Direct evidence for a deprotonated lysine serving as a H-bond "acceptor" in a photoreceptor protein.

Deprotonation or suppression of the pKa of the amino group of a lysine sidechain is a widely recognized phenomenon whereby the sidechain amino group transiently can act as a nucleophile at the active site of enzymatic reactions. However, a deprotonated lysine and its molecular interactions have not been directly experimentally detected. Here, we demonstrate a deprotonated lysine stably serving as an "acceptor" in a H-bond between the photosensor protein RcaE and its chromophore. Signal splitting and trans-H-bond J coupling observed by NMR spectroscopy provide direct evidence that Lys261 is deprotonated and serves as a H-bond acceptor for the chromophore NH group. Quantum mechanical/molecular mechanical calculations also indicate that this H-bond exists stably. Interestingly, the sidechain amino group of the lysine can act as both donor and acceptor. The remarkable shift in the H-bond characteristics arises from a decrease in solvation, triggered by photoisomerization. Our results provide insights into the dual role of this lysine. This mechanism has broad implications for other biological reactions in which lysine plays a role.

Molecular origins of absorption wavelength variation among phycocyanobilin-binding proteins.

Phycocyanobilin (PCB)-binding proteins, including cyanobacteriochromes and phytochromes, function as photoreceptors and exhibit a wide range of absorption maximum wavelengths. To elucidate the color-tuning mechanisms among these proteins, we investigated seven crystal structures of six PCB-binding proteins: Anacy_2551g3, AnPixJg2, phosphorylation-responsive photosensitive histidine kinase, RcaE, Sb.phyB(PG)-PCB, and Slr1393g3. Employing a quantum chemical/molecular mechanical approach combined with a polarizable continuum model, our analysis revealed that differences in absorption wavelengths among PCB-binding proteins primarily arise from variations in the shape of the PCB molecule itself, accounting for a ∼150 nm difference. Remarkably, calculated excitation energies sufficiently reproduced the absorption wavelengths of these proteins spanning ∼200 nm, including 728 nm for Anacy_2551g3. However, assuming the hypothesized lactim conformation resulted in a significant deviation from the experimentally measured absorption wavelength for Anacy_2551g3. The significantly red-shifted absorption wavelength of Anacy_2551g3 can unambiguously be explained by the significant overlap of molecular orbitals between the two pyrrole rings at both edges of the PCB chromophore without the need to hypothesize lactim formation.

Energetics of the H-Bond Network in Exiguobacterium sibiricum Rhodopsin.

Exiguobacterium sibiricum rhodopsin (ESR) functions as a light-driven proton pump utilizing Lys96 for proton uptake and maintaining its activity over a wide pH range. Using a combination of methodologies including the linear Poisson-Boltzmann equation and a quantum mechanical/molecular mechanical approach with a polarizable continuum model, we explore the microscopic mechanisms underlying its pumping activity. Lys96, the primary proton uptake site, remains deprotonated owing to the loss of solvation in the ESR protein environment. Asp85, serving as a proton acceptor group for Lys96, does not form a low-barrier H-bond with His57. Instead, deprotonated Asp85 forms a salt-bridge with protonated His57, and the proton is predominantly located at the His57 moiety. Glu214, the only acidic residue at the end of the H-bond network exhibits a pKa value of ∼6, slightly elevated due to solvation loss. It seems likely that the H-bond network [Asp85···His57···H2O···Glu214] serves as a proton-conducting pathway toward the protein bulk surface.

Genes for the type-I reaction center and galactolipid synthesis are required for chlorophyll a accumulation in a purple photosynthetic bacterium.

Anoxygenic photosynthesis is diversified into two classes: chlorophototrophy based on a bacterial type-I or type-II reaction center (RC). Whereas the type-I RC contains both bacteriochlorophyll and chlorophyll, type-II RC-based phototrophy relies only on bacteriochlorophyll. However, type-II phototrophic bacteria theoretically have the potential to produce chlorophyll a by the addition of an enzyme, chlorophyll synthase, because the direct precursor for the enzyme, chlorophyllide a, is produced as an intermediate of BChl a biosynthesis. In this study, we attempted to modify the type-II proteobacterial phototroph Rhodovulum sulfidophilum to produce chlorophyll a by introducing chlorophyll synthase, which catalyzes the esterification of a diterpenoid group to chlorophyllide a thereby producing chlorophyll a. However, the resulting strain did not accumulate chlorophyll a, perhaps due to absence of endogenous chlorophyll a-binding proteins. We further heterologously incorporated genes encoding the type-I RC complex to provide a target for chlorophyll a. Heterologous expression of type-I RC subunits, chlorophyll synthase, and galactolipid synthase successfully afforded detectable accumulation of chlorophyll a in Rdv. sulfidophilum. This suggests that the type-I RC can work to accumulate chlorophyll a and that galactolipids are likely necessary for the type-I RC assembly. The evolutionary acquisition of type-I RCs could be related to prior or concomitant acquisition of galactolipids and chlorophylls.

