Orientations of Iron–Sulfur Clusters F_A and F_B in the Homodimeric Type‑I Photosynthetic Reaction Center of <i>Heliobacterium modesticaldum</i>

Orientations of the F_A and F_B iron–sulfur (FeS) clusters in a structure-unknown type-I homodimeric heriobacterial reaction center (hRC) were studied in oriented membranes of the thermophilic anaerobic photosynthetic bacterium <i>Heliobacterium modesticaldum</i> by electron paramagnetic resonance (EPR), and compared with those in heterodimeric photosystem I (PS I). The Rieske-type FeS center in the cytochrome <i>b/c</i> complex showed a well-oriented EPR signal. Illumination at 14 K induced an F_B^– signal with <i>g</i>-axes of <i>g</i>_<i>z</i> = 2.066, <i>g</i>_<i>y</i> = 1.937, and <i>g</i>_<i>x</i> = 1.890, tilted at angles of 60°, 60°, and 45°, respectively, with respect to the membrane normal. Chemical reduction with dithionite produced an additional signal of F_A^–, which magnetically interacted with F_B^–, with <i>g</i>_<i>z</i> = 2.046, <i>g</i>_<i>y</i> = 1.942, and <i>g</i>_<i>x</i> = 1.911 at 30°, 60°, and 90°, respectively. The angles and redox properties of F_A^– and F_B^– in hRC resemble those of F_B^– and F_A^–, respectively, in PS I. Therefore, F_A and F_B in hRC, named after their <i>g</i>-value similarities, seem to be located like F_B and F_A, not like F_A and F_B, respectively, in PS I. The reducing side of hRC could resemble those in PS I, if the names of F_A and F_B are interchanged with each other

Menaquinone as the Secondary Electron Acceptor in the Type I Homodimeric Photosynthetic Reaction Center of <i>Heliobacterium modesticaldum</i>

The type I photosynthetic reaction center (RC) of heliobacteria (hRC) is a homodimer containing cofactors almost analogous to those in the plant photosystem I (PS I). However, its three-dimensional structure is not yet clear. PS I uses phylloquinone (PhyQ) as a secondary electron acceptor (A₁), while the available evidence has suggested that menaquinone (MQ) in hRC has no function as A₁. The present study identified a new transient electron spin-polarized electron paramagnetic resonance (ESP-EPR) signal, arising from the radical pair of the oxidized electron donor and the reduced electron acceptor (P800⁺MQ^–), in the hRC core complex and membranes from <i>Heliobacterium modesticaldum</i>. The ESP signal could be detected at 5–20 K upon flash excitation only after prereduction of the iron–sulfur center, F_X, and was selectively lost by extraction of MQ with diethyl ether. MQ was suggested to be located closer to F_X than PhyQ in PS I based on the simulation of the unique A/E (A, absorption; E, emission) ESP pattern, the reduction/oxidation rates of MQ, and the power saturation property of the static MQ^– signal. The result revealed the quinone usage as the secondary electron acceptor in hRC, as in the case of PS I

Experimental and Theoretical Mutation of Exciton States on the Smallest Type‑I Photosynthetic Reaction Center Complex of a Green Sulfur Bacterium Chlorobaclum tepidum

The exciton states on the smallest type-I photosynthetic reaction center complex of a green sulfur bacterium Chlorobaculum tepidum (GsbRC) consisting of 26 bacteriochlorophylls a (BChl a) and four chlorophylls a (Chl a) located on the homodimer of two PscA reaction center polypeptides were investigated. This analysis involved the study of exciton states through a combination of theoretical modeling and the genetic removal of BChl a pigments at eight sites. (1) A theoretical model of the pigment assembly exciton state on GsbRC was constructed using Poisson TrESP (P-TrESP) and charge density coupling (CDC) methods based on structural information. The model reproduced the experimentally obtained absorption spectrum, circular dichroism spectrum, and excitation transfer dynamics, as well as explained the effects of mutation. (2) Eight BChl a molecules at different locations on the GsbRC were selectively removed by genetic exchange of the His residue, which ligates the central Mg atom of BChl a, with the Leu residue on either one or two PscAs in the RC. His locations are conserved among all type-I RC plant polypeptide, cyanobacteria, and bacteria amino acid sequences. (3) Purified mutant-GsbRCs demonstrated distinct absorption and fluorescence spectra at 77 K, which were different from each other, suggesting successful pigment removal. (4) The same mutations were applied to the constructed theoretical model to analyze the outcomes of these mutations. (5) The combination of theoretical predictions and experimental mutations based on structural information is a new tool for studying the function and evolution of photosynthetic reaction centers

Experimental and Theoretical Mutation of Exciton States on the Smallest Type‑I Photosynthetic Reaction Center Complex of a Green Sulfur Bacterium Chlorobaclum tepidum

