The Photosynthetic Reaction Center of Blastochloris Viridis (PDB ID: 2X5U)
Created by Allison Plettner
Background and Related proteins:
Photosynthesis is the primary producer of oxygen and organic matter on earth. It is the process by which energy is captured from sunlight and converted into chemical energy (a transmembrane potential difference (10)) by plants, green algae and cyanobacteria. Photosynthetic animals are called photoautotrophs because they generate their own food. Photosynthesis also generates oxygen and assimilates carbon dioxide, both of which contribute to the composition of the Earth’s atmosphere. In addition to this vital function, nearly all life depends on directly or indirectly on this process (4). The function of the photosynthetic reaction center is to convert light energy into chemical energy by electron-transfer reactions which generate a charge separation across the membrane(1,3).
All photosynthetic reaction centers contain an identical functional core which has been evolutionarily conserved. The photosynthetic reaction center of the Blastochloris Viridis (also called Rhodopseudomonas viridis) contains four subunits C, H, L, and M which then support a closely associated “the special pair” (P960)(a pair of bacteriochlorophylls), four bacteriochlorophylls, two bacteriopheophytins, a bound menaquinone (QA), a mobile ubiquinone(QB), and a single non-heme iron (3,10). This overall structure creates an electron transfer chain spanning the membrane of the cell (10). The transfer of the electrons creates an electrochemical potential gradient which then drives ATP synthesis through the Calvin cycle. For each photon of light absorbed, two electrons (and therefore two protons) are pumped (10).
An e-score of 6e-111 indicates that the photosynthetic reaction center isolated from Thermochromatium Tepidum (PDB ID: 1EYS) has a similar primary structure, amino acid sequence, to the protein of interest (POI) (1). Additionally, a Z-score of 33.5 and an rmsd 1.9 indicate that 1EYS has similar three dimensional structure to the POI (4). Both proteins function as the sights of photosynthesis and depict the evolutionarily conserved structure and function of the photosynthetic reaction center protein(8,10). The molecular weights and isoelectric points of 2X5U and 1EYS are 132462.3 and 7.41 and 136734.5 and 7.20, respectively.
Structure:
The photosynthetic reaction center of the Blastochloris Viridis consists of four polypeptide subunits: C (cytochrome), H, L, and M.The L and M segments are composed of five helices forming the transmembrane heterodimer(2). The H subunit only has one transmembrane helix and facilitates water access to the electron transport chain. The C subunit contains four tightly bound prosthetic heme groups which act as the electron donors (2). The reaction center consists of four bacteriochlorophylls, two bacteriopheophytins, a bound menaquinone (QA), a mobile ubiquinone (QB) and a single non-heme Iron (II) ion (2,10). QA can only accept one electron while QB can accept two (7). QB is the final electron acceptor which binds weakly to the active site of the L subunit (near the cytoplasm) (2).The photosynthetic reaction cycle begins with a photon or energy transfer occurs between the light harvesting antenna and the special pair (3). The special pair then reduces one of the bacteriopheophytins which then transfers this electron to QA(3) (the primary quinine acceptor) (2). Overall ,this process consists of QB accepting two electrons from QAwhile in the active site and accepting two protons from the cytoplasm toproduce a dihydroquinol. The dihydroquinol (QH2) then dissociates and the neutral quinine then binds to the active site to complete the process (2).The result is that two protons (and two electrons in the opposite direction) are pumped across the membrane for every photon absorbed creating a transmembrane electrochemical potential gradient which is utilized to synthesize ATP from ADP (3,10).
The reaction center has a secondary structure containing: alpha helices, beta sheets, random coils, and turns. T2he C chain contains 336 residues which are arranged into 19 helices (containing 140 residues) and 10 beta strands (contain 12 residues). The H chain contains 258 residues of which 63 are contained within 9 helices and 62 are within 22 beta strands. The L chain contains 274 residues which are arranged into 15 helices (composed of 169 residues) and 9 beta strands (composed of 13 residues). Lastly, the M chain contains 324 residues of which 192 residues are arranged into 18 helices and 18 residues are arranged into 11 beta strands (6). Eleven of the helices within the reaction center complex are membrane spanning: five in L, five in M, and one in the H chain (7). Many water molecules are located near the subunit interfaces and play an important role in the subunit interactions (3)
The L and M chains (each with five membrane spanning helices) contain a 2-fold axis of symmetry perpendicular to the membrane plane (3).This L-M complex forms a flat surface parallel to the membrane surface. The C subunit and heme groups bind to the periplasmic side of the L-M complex while the globular domain of the H subunit binds to the cytoplasmic side (3). Neither the H nor the C subunits display symmetry. Both the L and M subunits contain two bacteriochlorophylls, one bacteriopheophytin, and one quinone arranged symmetrically. These form branches which extend from the special pair near the periplasmic side through the membrane to the cytoplasmic side (3). The surface of the reaction center complex is mainly hydrophobic lacking charged amino acid residues and only rarely containing polar residues or bound water molecules within the central region (3).