Energy transfer dynamics and the mechanism of biohybrid photosynthetic antenna complexes chemically linked with artificial chromophores.

A light-harvesting strategy is crucial for the utilisation of solar energy. In this study, we addressed the expanding light-harvesting (LH) wavelength of photosynthetic LH complex 2 (LH2, from Rhodoblastus acidophilus strain 10050) through covalent conjugation with extrinsic chromophores. To further understand the conjugation architecture and mechanism of excitation energy transfer (EET), we examined the effects of the linker length and spectral overlap integral between the emission and absorption spectra of the energy donor and acceptor pigments. In the former case, contrary to the intuition based on the Förster resonance energy transfer (FRET) theory, the observed energy transfer rate was similar regardless of the linker length, and the energy transfer efficiency increased with longer linkers. In the latter case, despite the energy transfer rate increases at higher spectral overlaps, it was quantitatively inconsistent with the FRET theory. The mechanism of EET beyond the FRET theory was discussed in terms of the higher-lying exciton state of B850, which mediates efficient EET despite the small spectral overlap. This systematic investigation provides insights for the development of efficient artificial photosynthetic systems.

Design of PG-Surfactants Bearing Polyacrylamide Polymer Chain to Solubilize Membrane Proteins in a Surfactant-Free Buffer.

The development of techniques capable of using membrane proteins in a surfactant-free aqueous buffer is an attractive research area, and it should be elucidated for various membrane protein studies. To this end, we examined a method using new solubilization surfactants that do not detach from membrane protein surfaces once bound. The designed solubilization surfactants, DKDKC12K-PAn (n = 5, 7, and 18), consist of two parts: one is the lipopeptide-based solubilization surfactant part, DKDKC12K, fand the other is the covalently connected linear polyacrylamide (PA) chain with different Mw values of 5, 7, or 18 kDa. Intermolecular interactions between the PA chains in DKDKC12K-PAn concentrated on the surfaces of membrane proteins via amphiphilic binding of the DKDKC12K part to the integral membrane domain was observed. Therefore, DKDKC12K-PAn (n = 5, 7, and 18) could maintain a bound state even after removal of the unbound by ultrafiltration or gel-filtration chromatography. We used photosystem I (PSI) from Thermosynecoccus vulcanus as a representative to assess the impacts of new surfactants on the solubilized membrane protein structure and functions. Based on the maintenance of unique photophysical properties of PSI, we evaluated the ability of DKDKC12K-PAn (n = 5, 7, and 18) as a new solubilization surfactant.

Efficient hydrogen production using photosystem I enhanced by artificial light harvesting dye.

In this study, we improved the hydrogen production efficiency by combining photosystem I with an artificial light harvesting dye, Lumogen Red. In the reaction system, Lumogen Red allows light absorption and energy transfer to photosystem I by Förster resonance energy transfer; therefore, the Pt nanoparticles act as active sites for hydrogen generation.

Design of New Extraction Surfactants for Membrane Proteins from Peptide Gemini Surfactants.

The development of additional extraction surfactants for membrane proteins is necessary for membrane protein research, since optimal combinations for the successful extraction of target membrane proteins from biological membranes that minimize protein denaturation are hard to predict. In particular, those that have a unique basal molecular framework are quite attractive and highly desired in this research field. In this study, we successfully constructed a new extraction surfactant for membrane proteins, NPDGC12KK, from the peptide-gemini-surfactant (PG-surfactant) molecular framework. The PG-surfactant is a U-shaped lipopeptide scaffold, consisting of a short linker peptide (-X-) between two long alkyl-chain-modified Cys residues and a peripheral peptide (Y-) at the N-terminal side of long alkyl-chain-modified Cys residues. Using photosystem I (PSI) and photosystem II (PSII) derived from Thermosynecoccus vulcanus as representative membrane proteins, we evaluated whether NPDGC12KK could solubilize membrane proteins while maintaining structure and functions. Neither the membrane integral domain nor the cytoplasmic domain of PSI and PSII suffered any damage upon the use of NPDGC12KK based on detailed photophysical measurements. Using thylakoid membranes of T. vulcanus as a representative biological membrane sample, we performed experiments to extract membrane proteins, such as PSI and PSII. Based on the extraction efficiency and maintenance of protein supramolecular structure established using clear native-PAGE analyses, we proved that NPDGC12KK functions as a novel class of peptide-containing extraction surfactants for membrane proteins.

