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Photosystem I (PSI) is one of the two photosystems in photosynthesis, and performs a series of electron transfer reactions leading to the reduction of ferredoxin. In higher plants, PSI is surrounded by four light-harvesting complex I (LHCI) subunits, which harvest and transfer energy efficiently to the PSI core. The crystal structure of PSI-LHCI supercomplex has been analyzed up to 2.6 Å resolution, providing much information on the arrangement of proteins and cofactors in this complicated supercomplex. Here we have optimized crystallization conditions, and analyzed the crystal structure of PSI-LHCI at 2.4 Å resolution. Our structure showed some shift of the LHCI, especially the Lhca4 subunit, away from the PSI core, suggesting the indirect connection and inefficiency of energy transfer from this Lhca subunit to the PSI core. We identified five new lipids in the structure, most of them are located in the gap region between the Lhca subunits and the PSI core. These lipid molecules may play important roles in binding of the Lhca subunits to the core, as well as in the assembly of the supercomplex. The present results thus provide novel information for the elucidation of the mechanisms for the light-energy harvesting, transfer and assembly of this supercomplex.

The function of proteins depends on their structural flexibility and conformational change. By utilizing silicon-nanowire-based single-molecule electrical circuits, here we present a label-free real-time measurement method that can directly monitor conformational changes of a photosynthetic LH1-RC complex, reaching the ultimate goal of analytic chemistry. These results manifest that the conformation of the LH1-RC complex vibrates among four conformations with strong temperature dependence. At the optimal temperature, States 2 and 3 occupy the main conformations of the LH1-RC complex, and its conformational variation mostly emerges as anharmonic vibration modes, which contributes to photon acquisition and heat transmission. The influence of light activation on occurrence percentage is observed, resulting from light-driven quivering of pigments. Therefore, this avenue proves to be an efficient platform for revealing the fundamental mechanisms of various biological processes in vitro.

To uncover the mechanism behind the high photo-electronic conversion efficiency in natural photosynthetic complexes it is essential to trace the dynamics of electronic and vibrational quantum coherences. Here we apply wavelet analysis to two-dimensional electronic spectroscopy data for three purple bacterial reaction centers with mutations that produce drastically different rates of primary charge separation. From the frequency distribution and dynamic evolution features of the quantum beating, electronic coherence with a dephasing lifetime of ~50 fs, vibronic coherence with a lifetime of ~150 fs and vibrational/vibronic coherences with a lifetime of 450 fs are distinguished. We find that they are responsible for, or couple to, different specific steps during the primary charge separation process, i.e., intradimer charge transfer inside the special bacteriochlorophyll pair followed by its relaxation and stabilization of the charge-transfer state. The results enlighten our understanding of how quantum coherences participate in, and contribute to, a biological electron transfer reaction.

The mildly thermophilic purple phototrophic bacterium Allochromatium tepidum provides a unique model for investigating various intermediate phenotypes observed between those of thermophilic and mesophilic counterparts. The core light-harvesting (LH1) complex from A. tepidum exhibits an absorption maximum at 890 nm and mildly enhanced thermostability, both of which are Ca

The light-harvesting-reaction center (LH1-RC) core complex of purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides is characterized by the presence of both a dimeric form and a monomeric form. Following structure determination of the monomeric LH1-RC including its previously unrecognized component designated protein-U (Nat. Commun. 12, 6300, 2021), here we present cryo-EM structures of the dimeric LH1-RC from native Rba. sphaeroides IL106 at 2.75 Å resolution and from an LH1-RC monomer lacking protein-U (ΔU) at 2.64 Å resolution. The native dimeric core complex reveals many asymmetric features in the arrangement of its two monomeric components including the structural integrity of protein-U, the overall LH1 organization, and the rigidities of the proteins and pigments that form the complex. PufX polypeptides play a critical role in connecting two monomers, with one PufX interacting at its N-terminus with another PufX and an LH1 β-polypeptide in another monomer, in good agreement with biochemical analyses. One of the proteins-U was only partially identified in the dimeric structure, signaling significantly different degrees of disorder in the two monomers. The ΔU LH1-RC monomer revealed a half-moon-shaped structure containing 11 α- and 10 β-polypeptides (compared with 14 of each in the wild type), indicating a critical role for protein-U in controlling the number of αβ-subunits required for correct assembly and stabilization of the LH1-RC dimer. The structural features are discussed in relation to the unusual topology of intracytoplasmic photosynthetic membranes and an assembly model proposed for the native Rba. sphaeroides dimeric LH1-RC complex in membranes of wild-type cells.

