Biochimica et Biophysica Acta 1322Ž. 1997 163±172 Structural analysis of the photosynthetic reaction center from the green sulfur bacterium Chlorobium tepidum Georgios Tsiotis a,), Christine Hager-Braun b, Bettina Wolpensinger a, Andreas Engel a, GunterÈ Hauska b a M.E. MullerÈ Institute for Microscopic Structural Biology, Biozentrum, UniÕersity of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland b Lehrstuhl furÈÈ Zellbiologie und Pflanzenphysiologie, UniÕersitat Regensburg, 93040 Regensburg, Germany Received 11 August 1997; accepted 18 August 1997 Abstract The reaction centerŽ. RC core complex was isolated from the green sulfur bacterium Chlorobium tepidum and characterized by gel electrophoresis, gel filtration, and analytical ultracentrifugation. The purified complex contained the PscA and PscC subunits and a small amount of the Fenna±Matthews±Olson proteinŽ. FMO protein as an impurity. The mass of the core complexes was found to be 248 kDa by scanning transmission electron microscopyŽ. STEM . This is compatible with the presence of two copies of the PscA subunit and at least one copy of the PscC subunit, and provides evidence that the isolated complex exists as a monomer. Digital images of negatively stained RC complexes were recorded by STEM and analyzed by single-particle averaging. The complex had a length of 14 nm and a width of 8 nm, comparable to the length and width of the monomeric cyanobacterial PSI complex. The averages revealed a pseudo two-fold symmetry axis, which is a prominent structural element of the monomeric form. q 1997 Elsevier Science B.V. Keywords: Electron microscopy; Green sulfur bacteria; Reaction center 1. Introduction higher plants. Iron±sulphur RCsŽ. type I reduce Fe±S centers, and occur in photosystem IŽ. PSI and in Photosynthetic reaction centersŽ. RCs are classi- green sulfur bacteria such as Chlorobia and in He- fied according to the nature of their terminal electron liobacteria wx1 . The similarity between the PSI type I acceptors. Q-type RCsŽ. type II reduce a mobile RCs of higher plants, green algae and cyanobacteria quinone. Examples are found in purple bacteria and and the type I RC of Chlorobia and Heliobacteria in the photosystem IIŽ. PSII of cyanobacteria and has been amply documentedwx 2±4 . The sequences of the genes coding for the large subunitsŽ PscA and PshA with a mass of 82 kDa; seewx 5. show their core Abbreviations: BChla, bacteriochlorophyll a; FMO, Fenna± to be composed of two identical transmembrane sub- Matthews±Olson; Psc, gene coded Chlorobia subunits; RC, reac- units which form a homodimerwx 6,7 . Analogous to tion center; SDS±PAGE, sodium dodecyl sulfate±polyacrylamide r gel electrophoresis; STEM, scanning transmission electron mi- the PsaA PsaB heterodimer of PSI, the homodimer croscopy of PscA of the Chlorobia carries the primary electron ) q Corresponding author. Fax: 41 61 2672 259. donor P840 Ž. a special pair of BChla , the primary 0005-2728r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S0005-2728Ž. 97 00073-X 164 G. Tsiotis et al.rBiochimica et Biophysica Acta 1322() 1997 163±172 wx electron acceptor A0 Ž. a Chla monomer , an interme- crystallography 20,21 . Knowledge about Fe±S-type diate acceptor A1 Ž speculated to be a menaquinone, RCs is lagging behind, but the PSI RC which has a wx but see 8. and an Fe±S cluster analogous to FX of heterodimeric nature has been crystallized in 2D and PSI. The PscA protein contains 20 histidine residues 3D crystalswx 22±27 , and structural resolution is in compared to 42 and 37 in PsaA and PsaB, respec- progresswx 28,29 . The arrangement of the core in tivelywx 6,7 . In analogy to PSI it was proposed that green sulfur bacteria may aid a better understanding these are candidates for binding the BChla molecules. of RCs in general, in particular of their evolution and For Chlorobium limicola a transcription unit has been of the selection pressure for the formation of het- identified which contains a second gene Ž.pscB erodimeric cores in aerotolerant organisms. downstream of that coding for the large core subunit. In the present communication, we report structural It corresponds to a 23 kDa protein which contains studies of a RC complex containing the PscA and two Fe±S clusters, analogous to the terminal electron PscC subunits from the green sulfur bacterium acceptor FAB and F of PSI which are located on the Chlorobium tepidum. Structural information was ob- psaC gene productwx 9 . An additional protein, denoted tained by gel filtration, analytical ultracentrifugation, PscD, has been detected in the Chlorobia RC com- mass determination using the scanning transmission plex. The gene coding for PscD has been sequenced microscopeŽ. STEM , and image processing. The mass and represents a 16.5 kDa polar proteinwx 10 which is analysis provided solid evidence for the homodimeric thought to be the analog of the PsaD protein. Similar nature of the RC complex in the green sulfur bac- to PSI which binds the electron donor