Additionally, the reaction center protein has 13 ligands associated with it. These consist of: four Protoporphyrin IX containing Fe (which are the heme groups with an iron core), four bacteriochlorophyll A molecules, two bacteriopheophytin A molecules, an Fe (II) ion, and a menaquinone-7. All of the heme groups are covalently bound to the C chain (3,7,9) by thioester bonds (3). Two of the bacteriochlorophylls and one bacteriopheophytins are non-covalently bound on the L chain. The remaining ligands are non-covalently bound on the M chain (9).
There are 216 α-carbon atoms of the M subunit which are superimposable on the L subunit. Approximately 40% of each of these subunits is composed of helices. The transmembrane helices of L and M chains contain 74 residues with polar side chains; however, there are only 7 inter-helix hydrogen bonds (3). The remaining polar residues interact with segments near the membrane surface or with cofactors (3). The H subunit can be divided into three distinct segments: a membrane spanning (H1-40), surface segment (H41-106) and a globular segment formed by the remaining residues. There is only one transmembrane helix contained with the H subunit (residues H12-H35) (3). The other major secondary structures of the H subunit are parallel and anti-parallel beta strands within the globular region. Within the H subunit, 65 residues are in contact with the L and M subunit complex. The C subunit is composed predominantly of helices with 4 disordered residues at the end (3). The C structure can be divided into five segments: the N-terminal (C1-C66), the first heme-binding segment for hemes 1 and 2 (C67-C142), a connecting segment (C143 to C225), the second heme binding segment for the third and fourth heme groups (C226 to C315) and lastly the C-terminal segment (3). The two heme-binding segments show local symmetry. There are many hydrogen bond sbetween the polar residues of the C subunit. The first heme is bound via thioester bridges to CysC87 and CysC90. The second heme is bound via CysC132 and CysC135. Heme three is the closest to the special pair and is bound via CysC244 and CysC247. Lastly, heme four is bound via HisC124 and HisC309 (3).
Currently, the overall protein structure of the B. viridis reaction center can be modeled at cryogenic temperatures. However, the final four residues of the C subunit (C333-336) and the flexible loop (H45-H54) within the H subunit cannot (2). Primarily the orientation of the secondary ubiquinone, QB, has been studied. QB can be located either deep within its binding pocket forming a hydrogen bond with HisL190, called the proximal position, or can be located 5Å closer to the proteins surface (out of the binding pocket), called the distal position (2). Predominantly, QB occupies the proximal position (2). There are three conformations of the QB binding site: QB in the proximal position, QB in the distal position, or an empty site occupied by several ordered water molecules. There is evidence that the transfer of an electron from QA to QB involves a conformation change of QB, a ‘conformation gate’ (2). However, the evidence across various experiments differ in conclusions. Several statethat QB predominantly occupies the distal position when in the dark, while other studies suggest that the distal position is never predominantly favored. Therefore, there is not a consensus determining the existence or effect of a ‘conformation gate’ (2).
Conformationa changes impart functionality due to the movement of QB, H and the TyrL162 of the L subunit (TyrL162) towards the activated special pair. Tyr162 is highly conserved in the bacterial photosynthetic reaction centers and located between the special pair of bacteriochlorophylls and the highest potential heme c59(10). The hydroxyl group side chain of TyrL162 hydrogen bonds to water (Wat501).This water molecule is coordinated to two other water molecules and to the hydroxyl group of SerM188 (10). The environment change, which results from photoxidation of the special pair, causes the oxygen of the hydroxyl group of TyrL162 to move 1.3Å to bring it into steric contact with the L branch of the bacteriochlorophyll of the special pair. This motion results in a new hydrogen bond between the hydroxyl group of threonine (ThrM185) of the M chain and a water molecule (Wat10) (10). The hydrogen bonds are especially important to provide stability to the deprotonated form of TyrL162. The excess positive charge on the special pair induces a proton transfer from TyrL162 to GluC254 in the direction opposite of electron transfer (10). It has been tested that the electron transfer reaction is inhibited at low temperatures. It is assumed tha tthis is due to the inhibition of movement of TyrL162. This suggests that the specific orientation of TyrL162 and the protonation state are key for electron transfer (10).
The hydroxyl proton of TyrL162 is transferred to the carboxylate moiety of GluC254 upon photoactivation. This occurs due to the positive charge on the special pair (P+) which drives a proton in the direction opposite of electron transfer(10). The water mediated hydrogen bond network which connects the two amino acids, TyrL162 and GluC254, allows the transfer of protons in bulk. GluC254 is exposed to the solvent allowing TyrL162 to spontaneously deprotonate upon photoactivation (10).
During the electron transfer process, the special pair is oxidized to generate P+. The special pair is associated with many amino acid side chains through hydrogen bonding. The hydrogen bonds act to stabilize the neutral P state of the special pair relative to the oxidize P+ state to increase the redox potential (7). The hydrogen of HisL168 is associated with the acetyl oxygen of the bacteriochlorophyll. In particular, HisL168 has been studied by a mutation toPheL168. This mutation eliminates one hydrogen bond allows movement of the P+ towards the Mg 2+ ion contributing to the stabilization of the P+ state (7).This lowers the redox potential and results in a faster electron transfer. However ,the mutation from His to PheL168 results in decreased efficiency of the energy transfer from the B1015 antennae. Therefore, the HisL168 amino acid hydrogen bond is key to maintaining the efficiency of electron transport chain (7).