In this work, the 3D printing technique based on selective laser melting (SLM) was applied to fabricate a Ni-free and Ag-bearing Zr-based bulk metallic glass (BMG) and porous architectures with complex geometries and various functions. The 3D-printed Zr-based BMG was systematically examined by a set of experiments, including microstructure characterizations, mechanical testing, wear and corrosion resistance measurements, in vitro biocompatibility and antibacterial capability assessments. We revealed that the 3D-printed BMG presents almost fully amorphous structure with a high strength (>1700 MPa) and relatively low Young's modulus (∼80 GPa). The wear resistance was comparable to the commercial Ti6Al4V medical alloy in simulated body fluid (SBF), and the corrosion resistance surpassed Ti6Al4V due to lower corrosion rate as well as smaller passive current density. In addition, the 3D-printed BMG showed excellent biocompatibility, as indicated by superior cell proliferation support and ppb level of ion release. More importantly, the 3D printed BMG also exhibited excellent antibacterial ability against E. coli due to the inclusion of a trace amount of Ag. Furthermore, for application purposes, porous BMG components with pore size of 500 μm were printed by SLM technique, which displayed an interconnected porosity of 70% and extremely low Young's modulus of 13 GPa (being comparable to the human bone tissue), while maintaining a high yield strength of 350 MPa. The findings in this work suggested that the SLM-based 3D printing technique could be promising for manufacturing Zr-based BMGs with complex geometries in a wide range of biomedical applications.

© 2021 American Chemical Society.Despite extensive study, mysteries remain regarding the highly efficient ultrafast charge separation processes in photosynthetic reaction centers (RCs). In this work, transient Stark signals were found to be present in ultrafast two-dimensional electronic spectra recorded for purple bacterial RCs at 77 K. These arose from the electric field that is inherent to the intradimer charge-transfer intermediate of the bacteriochlorophyll pair (P), PA+PB-. By comparing three mutated RCs, a correlation was found between the efficient formation of PAA+PB- and a fast charge separation rate. Importantly, the energy level of P∗ was changed due to the Stark shift, influencing the driving force for P∗ → P+BA- electron transfer and hence its rate. Furthermore, the orientation and amplitude of the inherent electric field varied in different ways upon different mutation, leading to contrasting changes in the rates. This mechanism of modulation provides a solution to a long-lasting inconsistency between experimental observations and activation energy theory.

Rhodospirillum (Rsp.) rubrum is one of the most widely used model organisms in bacterial photosynthesis. This purple phototroph is characterized by the presence of both rhodoquinone (RQ) and ubiquinone as electron carriers and bacteriochlorophyll (BChl) a esterified at the propionic acid side chain by geranylgeraniol (BChl aG) instead of phytol. Despite intensive efforts, the structure of the light-harvesting-reaction center (LH1-RC) core complex from Rsp. rubrum remains at low resolutions. Using cryo-EM, here we present a robust new view of the Rsp. rubrum LH1-RC at 2.76 A resolution. The LH1 complex forms a closed, slightly elliptical ring structure with 16 αβ-polypeptides surrounding the RC. Our biochemical analysis detected RQ molecules in the purified LH1-RC, and the cryo-EM density map specifically positions RQ at the QA site in the RC. The geranylgeraniol side chains of BChl aG coordinated by LH1 β-polypeptides exhibit a highly homologous tail-up conformation that allows for interactions with the bacteriochlorin rings of nearby LH1 α-associated BChls aG. The structure also revealed key protein-protein interactions in both N- and C-terminal regions of the LH1 αβ-polypeptides, mainly within a face-to-face structural subunit. Our high-resolution Rsp. rubrum LH1-RC structure provides new insight for evaluating past experimental and computational results obtained with this old organism over many decades and lays the foundation for more detailed exploration of light-energy conversion, quinone transport, and structure-function relationships in this pigment-protein complex.

Halorhodospira (Hlr.) halochloris is a unique phototrophic purple bacterium because it is a triple extremophile-the organism is thermophilic, alkalophilic, and halophilic. The most striking photosynthetic feature of Hlr. halochloris is that the bacteriochlorophyll (BChl) b-containing core light-harvesting (LH1) complex surrounding its reaction center (RC) exhibits its LH1 Q

One of the primary objectives in chemistry research is to observe atomic motions during reactions in real time. Although X-ray free-electron lasers (XFELs) have facilitated the capture of reaction intermediates using time-resolved serial femtosecond crystallography (TR-SFX), only a few natural photoactive proteins have been investigated using this method, mostly due to the lack of suitable phototriggers. Here we report the genetic encoding of a xanthone amino acid (FXO), as an efficient phototrigger, into a rationally designed human liver fatty-acid binding protein mutant (termed XOM), which undergoes photo-induced C-H bond transformation with high selectivity and quantum efficiency. We solved the structures of XOM before and 10-300 ns after flash illumination, at 1.55-1.70 Å resolutions, and captured the elusive excited-state intermediates responsible for precise C-H bond activation. We expect that most redox enzymes can now be investigated by TR-SFX, using our method, to reveal reaction intermediates key for their efficiency and selectivity.