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.

Extension of Light-Harvesting Ability of Photosynthetic Light-Harvesting Complex 2 (LH2) through Ultrafast Energy Transfer from Covalently Attached Artificial Chromophores.

Introducing appropriate artificial components into natural biological systems could enrich the original functionality. To expand the available wavelength range of photosynthetic bacterial light-harvesting complex 2 (LH2 from Rhodopseudomonas acidophila 10050), artificial fluorescent dye (Alexa Fluor 647: A647) was covalently attached to N- and C-terminal Lys residues in LH2 α-polypeptides with a molar ratio of A647/LH2 ≃ 9/1. Fluorescence and transient absorption spectroscopies revealed that intracomplex energy transfer from A647 to intrinsic chromophores of LH2 (B850) occurs in a multiexponential manner, with time constants varying from 440 fs to 23 ps through direct and B800-mediated indirect pathways. Kinetic analyses suggested that B800 chromophores mediate faster energy transfer, and the mechanism was interpretable in terms of Förster theory. This study demonstrates that a simple attachment of external chromophores with a flexible linkage can enhance the light harvesting activity of LH2 without affecting inherent functions of energy transfer, and can achieve energy transfer in the subpicosecond range. Addition of external chromophores, thus, represents a useful methodology for construction of advanced hybrid light-harvesting systems that afford solar energy in the broad spectrum.

Harvesting Far-Red Light by Chlorophyll f in Photosystems I and II of Unicellular Cyanobacterium strain KC1.

Cells of a unicellular cyanobacterium strain KC1, which were collected from Japanese fresh water Lake Biwa, formed chlorophyll (Chl) f at 6.7%, Chl a' at 2.0% and pheophytin a at 0.96% with respect to Chl a after growth under 740 nm light. The far-red-acclimated cells (Fr cells) formed extra absorption bands of Chl f at 715 nm in addition to the major Chl a band. Fluorescence lifetimes were measured. The 405-nm laser flash, which excites mainly Chl a in photosystem I (PSI), induced a fast energy transfer to multiple fluorescence bands at 720-760 and 805 nm of Chl f at 77 K in Fr cells with almost no PSI-red-Chl a band. The 630-nm laser flash, which mainly excited photosystem II (PSII) through phycocyanin, revealed fast energy transfer to another set of Chl f bands at 720-770 and 810 nm as well as to the 694-nm Chl a fluorescence band. The 694-nm band did not transfer excitation energy to Chl f. Therefore, Chl a in PSI, and phycocyanin in PSII of Fr cells transferred excitation energy to different sets of Chl f molecules. Multiple Chl f forms, thus, seem to work as the far-red antenna both in PSI and PSII. A variety of cyanobacterial species, phylogenically distant from each other, seems to use a Chl f antenna in far-red environments, such as under dense biomats, in colonies, or under far-red LED light.

Absence of a link between stabilized charge-separated state and structural changes proposed from crystal structures of a photosynthetic reaction center.

Structural differences between illuminated and unilluminated crystal structures led to the proposal that the charge-separated state was stabilized by structural changes in its membrane extrinsic protein subunit H in a bacterial photosynthetic reaction center [Katona, G. et al. Nat. Struct. Mol. Biol. 2005, 12, 630-631]. Here, we explored the proposal by titrating all titratable sites and calculating the redox potential (Em) values in these crystal structures. Contrary to the expected charge-separated states, Em for quinone, Em(QA/QA•-), is even lower in the proposed charge-separated structure than in the ground-state structure. The subunit-H residues, which were proposed to exhibit electron-density changes in the two crystal structures, contribute to an Em(QA/QA•-) difference of only <0.5 mV. Furthermore, the protonation states of the titratable residues in the entire reaction center are practically identical in the two structures. These findings indicate that the proposed structural differences are irrelevant to explaining the significant prolongation of the charge-separated-state lifetime.

