Biology subjects

Rutherford, A. W.

Publications and source records attributed to Rutherford, A. W..

15 recordsLinked to original sources

bioRxiv2025

Far-red light absorption strategies and their structural basis in Photosystem I of Acaryochloris marina NIES-2412

The marine cyanobacterium, Acaryochloris marina, uses the red-shifted chlorophyll d as its primary pigment, allowing it to absorb photons >700 nm. However, the widely studied type strain, A. marina MBIC11017, is atypical compared to most cyanobacteria, due to the absence of low energy chlorophylls ( red forms) within its Photosystem I complex. Consequently, Photosystem I and Photosystem II in the MBIC11017 strain share similar absorption spectra and are incapable of absorbing photons >740 nm. Recently, it has been discovered that the absorption and emission spectra from other A. marina strains are significantly more red-shifted than the MBIC11017 strain. Here, we have combined advanced spectroscopy and high-resolution cryo-EM to characterize Photosystem I from Acaryochloris marina NIES-2412, a red-shifted strain that is more representative of the A. marina species. The structure resolves all 96 chlorophylls and cofactors and indicates the location of the red chlorophyll forms. Spectroscopic analysis reveals two distinct types of red forms: one arising from the classical mechanism of charge transfer-exciton mixing, and the other from purely excitonic interactions. Furthermore, we have identified PsaX2 as a critical subunit that fine-tunes the pigment geometries and energies to enable the formation of these red forms. Together, these findings reveal how NIES-2412 PSI balances far-red light harvesting and energy trapping, highlighting its distinct strategy for adaptation in far-red light environments and redefining A. marina MBIC11017 as an atypical representative of the species.

biophysics↗

bioRxiv2025

Structure of far-red allophycocyanin: stripped down and tuned up for low energy photosynthesis.

A diverse subset of cyanobacteria is capable of transiently modifying their photosynthetic machinery in a process known as far-red light photoacclimation to drive photosynthesis with less energetic photons (700 nm - 800 nm). To achieve this all the main light-driven components of the photosynthetic apparatus, including their allophycocyanin antenna, are replaced with red-shifted paralogues. Recent studies based on the structure of an incomplete complex provided some insights into the tuning of the far-red phycobiliproteins. Here, we solved the structure of the intact bicylindrical allophycocyanin complex from the cyanobacterium Chroococcidiopsis thermalis PCC 7203 at a resolution of 2.61 [A] determined by Cryo-electron microscopy single particle analysis. A comparison between far-red and white light allophycocyanin cores provides insight on the evolutionary adaptations needed to optimize excitation energy transfer in the far-red light adapted photosynthetic apparatus. The reduction in antenna size in far-red photosynthesis, suggests a need to optimize membrane packing to increase the number of photosystems, while the wider spread in the absorption range of the bilins suggests faster and more efficient excitation energy transfer to far-red Photosystem II by limiting backflow of excitation from the reaction centres to the far-red bilin pigments.

biophysics↗

bioRxiv2024

Investigation of electrochromic band-shifts in the Soret region induced by the formation of TyrD, TyrZ, and QA- in Photosystem II

The effects of TyrD*, TyrZ*, and QA*- radical formation on the absorption spectrum in the Soret region were studied in Mn-depleted Photosystem II at pH 8.6 (in order to be in the TyrD state after dark adaptation). Flash-induced difference spectra were recorded in several PSII samples from: i) Thermosynechococcus vestitus (formerly T. elongatus), ii) Synechocystis sp. PCC 6803, iii) Chroococcidiopsis thermalis PCC 7203 grown under far-red light, and iv) Acaryochloris marina. In the case of T. vestitus, mutants D1/H198Q, D1/T1789H, D2/I178H, and D2/Y160F, with PsbA1/Q130 instead of PsbA3/E130, were also studied for possible contributions from PD1, ChlD1, ChlD2, and PheD1, respectively. For a possible contribution from PD2, the D2/H197A mutant was studied in S. 6803. While PD1 is clearly the species whose spectrum is blue-shifted by [~]3nm in the presence of QA*-, as has already been well documented in the literature, the species whose spectra shift upon the formation of TyrD* and TyrZ* remain to be clearly identified, as they appear different from PD1, PD2, PheD1, ChlD1, and ChlD2, as concluded by the lack of different light-induced difference spectra in the mutants listed above. Although we cannot rule out a weak effect, considering the accuracy of the experiments, it is proposed that other pigments, such as antenna Chl and/or Car, near the reaction center are involved. Additionally, it is shown that: i) there is no proton release into the bulk upon the oxidation of TyrD at pH 8.6, and ii) the rearrangement of the electrostatic environment of the pigments involved in the light-induced difference spectra in the samples studied, upon the formation of TyrD*, TyrZ*, and QA*-, likely occurs differently from both a kinetic and structural perspective.