The core light-harvesting complexes (LH1) in bacteriochlorophyll (BChl) b-containing purple phototrophic bacteria are characterized by a near-infrared absorption maximum around 1010 nm. The determinative cause for this ultra-redshift remains unclear. Here, we present results of circular dichroism (CD) and resonance Raman measurements on the purified LH1 complexes in a reaction center-associated form from a mesophilic and a thermophilic Blastochloris species. Both the LH1 complexes displayed purely positive CD signals for their Qy transitions, in contrast to those of BChl a-containing LH1 complexes. This may reflect differences in the conjugation system of the bacteriochlorin between BChl b and BChl a and/or the differences in the pigment organization between the BChl b- and BChl a-containing LH1 complexes. Resonance Raman spectroscopy revealed remarkably large redshifts of the Raman bands for the BChl b C3-acetyl group, indicating unusually strong hydrogen bonds formed with LH1 polypeptides, results that were verified by a published structure. A linear correlation was found between the redshift of the Raman band for the BChl C3-acetyl group and the change in LH1-Qy transition for all native BChl a- and BChl b-containing LH1 complexes examined. The strong hydrogen bonding and π–π interactions between BChl b and nearby aromatic residues in the LH1 polypeptides, along with the CD results, provide crucial insights into the spectral and structural origins for the ultra-redshift of the long-wavelength absorption maximum of BChl b-containing phototrophs.

Light-harvesting-1 (LH1)-reaction center (RC) super-complex is a membrane protein-pigment complex existing in purple photosynthetic bacteria, where LH1 absorbs light energy and transfers them rapidly and efficiently to RC to initiate the charge separation and electron transfer reactions. The structure of LH1-RC has been reported at relatively low resolutions from several different species of bacteria previously, but was solved at an atomic resolution recently from a thermophilic photosynthetic bacterium Thermochromatium tepidum. This high-resolution structure revealed the detailed organization of the super-complex including a number of unique features that are important for its functioning, such as a more intact RC structure, transporting routes for quinones to replace the bound QB as well as for the in-and-out of the closed LH1 ring, detailed coordinating environment of the Ca2+ ions in LH1 important for the remarkable red shift of the absorption spectrum, as well as for the enhanced thermostability. These results thus greatly advance our understanding on the mechanisms of energy transfer, quinone exchange, the red shift in the LH1-Qy transition and the enhanced thermal stability, in this super-complex.

Light-harvesting complex 1 (LH1) and the reaction centre (RC) form a membrane-protein supercomplex that performs the primary reactions of photosynthesis in purple photosynthetic bacteria. The structure of the LH1-RC complex can provide information on the arrangement of protein subunits and cofactors; however, so far it has been resolved only at a relatively low resolution. Here we report the crystal structure of the calcium-ion-bound LH1-RC supercomplex of Thermochromatium tepidum at a resolution of 1.9 A. This atomic-resolution structure revealed several new features about the organization of protein subunits and cofactors. We describe the loop regions of RC in their intact states, the interaction of these loop regions with the LH1 subunits, the exchange route for the bound quinone QB with free quinone molecules, the transport of free quinones between the inside and outside of the LH1 ring structure, and the detailed calcium-ion-binding environment. This structure provides a solid basis for the detailed examination of the light reactions that occur during bacterial photosynthesis.

Rhodobacter (Rba.) sphaeroides is the most widely used model organism in bacterial photosynthesis. The light-harvesting-reaction center (LH1-RC) core complex of this purple phototroph is characterized by the co-existence of monomeric and dimeric forms, the presence of the protein PufX, and approximately two carotenoids per LH1 αβ-polypeptides. Despite many efforts, structures of the Rba. sphaeroides LH1-RC have not been obtained at high resolutions. Here we report a cryo-EM structure of the monomeric LH1-RC from Rba. sphaeroides strain IL106 at 2.9 Å resolution. The LH1 complex forms a C-shaped structure composed of 14 αβ-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its channels. Our findings offer an exciting new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general., Rhodobacter (Rba.) sphaeroides is a model organism for studying bacterial photosynthesis. Here, the authors present the 2.9 Å cryo-EM structure of the monomeric light-harvesting-reaction center core complex from Rba. sphaeroides strain IL106, which revealed the position and conformation of PufX and the presence of an additional component protein-U, an integral membrane protein.