Molecular assembly of zinc chlorophyll derivatives by using recombinant light-harvesting polypeptides with His-tag and immobilization on a gold electrode.

LH1-α and -ß 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.

Creation of cross-linked bilayer membranes that can incorporate membrane proteins from oligo-Asp-based peptide gemini surfactants.

We designed novel bilayer-forming amphiphiles based on the cyclic oligo-Asp-based peptide gemini (PG) surfactants cr-D2C12 and cr-D3C12, which consist of -Cys(Asp)nCys- (n = 2 or 3) as a core peptide and two Cys residues containing a dodecylamidomethyl group. Dynamic light scattering and transmission electron microscopy measurements revealed the formation of spherical bilayer membranes that could incorporate the light-harvesting antenna complex 2 (LH2) from Rhodopseudomonas acidophila . Furthermore, this proteoliposome-like conjugate could be assembled onto cationized glass and mica to form planar bilayer membranes incorporating LH2. Using atomic force microscopy, we observed LH2 protruding (ca. 1.2-1.5 nm) from flat terraces of the planar bilayer membranes formed from cr-D2C12 or cr-D3C12. Thus, our designed PG surfactants are a new class of bilayer-forming amphiphiles that may be applied to the study of various membrane proteins.

Application of peptide gemini surfactants as novel solubilization surfactants for photosystems I and II of cyanobacteria.

We designed novel peptide gemini surfactants (PG-surfactants), DKDKC12K and DKDKC12D, which can solubilize Photosystem I (PSI) of Thermosynecoccus elongatus and Photosystem II (PSII) of Thermosynecoccus vulcanus in an aqueous buffer solution. To assess the detailed effects of PG-surfactants on the original supramolecular membrane protein complexes and functions of PSI and PSII, we applied the surfactant exchange method to the isolated PSI and PSII. Spectroscopic properties, light-induced electron transfer activity, and dynamic light scattering measurements showed that PSI and PSII could be solubilized not only with retention of the original supramolecular protein complexes and functions but also without forming aggregates. Furthermore, measurement of the lifetime of light-induced charge-separation state in PSI revealed that both surfactants, especially DKDKC12D, displayed slight improvement against thermal denaturation below 60 °C compared with that using ß-DDM. This degree of improvement in thermal resistance still seems low, implying that the peptide moieties did not interact directly with membrane protein surfaces. By conjugating an electron mediator such as methyl viologen (MV(2+)) to DKDKC12K (denoted MV-DKDKC12K), we obtained derivatives that can trap the generated reductive electrons from the light-irradiated PSI. After immobilization onto an indium tin oxide electrode, a cathodic photocurrent from the electrode to the PSI/MV-DKDKC12K conjugate was observed in response to the interval of light irradiation. These findings indicate that the PG-surfactants DKDKC12K and DKDKC12D provide not only a new class of solubilization surfactants but also insights into designing other derivatives that confer new functions on PSI and PSII.

Difference in the Charge-Separation Energetics between Distinct Conformers in the PixD Photoreceptor.

Blue light using flavin (BLUF) domain proteins are photoreceptors in various organisms. The PixD BLUF domain can adopt two conformations, W91out and W91in, with Trp91 either proximal or distal to flavin (FMN). Using a quantum mechanical/molecular mechanical/polarizable continuum model approach, the energetics of charge-separated and biradical states in the two conformations were investigated. In the W91out conformation, the charge-separated state (FMN•-) is more stable than the photoexcited state (FMN*), whereas it is less stable due to an electrostatic repulsive interaction with the Ser28 side chain in the W91in conformation. This leads to a lower activation energy for the charge separation in the W91out conformation, resulting in a faster charge separation compared to that in the W91in conformation. In the W91out conformation, the radical state (FMNH•) is more stable than FMN•- and forms from FMN•-, leading to reorientation of the Gln50 side chain adjacent to FMN and formation of a hydrogen bond between Gln50 and FMN. Subsequently, a signaling state forms through charge recombination. In contrast, in the W91in conformation, FMN•- cannot proceed further, returning to the dark-adapted state, as FMNH• is less stable. Thus, formation of the signaling state exclusively occurs in the W91out conformation.

Mechanism of Absorption Wavelength Shift of Bacteriorhodopsin During Photocycle.