biophysics↗

bioRxiv2024

Locating the Missing Chlorophylls f in Far-red Photosystem I

The discovery of chlorophyll f-containing oxygenic photosynthesis, with its long-wavelength photochemistry, represented a new low-energy paradigm. However, subsequent structural studies on chlorophyll f-containing Photosystem I (PSI) found five chlorophylls f but none among the photochemically active pigments and concluded that chlorophyll f plays no photochemical role. Here we report a cryo-EM structure (2.01 [A]) of far-red PSI from Chroococcidiopsis thermalis PCC 7203, showing all eight chlorophylls f, including the redox active A-1B. Simulations of absorption difference spectra induced by charge separation indicate that the A-1B chlorophyll f absorbs at 755 nm. The chlorophyll f sites, some wavelength assignments, and conserved far-red-specific amino acids, provide functional insights, including redox tuning of chlorophyll f as the primary donor and far-red excitation energy-sharing over the PSI trimer.

biochemistry↗

bioRxiv2024

Modified Chlorophyll Pigment at ChlD1 Tunes Photosystem II Beyond the Red-Light Limit

Photosystem II (PSII) is powered by the light-capturing properties of chlorophyll a pigments that define the spectral range of oxygenic photosynthesis. Some photosynthetic cyanobacteria can acclimate to growth in longer wavelength light by replacing five chlorophylls for long wavelength pigments in specific locations, including one in the reaction center (RC). However, the exact location and the nature of this long wavelength pigment still remain uncertain. Here we have addressed the color-tuning mechanism of the farred light PSII (FRL-PSII) by excited state calculations at both the ab initio correlated (ADC2) and linear-response time-dependent density functional theory (LR-TDDFT) levels in combination with large-scale hybrid quantum/classical (QM/MM) simulations and atomistic molecular dynamics. We show that substitution of a single chlorophyll pigment (ChlD1) at the RC by chlorophyll d leads to a spectral shift beyond the far-red light limit, as a result of the protein electrostatic, polarization and electronic coupling effects that reproduce key structural and spectroscopic observations. Pigment substitution at the ChlD1 site further results in a low site energy within the RC that could function as a sink for the excitation energy and initiate the primary charge separation reaction, driving the water oxidation. Our findings provide a basis for understanding color-tuning mechanisms and bioenergetic principles of oxygenic photosynthesis at the far-red light limit.

biophysics↗

bioRxiv2024

Mechanism of Proton Release during Water Oxidation in Photosystem II

Photosystem II (PSII) catalyzes the light-driven water oxidation that releases dioxygen into our atmosphere and provides the electrons needed for the synthesis of biomass. The catalysis occurs in the oxygen-evolving oxo-manganese-calcium (Mn4O5Ca) cluster that drives the stepwise oxidation and deprotonation of substrate water molecules leading to the O2 formation. However, despite recent advances, the mechanism of these reactions remains unclear and much debated. Here we show that the light-driven Tyr161D1 oxidation adjacent to the Mn4O5Ca cluster, significantly decreases the barrier for proton transfer from the putative substrate water molecule (W3/Wx) to Glu310D2, which is accessible to the luminal bulk. By combining hybrid quantum/classical (QM/MM) free energy calculations with atomistic molecular dynamics (MD) simulations, we probe the energetics of the proton transfer along the Cl1 pathway. We demonstrate that the proton transfer occurs via water molecules and a cluster of conserved carboxylates, driven by redox-triggered electric fields directed along the pathway. Glu65D1 establishes a local molecular gate that controls the proton transfer to the luminal bulk, whilst Glu312D2 acts as a local proton storage site. The identified gating region could be important in preventing back-flow of protons to the Mn4O5Ca cluster. The structural changes, derived here based on the dark-state PSII structure, strongly support recent time-resolved XFEL data of the S3[->]S4 transition (Nature 617, 2023), and reveal the mechanistic basis underlying deprotonation of the substrate water molecules. Our combined findings provide insight into the water oxidation mechanism of PSII and show how the interplay between redox-triggered electric fields, ion-pairs, and hydration effects control proton transport reactions. Significance StatementPhotosystem II is natures water splitting enzyme that produces the oxygen in the atmosphere and drives the synthesis of biomass. The water splitting reaction releases protons to the luminal bulk contributing to the protonmotive force that drives the synthesis of ATP. Key mechanistic principles of the light-driven water splitting reaction remain debated, amongst them the catalytically important deprotonation steps. Here we show how the oxygen-evolving oxo-manganese-calcium cluster transports protons via conserved carboxylates and water molecules in proton arrays that lead to the luminal bulk. We identify a local proton storage site and molecular gates that prevent wasteful back reactions by undergoing conformational changes, and we show how electric field effects control the protonation dynamics in Photosystem II.