plastocyanin or terium Chlorobium tepidum. cyt553 , the RC of green sulfur bacteria binds cyt c 551 q Ž.pscC gene product , a 20 kDa protein, to reduce P840 wx11±14 . However, there is a controversy about the 2. Materials and methods nature of this proteinwx 12,14,15 . The association of the PscA protein with a 42 kDa proteinŽ the FMO 2.1. Analytical methods protein. has also been observed. This protein is not required for the primary charge separation but seems The RC of Chlorobium tepidum was isolated as to stabilize the RCwx 10,12,13,15 . It carries 7 BChla describedwx 10 . Photosynthetic membranes were solu- molecules and was the first chlorophyll protein struc- bilized using Triton X-100 under anaerobic condi- ture to be solved by X-ray crystallographywx 16 . tions. The extract was fractionated on a sucrose Although the FMO protein has been found in various density gradient containing Triton X-100 and yielded RC preparations, the exact number of copies per RC a brownish and a bluish green band. The brownish is not knownwx 4 . band which contained the PscA and PscC proteins, A purified preparation of the P840 RC from was used for structural analysis. Gel electrophoresis Chlorobium with only three subunits, has been ob- was carried out as outlined inwx 26 and stained with tained using the detergent octyl glucoside. This ex- silver nitrate. A Superdex 200 PC 3.2r30Ž. Pharmacia hibited labile photoactivitywx 11 . Recently, several analytical molecular sieve column attached to a Phar- reports of the isolation of more efficient RCs from macia SMART system was used for gel filtration. Chlorobia have been publishedwx 10,14,15,17,18 . The column was equilibrated with 10 mM Tris, 0.1% s Chlorobium tepidum RC complexes which show NaN3 , 0.15% Triton X-100, pH 8Ž. buffer A at a steady state, light induced, electron transfer contain flow rate of 0.1 mlrmin. The filtrations were per- the PscA, PscB, PscC, PscD and FMO proteins. formed using two wavelengths of light, 435 and Recently, the isolation of a subcomplex including the 598 nm, to detect the eluted proteins. The column PscA and PscC proteins was reported which showed temperature was 88C. The sedimentation velocity of light induced charge separation and recombination PscArPscCrdetergent particles was determined at between the P840 and the primary electron acceptor 208C with the Beckman model XLA analytical ultra- wx19 . centrifuge equipped with absorption optics. Sedimen- The structure of the Q-type RCs in purple bacteria tation velocity runs were carried out at 52 000 rpm in has been elucidated at atomic resolution by X-ray a 12 mm double sector Epon cell. G. Tsiotis et al.rBiochimica et Biophysica Acta 1322() 1997 163±172 165 2.2. Scanning transmission electron microscopy and Intermediate references were constructed from the image processing sums of those particles whose correlation coefficients with the reference were above a threshold value. In Detergent solubilized particles were adsorbed to each run, particles contributing to the final average thin carbon films supported by thicker fenestrated were accumulated into two independent averages from carbon layers, washed on 3 droplets of quartz bi-dis- which the resolution was assessed using the ring tilled water and either freeze-dried without staining, correlation functionwx 31 , the phase residual wx 32 , and or negatively stained with a 1% uranyl formate solu- the spectral signal-to-noise ratiowx 33 . Subsequently, tion and air dried. For mass analysis, elastic dark multivariate statistical analysiswx 34,35 was applied. field images of unstained complexes were recorded After classification of the selected particles using an using a VG-HB5 STEM at defined electron doses and algorithm kindly provided by Dr. J.P. Bretaudiere 80 kV acceleration voltage. The particle mass was wx36 and implemented in IMPSYS, a dendrogram evaluated by measuring the number of electrons elas- representing the likeness of the different clusters was tically scattered by a circular region enclosing the obtained. Closely related clusters were then added to particle and subtracting the background contribution improve the signal-to-noise ratio of the final aver- due to the thin carbon film using the IMPSYS soft- ages. warewx 30 . Gauss curves were fitted to the mass histogram peakswx 30 . Dark-field images of negatively stained particles 3. Results were recorded in the same instrument at 100 kV acceleration voltage. Well preserved particles sam- 3.1. Characterization of the purified complexes by pled at 0.35 nm were selected interactively, using the SDS±PAGE, HPLC gel filtration and analytical ul- SEMPER image processing system installed on a tracentrifugation VAX 3100 work station. As a first step 64 = 64 pixel regions including RC complexes were selected. The The RC complex was isolated from Chlorobium particles were aligned using correlation techniques, tepidum using Triton X-100 to solubilize the photo- starting with a single projection as first reference.