The exciton states on the smallest type-I photosynthetic reaction center complex of a green sulfur bacterium Chlorobaculum tepidum (GsbRC) consisting of 26 bacteriochlorophylls a (BChl a) and four chlorophylls a (Chl a) located on the homodimer of two PscA reaction center polypeptides were investigated. This analysis involved the study of exciton states through a combination of theoretical modeling and the genetic removal of BChl a pigments at eight sites. (1) A theoretical model of the pigment assembly exciton state on GsbRC was constructed using Poisson TrESP (P-TrESP) and charge density coupling (CDC) methods based on structural information. The model reproduced the experimentally obtained absorption spectrum, circular dichroism spectrum, and excitation transfer dynamics, as well as explained the effects of mutation. (2) Eight BChl a molecules at different locations on the GsbRC were selectively removed by genetic exchange of the His residue, which ligates the central Mg atom of BChl a, with the Leu residue on either one or two PscAs in the RC. His locations are conserved among all type-I RC plant polypeptide, cyanobacteria, and bacteria amino acid sequences. (3) Purified mutant-GsbRCs demonstrated distinct absorption and fluorescence spectra at 77 K, which were different from each other, suggesting successful pigment removal. (4) The same mutations were applied to the constructed theoretical model to analyze the outcomes of these mutations. (5) The combination of theoretical predictions and experimental mutations based on structural information is a new tool for studying the function and evolution of photosynthetic reaction centers

Experimental and Theoretical Mutation of Exciton States on the Smallest Type‑I Photosynthetic Reaction Center Complex of a Green Sulfur Bacterium Chlorobaclum tepidum

The exciton states on the smallest type-I photosynthetic reaction center complex of a green sulfur bacterium Chlorobaculum tepidum (GsbRC) consisting of 26 bacteriochlorophylls a (BChl a) and four chlorophylls a (Chl a) located on the homodimer of two PscA reaction center polypeptides were investigated. This analysis involved the study of exciton states through a combination of theoretical modeling and the genetic removal of BChl a pigments at eight sites. (1) A theoretical model of the pigment assembly exciton state on GsbRC was constructed using Poisson TrESP (P-TrESP) and charge density coupling (CDC) methods based on structural information. The model reproduced the experimentally obtained absorption spectrum, circular dichroism spectrum, and excitation transfer dynamics, as well as explained the effects of mutation. (2) Eight BChl a molecules at different locations on the GsbRC were selectively removed by genetic exchange of the His residue, which ligates the central Mg atom of BChl a, with the Leu residue on either one or two PscAs in the RC. His locations are conserved among all type-I RC plant polypeptide, cyanobacteria, and bacteria amino acid sequences. (3) Purified mutant-GsbRCs demonstrated distinct absorption and fluorescence spectra at 77 K, which were different from each other, suggesting successful pigment removal. (4) The same mutations were applied to the constructed theoretical model to analyze the outcomes of these mutations. (5) The combination of theoretical predictions and experimental mutations based on structural information is a new tool for studying the function and evolution of photosynthetic reaction centers

Light-Induced Electron Spin-Polarized (ESP) EPR Signal of the P800⁺ Menaquinone^– Radical Pair State in Oriented Membranes of Heliobacterium modesticaldum: Role/Location of Menaquinone in the Homodimeric Type I Reaction Center

Function/location of menaquinone (MQ) was studied in the photosynthetic reaction center of Heliobacterium (Hbt.) modesticaldum (hRC), which is one of the most primitive homodimeric type I RCs. The spin-polarized electron paramagnetic resonance signals of light-induced radical pair species, which are made of oxidized electron donor bacteriochlorophyll <i>g</i> (P800⁺) and reduced menaquinone (MQ^–) or iron–sulfur cluster (F_X^–), were measured in the oriented membranes of Hbt. modesticaldum at cryogenic temperature. The spectral shape of transient electron spin-polarized signal of P800⁺F_X^– radical pair state varied little with respect to the direction of the external magnetic field. It suggested a dominant contribution of the spin evolution on the precursor primary radical pair P800⁺A₀^– state with the larger isotropic magnetic exchange interaction <i>J</i> than the anisotropic dipole interaction <i>D</i>. The pure P800⁺MQ^– signal was simulated by subtracting the effects of spin evolution during the electron-transfer process. It was concluded that the <i>J</i> value of the P800⁺MQ^– radical pair is negative with an amplitude almost comparable to |<i>D</i>|. It is in contrast to a positive and small <i>J</i> value of the P700⁺PhyQ^– state in photosystem I (PS I). The results indicate similar but somewhat different locations/binding sites of quinones between hRC and PS I

Lipid-Controlled Stabilization of Charge-Separated States (P⁺Q_B^–) and Photocurrent Generation Activity of a Light-Harvesting–Reaction Center Core Complex (LH1-RC) from <i>Rhodopseudomonas palustris</i>