biophysics↗

bioRxiv2024

In vivo ElectroChromic Shift measurements of photosynthetic activity in far-red absorbing cyanobacteria

Some cyanobacteria can do photosynthesis using not only visible but also far-red light that is unused by most other oxygenic photoautotrophs because of its lower energy content. These species have a modified photosynthetic apparatus containing red-shifted pigments. The incorporation of red-shifted pigments decreases the photochemical efficiency of photosystem I and, especially, photosystem II, and it might affect the distribution of excitation energy between the two photosystems with possible consequences on the activity of the entire electron transport chain. To investigate the in vivo effects on photosynthetic activity of these pigment changes, we present here the adaptation of a spectroscopic method, based on a physical phenomenon called ElectroChromic Shift (ECS), to the far-red absorbing cyanobacteria Acaryochloris marina and Chroococcidiopsis thermalis PCC7203. ECS measures the electric field component of the trans-thylakoid proton motive force generated by photosynthetic electron transfer. We show that ECS can be used in these cyanobacteria to investigate in vivo the stoichiometry of photosystem I and photosystem II and their absorption cross-section, as well as the overall efficiency of light energy conversion into electron transport. Our results indicate that both species use visible and far-red light with similar efficiency, despite significant differences in their light absorption characteristics. ECS thus represents a new non-invasive tool to study the performance of naturally occurring far-red photosynthesis.

biophysics↗

bioRxiv2024

Contributions of PD1 and PD2 to the difference spectrum in the Soret region in Photosystem II

Flash-induced absorption changes in the Soret region, which originate from the [PD1PD2]+ state, the chlorophyll cation radical formed upon Photosystem II (PSII) excitation, were investigated in Mn-depleted Photosystem II. In wild-type PSII from Thermosynechococcus elongatus, the [PD1PD2]+-minus-[PD1PD2] difference spectrum shows a main negative feature at 434 nm and a smaller negative feature at 446 nm [Boussac et al. Photosynth Res (2023), https://doi.org/10.1007/s11120-023-01049-3]. While the main feature at 434 nm is associated with PD1+ formation, the origin of the dip at 446 nm remains to be identified. For that, we have compared the [PD1PD2]+-minus-[PD1PD2] difference spectra from the PsbA3/H198Q PSII mutant in T. elongatus and D2/H197A PSII mutant in Synechocystis sp. PCC 6803 with their respective wild type strains. By modifying the PD1 axial ligand with the H198Q mutation in the D1 protein in T. elongatus, the contribution at 434 nm was shifted to 431 nm, while the contribution at 446 nm was hardly affected. In Synechocystis sp. PCC 6803, by modifying the PD2 axial ligand with the H197A mutation in the D2 protein, the contribution at 446 nm was downshifted by [~] 3 nm to [~] 443 nm, while the main contribution at 432 nm was only slightly shifted upwards to 433 nm. This result suggests that the bleaching seen at 446 nm involves PD2. This could reflects a change in the [PD1+PD2]{longleftrightarrow}[PD1PD2+] equilibrium or a more complex mechanism.

biophysics↗

bioRxiv2022

Absorption changes in Photosystem II in the Soret band region upon the formation of the chlorophyll cation radical +