Bacteriorhodopsin, a light-driven proton pump, alters the absorption wavelengths in the range of 410-617 nm during the photocycle. Here, we report the absorption wavelengths, calculated using 12 bacteriorhodopsin crystal structures (including the BR, BR13-cis, J, K0, KE, KL, L, M, N, and O state structures) and a combined quantum mechanical/molecular mechanical/polarizable continuum model (QM/MM/PCM) approach. The QM/MM/PCM calculations reproduced the experimentally measured absorption wavelengths with a standard deviation of 4 nm. The shifts in the absorption wavelengths can be explained mainly by the following four factors: (i) retinal Schiff base deformation/twist induced by the protein environment, leading to a decrease in the electrostatic interaction between the protein environment and the retinal Schiff base; (ii) changes in the protonation state of the protein environment, directly altering the electrostatic interaction between the protein environment and the retinal Schiff base; (iii) changes in the protonation state; or (iv) isomerization of the retinal Schiff base, where the absorption wavelengths of the isomers originally differ.

Photosynthetic oxygen evolution in mesoporous silica material: adsorption of photosystem II reaction center complex into 23 nm nanopores in SBA.

An oxygen-evolving photosynthetic reaction center complex (PSII) was adsorbed into nanopores in SBA, a mesoporous silica compound. We purified the dimer of PSII complex from a thermophilic cyanobacterium, Thermosynechococcus vulcanus, which grows optimally at 57 °C. The thermally stable PSII dimeric complex has a diameter of 20 nm and a molecular mass of 756 kDa and binds more than 60 chlorophylls. The SBA particles, with average internal pore diameters of 15 nm (SBA(15)) and 23 nm (SBA(23)), adsorbed 4.7 and 15 mg of PSII/g SBA, respectively. Measurement with a confocal laser-scanning microscope indicated the adsorption of PSII to the surface and the inner space of the SBA(23) particles, indicating the adsorption of PSII into the 23 nm silica nanopores. PSII did not bind to the inner pores of SBA(15). PSII bound to SBA(23) showed the high and stable activity of a photosynthetic oxygen-evolving reaction, indicating the light-driven electron transport from water to the quinone molecules added in the outer medium. The PSII-SBA conjugate can be a new material for photosensors and artificial photosynthetic systems.

Direct Energy Transfer from Allophycocyanin-Free Rod-Type CpcL-Phycobilisome to Photosystem I.

Phycobilisomes (PBSs) are photosynthetic antenna megacomplexes comprising pigment-binding proteins (cores and rods) joined with linker proteins. A rod-type PBS that does not have a core is connected to photosystem I (PSI) by a CpcL linker protein, which stabilizes a red-form of the phycocyanobilin (red-PCB) in the rod. However, quantitative information on the energy transfer from red-type PBS to PSI has not been determined. Herein, the isolated supercomplex of the rod-type PBS and the PSI tetramer from Anabaena sp. PCC 7120 were probed by time-resolved spectroscopy at 77 K and by decay-associated spectral analysis to show that red-PCB mediates the fast and efficient (time constant = 90 ps, efficiency = 95%) transfer of excitation energy from PCB to chlorophyll a (Chl a). According to the Förster energy transfer mechanism, this high efficiency corresponds to a 4 nm distance between red-PCB and Chl a, suggesting that ß-84 PCB in the rod acts as red-PCB.

Mechanism of absorption wavelength shifts in anion channelrhodopsin-1 mutants.

Using a quantum mechanical/molecular mechanical approach, we show the mechanisms of how the protein environment of Guillardia theta anion channelrhodopsin-1 (GtACR1) can shift the absorption wavelength. The calculated absorption wavelengths for GtACR1 mutants, M105A, C133A, and C237A are in agreement with experimentally measured wavelengths. Among 192 mutant structures investigated, mutations at Thr101, Cys133, Pro208, and Cys237 are likely to increase the absorption wavelength. In particular, T101A GtACR1 was expressed in HEK293T cells. The measured absorption wavelength is 10 nm higher than that of wild type, consistent with the calculated wavelength. (i) Removal of a polar residue from the Schiff base moiety, (ii) addition of a polar or acidic residue to the ß-ionone ring moiety, and (iii) addition of a bulky residue to increase the planarity of the ß-ionone and Schiff base moieties are the basis of increasing the absorption wavelength.