur bacterium Chlorobium limicola f.sp. thiosulfatophilum PS1 subunits (8, 18). Of the 11 putative transmembrane was purified some years ago (10, 11), and more recently it was segments, I-III and VI-XI of the P840 protein fit the corre- also purified from Heliobacteria (12, 23). Fig. 1 shows the sponding segments in the PS1 subunits, but the region of the polypeptide pattern of the P840 reaction center from Chlo- putative transmembrane helix V in psaA and -B products robium. Most prominent is a diffuse band of about 65 kDa, carries one negative and two positive charges in the P840 from which four partial peptide sequences were obtained. It protein. On the other hand, shifting the transmembrane spans corresponds to a similar band seen in PS1 preparations (17), in this part of the P840 protein toward the N terminus would which represents the two large reaction center subunits of make the corresponding regions IV in the P840 protein and in PS1 ofabout 83 and 82 kDa (7, 8, 18). Fig. 2 shows the general thepsaA and -B products run in opposite directions. Possibly arrangement of the DNA fragment containing the reaction segments IV and V are not embedded in the membrane. center genes. It is 4.8 kb long and reveals three open reading However, the distribution ofcharges in the interhelical loops, frames (GenBank, accession no. M94675, gene description which follows the "positive inside rule" (28) when the N CHLORC).