The photosynthetic light-harvesting–reaction center core complex (LH1-RC) is a natural excitonic and photovoltaic device embedded in a lipid membrane. In order to apply LH1-RCs as a biohybrid energy-producing material, some important issues must be addressed, including how to make LH1-RCs function as efficiently as possible. In addition, they should be characterized to evaluate how many active LH1-RCs efficiently work in artificial systems. We report here that an anionic phospholipid, phosphatidylglycerol (PG), stabilizes the charge-separated state (a photooxidized electron donor and reduced quinone pair, P⁺Q_B^–) of LH1-RC (from <i>Rhodopseudomonas palustris</i>) and enhances its activity in photocurrent generation. Steady-state fluorometric analysis demonstrated that PG enhances the formation of the P⁺Q_B^– state at lower irradiances. The photocurrent generation activity was analyzed via Michaelis–Menten kinetics, revealing that 38% of LH1-RCs reconstituted into the PG membrane generated photocurrent at a turnover frequency of 46 s^–1. PG molecules, which interact with LH1-RC in vivo, play the role of an active effector component for LH1-RC to enhance its function in the biohybrid system

Oxygen-Evolving Porous Glass Plates Containing the Photosynthetic Photosystem II Pigment–Protein Complex

The development of artificial photosynthesis has focused on the efficient coupling of reaction at photoanode and cathode, wherein the production of hydrogen (or energy carriers) is coupled to the electrons derived from water-splitting reactions. The natural photosystem II (PSII) complex splits water efficiently using light energy. The PSII complex is a large pigment–protein complex (20 nm in diameter) containing a manganese cluster. A new photoanodic device was constructed incorporating stable PSII purified from a cyanobacterium Thermosynechococcus vulcanus through immobilization within 20 or 50 nm nanopores contained in porous glass plates (PGPs). PSII in the nanopores retained its native structure and high photoinduced water splitting activity. The photocatalytic rate (turnover frequency) of PSII in PGP was enhanced 11-fold compared to that in solution, yielding a rate of 50–300 mol e^–/(mol PSII·s) with 2,6-dichloroindophenol (DCIP) as an electron acceptor. The PGP system realized high local concentrations of PSII and DCIP to enhance the collisional reactions in nanotubes with low disturbance of light penetration. The system allows direct visualization/determination of the reaction inside the nanotubes, which contributes to optimize the local reaction condition. The PSII/PGP device will substantively contribute to the construction of artificial photosynthesis using water as the ultimate electron source

Light-Driven Hydrogen Production by Hydrogenases and a Ru-Complex inside a Nanoporous Glass Plate under Aerobic External Conditions

Hydrogenases are powerful catalysts for light-driven H₂ production using a combination of photosensitizers. However, except oxygen-tolerant hydrogenases, they are immediately deactivated under aerobic conditions. We report a light-driven H₂ evolution system that works stably even under aerobic conditions. A [NiFe]-hydrogenase from <i>Desulfovibrio vulgaris</i> Miyazaki F was immobilized inside nanoporous glass plates (PGPs) with a pore diameter of 50 nm together with a ruthenium complex and methyl viologen as a photosensitizer and an electron mediator, respectively. After immersion of PGP into the medium containing the catalytic components, an anaerobic environment automatically established inside the nanopores even under aerobic external conditions upon irradiation with solar-simulated light; this system constantly evolved H₂ with an efficiency of 3.7 μmol H₂ m^–2 s^–1. The PGP system proposed in this work represents a promising first step toward the development of an O₂-tolerant solar energy conversion system

Molecular Assembly of Zinc Chlorophyll Derivatives by Using Recombinant Light-Harvesting Polypeptides with His-tag and Immobilization on a Gold Electrode

LH1-α and -β polypeptides, which make up the light-harvesting 1 (LH1) complex of purple photosynthetic bacteria, along with bacteriochlorophylls, have unique binding properties even for various porphyrin analogs. Herein, we used the porphyrin analogs, Zn-Chlorin and the Zn-Chlorin dimer, and examined their binding behaviors to the LH1-α variant, which has a His-tag at the C-terminus (MBP-rubα-YH). Zn-Chlorin and the Zn-Chlorin dimer could bind to MBP-rubα-YH and form a subunit-type assembly, similar to that from the native LH1 complex. These complexes could be immobilized onto Ni-nitrilotriacetic acid-modified Au electrodes, and the cathodic photocurrent was successfully observed by photoirradiation. Since Zn-Chlorins in this complex are too far for direct electron transfer from the electrode, a contribution of polypeptide backbone for efficient electron transfer was implied. These findings not only show interesting properties of LH1-α polypeptides but also suggest a clue to construct artificial photosynthesis systems using these peptide materials