Flash-induced absorption changes in the Soret region arising from the [PD1PD2]+ state, the chlorophyll cation radical formed upon light excitation of Photosystem II (PSII), were measured in Mn-depleted PSII cores at pH 8.6. Under these conditions, TyrD is i) reduced before the first flash, and ii) oxidized before subsequent flashes. In wild-type PSII, when TyrD[bullet] is present, an additional signal in the [PD1PD2]+-minus-[PD1PD2] difference spectrum was observed when compared to the first flash when TyrD is not oxidized. The additional feature was "W-shaped" with troughs at 434 nm and 446 nm. This feature was absent when TyrD was reduced, but was present i) when TyrD was physically absent (and replaced by phenylalanine) or ii) when its H-bonding histidine (D2-His189) was physically absent (replaced by a Leucine). Thus, the simple difference spectrum without the double trough feature at 434 nm and 446 nm, seemed to require the native structural environment around the reduced TyrD and its H bonding partners to be present. We found no evidence of involvement of PD1, ChlD1, PheD1, PheD2, TyrZ, and the Cytb559 heme in the W-shaped difference spectrum. However, the use of a mutant of the PD2 axial His ligand, the D2-His197Ala, shows that the PD2 environment seems involved in the formation of "W-shaped" signal.

biophysics↗

bioRxiv2022

Impact of energy limitations on function and resilience in long-wavelength Photosystem II

Photosystem II (PSII) uses the energy from red light to split water and reduce quinone, an energy-demanding process based on chlorophyll a (Chl-a) photochemistry. Two kinds of cyanobacterial PSII can use Chl-d and Chl-f to perform the same reactions using lower energy, far-red light. PSII from Acaryochloris marina has Chl-d replacing all but one of its 35 Chl-a, while PSII from Chroococcidiopsis thermalis, a facultative far-red species, has just 4 Chl-f and 1 Chl-d and 30 Chl-a. From bioenergetic considerations, the far-red PSII were predicted to lose photochemical efficiency and/or resilience to photodamage. Here, we compare enzyme turnover efficiency, forward electron transfer, back-reactions and photodamage in Chl-f-PSII, Chl-d-PSII and Chl-a-PSII. We show that: i) all types of PSII have a comparable efficiency in enzyme turnover; ii) the modified energy gaps on the acceptor side of Chl-d-PSII favor recombination via PD1+Phe- repopulation, leading to increased singlet oxygen production and greater sensitivity to high-light damage compared to Chl-a-PSII and Chl-f-PSII; ii) the acceptor-side energy gaps in Chl-f-PSII are tuned to avoid harmful back reactions, favoring resilience to photodamage over efficiency of light usage. The results are explained by the differences in the redox tuning of the electron transfer cofactors Phe and QA and in the number and layout of the chlorophylls that share the excitation energy with the primary electron donor. PSII has adapted to lower energy in two distinct ways, each appropriate for its specific environment but with different functional penalties.

biochemistry↗

bioRxiv2022

Bicarbonate Activation of Monomeric Photosystem II-PsbS/Psb27 Complex

In thylakoid membranes, Photosystem II monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while Photosystem II monomers from granal regions (PSIIm) lack these subunits. Here, we have isolated and characterised these two types of Photosystem II complexes. The PSIIm-S/27 showed enhanced fluorescence, the near-absence of oxygen evolution, as well as limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to the PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for the bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA*- couple, which controls the charge recombination route, and this limits chlorophyll triplet mediated 1O2 formation (Brinkert K et al. (2016) Proc Natl Acad Sci U S A. 113(43):12144-12149). These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit, by using a bicarbonate-mediated switch and protective mechanism. One sentence summaryA photosystem II monomer with PsbS and Psb27 as additional subunits, is inactive as isolated but activated by bicarbonate, and is attributed to be a late-stage intermediate in photoassembly.

plant biology↗

bioRxiv2020

The primary donor of far-red Photosystem II: ChlD1 or PD2?