Halorhodospira (Hlr.) halochloris is a unique phototrophic purple bacterium because it is a triple extremophile—the organism is thermophilic, alkalophilic, and halophilic. The most striking photosynthetic feature of Hlr. halochloris is that the bacteriochlorophyll (BChl) b-containing core light-harvesting (LH1) complex surrounding its reaction center (RC) exhibits its LH1 Qy absorption maximum at 1016 nm, which is the lowest transition energy among phototrophic organisms. Here we report that this extraordinarily red-shifted LH1 Qy band of Hlr. halochloris exhibits interconvertible spectral shifts depending on the electrostatic charge distribution around the BChl b molecules. The 1016 nm band of the Hlr. halochloris LH1-RC complex was blue-shifted to 958 nm upon desalting or pH decrease but returned to its original position when supplemented with salts or pH increase. Resonance Raman analysis demonstrated that these interconvertible spectral shifts are not associated with the strength of hydrogen-bonding interactions between BChl b and LH1 polypeptides. Furthermore, circular dichroism signals for the LH1 Qy transition of Hlr. halochloris appeared with a positive sign (as in BChl b-containing Blastochloris species) and opposite those of BChl a-containing purple bacteria, possibly due to a combined effect of slight differences in the transition dipole moments between BChl a and BChl b and in the interactions between adjacent BChls in their assembled state. Based on these findings and LH1 amino acid sequences, it is proposed that Hlr. halochloris evolved its unique and tunable light-harvesting system with electrostatic charges in order to carry out photosynthesis and thrive in its punishing hypersaline and alkaline habitat.

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex.

Ultrafast excitation relaxation within a manifold exciton state and long-lived vibrational coherence are two universal characteristics of photosynthetic antenna complexes. In this work, we studied the two-dimensional electronic spectra of two core light-harvesting (LH1) complexes of Thermochromatium (Tch.) tepidum, native Ca2+-LH1 and modified Ba2+-LH1. The role of the vibrational coherence in the exciton relaxation was revealed by comparing the two LH1 with similar structures but different electronic properties and by the evolution of the exciton and vibrational coherence as a function of temperature.

The light-harvesting 1 reaction center (LH1-RC) complex in the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum binds Ca ions as cofactors, and Ca-binding is largely involved in its characteristic Q y absorption at 915 nm and enhanced thermostability. Ca2+ can be biosynthetically replaced by Sr2+ in growing cultures of Tch. tepidum. However, the resulting Sr2+-substituted LH1-RC complexes in such cells do not display the absorption maximum and thermostability of those from Ca2+-grown cells, signaling that inherent structural differences exist in the LH1 complexes between the Ca2+- and Sr2+-cultured cells. In this study, we examined the effects of the biosynthetic Sr2+-substitution and limited proteolysis on the spectral properties and thermostability of the Tch. tepidum LH1-RC complex. Preferential truncation of two consecutive, positively charged Lys residues at the C-terminus of the LH1 α-polypeptide was observed for the Sr2+-cultured cells. A proportion of the truncated LH1 α-polypeptide increased during repeated subculturing in the Sr2+-substituted medium. This result suggests that the truncation is a biochemical adaptation to reduce the electrostatic interactions and/or steric repulsion at the C-terminus when Sr2+ substitutes for Ca2+ in the LH1 complex. Limited proteolysis of the native Ca2+-LH1 complex with lysyl protease revealed selective truncations at the Lys residues in both C- and N-terminal extensions of the α- and β-polypeptides. The spectral properties and thermostability of the partially digested native LH1-RC complexes were similar to those of the biosynthetically Sr2+-substituted LH1-RC complexes in their Ca2+-bound forms. Based on these findings, we propose that the C-terminal domain of the LH1 α-polypeptide plays important roles in retaining proper structure and function of the LH1-RC complex in Tch. tepidum.

Low molecular weight (MW) polyols are organic osmolytes influencing protein structure and activity. We have intended to investigate the effects of low MW polyols on the optical and the excited-state properties of the light-harvesting complex 2 (LH2) isolated from the photosynthetic bacterium Thermochromatium (Tch.) tepidum, a thermophile growing at ∼50 °C. Steady state spectroscopy demonstrated that, on increasing glycerol or sorbitol fractions up to 60% (polyol/water, v/v), the visible absorption of carotenoids (Crts) remained unchanged, while the near infrared Qy absorption of bacteriochlorophyll a (BChl) at 800 nm (B800) and 850 nm (B850) varied slightly. Further increasing the fraction of glycerol (but not sorbitol) to 80% (v/v) induced distinct changes of the near infrared absorption and fluorescence spectra. Transient absorption spectroscopy revealed that, following the fast processes of BChl-to-Crt triplet energy transfer, rather weak Qy signals of B800 and B850 remained and evolved in phase with the kinetics of triplet excited state Crt (3Crt*), which are attributed to the Qy band shift as a result of 3Crt*-BChl interaction. The steady state and the transient spectral responses of the Qy bands are found to correlate intimately with the water activity varying against polyol MW and mixing ratio, which are rationalized by the change of the hydration status of the C- and N-termini of LH2. Our results suggest that, with reference to the mesophilic purple bacterium Rhodobacter sphaeroides 2.4.1, Tch. tepidum adopts substantially more robust LH2 hydration against the osmotic effects from the low MW polyols.

Methylation of nucleotides in ribosomal RNAs (rRNAs) is a ubiquitous feature that occurs in all living organisms. The formation of methylated nucleotides is performed by a variety of RNA-methyltransferases. Chloroplasts of plant cells result from an endosymbiotic event and possess their own genome and ribosomes. However, enzymes responsible for rRNA methylation and the function of modified nucleotides in chloroplasts remain to be determined. Here, we identified an rRNA methyltransferase, CMAL (Chloroplast MraW-Like), in the Arabidopsis chloroplast and investigated its function. CMAL is the Arabidopsis ortholog of bacterial MraW/ RsmH proteins and accounts to the N4-methylation of C1352 in chloroplast 16S rRNA, indicating that CMAL orthologs and this methyl-modification nucleotide is conserved between bacteria and the endosymbiont-derived eukaryotic organelle. The knockout of CMAL in Arabidopsis impairs the chloroplast ribosome accumulation and accordingly reduced the efficiency of mRNA translation. Interestingly, the loss of CMAL leads not only to defects in chloroplast function, but also to abnormal leaf and root development and overall plant morphology. Further investigation showed that CMAL is involved in the plant development probably by modulating auxin derived signaling pathways. This study uncovered the important role of 16S rRNA methylation mediated by CMAL in chloroplast ribosome biogenesis and plant development.

Photosynthetic bacteria have been proven to be excellent model organisms because they own the relatively simplified model systems for us to study the reactions that occurs at the initial stage of photosynthesis, compared with the oxygen-evolving cyanobacteria, algae, and higher plants. In purple bacteria, there are usually two kinds of light-harvesting (LH) complexes, named LH1 and LH2, respectively. LH2 is the peripheral antenna complex, and LH1 is the core antenna complex that surrounds the reaction center (RC) to form the LH1-RC supercomplex. Solar energy is first absorbed by the LH complex and then transferred rapidly and efficiently to the RC, where the charge separation and electron transfer take place. Several high-resolution structures are available for the RC and LH2 for a long time; for LH1-RC complex, its structure was solved and improved to an atomic resolution recently with a thermophilic purple photosynthetic bacterium Thermochromatium tepidum. The high-resolution structure provided much more detailed structural information of this supercomplex including the arrangements of protein subunits, pigments, and cofactors; a much more intact RC complex due to the protection of LH1 complex; the detailed coordination of the Ca2+ ions in the LH1 that are important for the absorption maximum at 915 nm as well as for the enhanced thermostability; the possible ubiquinone exchange pathway in the closed LH1 ring; and so on. In addition, the dynamic processes involved in this complex were also discussed. All these results greatly advance our understanding on the molecular mechanism of bacterial photosynthesis, which could be essential for designing artificial photoelectronic conversion materials with enhanced performance.

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously

While the majority of the core light-harvesting complexes (LH1) in purple photosynthetic bacteria exhibit a Qy absorption band in the range of 870–890 nm, LH1 from the thermophilic bacterium Thermochromatium tepidum displays the Qy band at 915 nm with an enhanced thermostability. These properties are regulated by Ca2+ ions. Substitution of the Ca2+ with other divalent metal ions results in a complex with the Qy band blue-shifted to 880–890 nm and a reduced thermostability. Following the recent publication of the structure of the Ca-bound LH1-reaction center (RC) complex [Niwa, S., et al. (2014) Nature 508, 228], we have determined the crystal structures of the Sr- and Ba-substituted LH1-RC complexes with the LH1 Qy band at 888 nm. Sixteen Sr2+ and Ba2+ ions are identified in the LH1 complexes. Both Sr2+ and Ba2+ are located at the same positions, and these are clearly different from, though close to, the Ca2+-binding sites. Conformational rearrangement induced by the substitution is limited to the metal-b...