Far-red light (FRL) Photosystem II (PSII) isolated from Chroococcidiopsis thermalis is studied using parallel analyses of low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies in conjunction with fluorescence measurements. This extends earlier studies (Nurnberg et al 2018 Science 360 (2018) 1210-1213). We confirm that the chlorophyll absorbing at 726 nm is the primary electron donor. At 1.8 K efficient photochemistry occurs when exciting at 726 nm and shorter wavelengths; but not at wavelengths longer than 726 nm. The 726 nm absorption peak exhibits a 21 {+/-} 4 cm-1 electrochromic shift due to formation of the semiquinone anion, QA*-. Modelling indicates that no other FRL pigment is located among the 6 central reaction center chlorins: PD1, PD2 ChlD1, ChlD2, PheoD1 and PheoD2. Two of these chlorins, ChlD1 and PD2, are located at a distance and orientation relative to QA*- so as to account for the observed electrochromic shift. Previously, ChlD1 was taken as the most likely candidate for the primary donor based on spectroscopy, sequence analysis and mechanistic arguments. Here, a more detailed comparison of the spectroscopic data with exciton modelling of the electrochromic pattern indicates that PD2 is at least as likely as ChlD1 to be responsible for the 726 nm absorption. The correspondence in sign and magnitude of the CD observed at 726 nm with that predicted from modelling favors PD2 as the primary donor. The pros and cons of PD2 vs ChlD1 as the location of the FRL-primary donor are discussed. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/021097v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@159ea46org.highwire.dtl.DTLVardef@23c04forg.highwire.dtl.DTLVardef@126c235org.highwire.dtl.DTLVardef@1982d58_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPrimary Donor confirmed at 726 nm C_LIO_LIDetermination of far-red chl pigment Qy excitation positions, widths, CD and MCD amplitudes C_LIO_LIQuantification of electrochromic shifts and QA*- photoconversion yield C_LIO_LIElectrochromic shift consistent with primary donor at either ChlD1 or PD2 C_LIO_LIThe CD amplitude favors the primary donor at PD2 C_LI

biophysics↗

bioRxiv2020

Origin of photosynthetic water oxidation at the dawn of life

Oxygenic photosynthesis starts with the oxidation of water to O2, a light-driven reaction catalysed by photosystem II. Cyanobacteria are the only prokaryotes capable of water oxidation and therefore, it is assumed that relative to the origin of life and bioenergetics, the origin of oxygenic photosynthesis is a late innovation. However, when exactly water oxidation originated remains an unanswered question. Here we use relaxed molecular clocks to compare one of the two ancestral core duplications that are unique to water-oxidizing photosystem II, that leading to CP43 and CP47, with some of the oldest well-described events in the history of life. Namely, the duplication leading to the Alpha and Beta subunits of the catalytic head of ATP synthase, and the divergence of archaeal and bacterial RNA polymerases and ribosomes. We also compare it with more recent events such as the duplication of cyanobacteria-specific FtsH metalloprotease subunits, of CP43 variants used in a variety of photoacclimation responses, and the speciation events leading to Margulisbacteria, Sericytochromatia, Vampirovibrionia, and other clades containing anoxygenic phototrophs. We demonstrate that the ancestral core duplication of photosystem II exhibits patterns in the rates of protein evolution through geological time that are nearly identical to those of the ATP synthase, RNA polymerase, or the ribosome. Furthermore, we use ancestral sequence reconstruction in combination with comparative structural biology of photosystem subunits, to provide additional evidence supporting the premise that water oxidation had originated before the ancestral core duplications. Our work suggests that photosynthetic water oxidation originated closer to the origin of life and bioenergetics than can be documented based on species trees alone.

evolutionary biology↗

bioRxiv2018

Evolution of photochemical reaction centres: more twists?

The earliest event recorded in the molecular evolution of photosynthesis is the structural and functional specialisation of Type I (ferredoxin-reducing) and Type II (quinone-reducing) reaction centres. Here we point out that the homodimeric Type I reaction centre of Heliobacteria has a Ca2+-binding site with a number of striking parallels to the Mn4CaO5 cluster of cyanobacterial Photosystem II. This structural parallels indicate that water oxidation chemistry originated at the divergence of Type I and Type II reaction centres. We suggests that this divergence was triggered by a structural rearrangement of a core transmembrane helix resulting in a shift of the redox potential of the electron donor side and electron acceptor side at the same time and in the same redox direction.

evolutionary biology↗

bioRxiv2017

Molecular evidence for the early evolution of photosynthetic water oxidation

Photosystem II is a photochemical reaction center that catalyzes the light-driven oxidation of water to molecular oxygen. Water oxidation is the distinctive photochemical reaction that permitted the evolution of oxygenic photosynthesis and the eventual rise of Eukaryotes. At what point during the history of life an ancestral photosystem evolved the capacity to oxidize water still remains unknown. Here we study the evolution of the core reaction center proteins of Photosystem II using sequence and structural comparisons in combination with Bayesian relaxed molecular clocks. Our results indicate that a homodimeric photosystem with sufficient oxidizing power to split water had already appeared in the early Archean about a billion years before the most recent common ancestor of all described Cyanobacteria capable of oxygenic photosynthesis, and well before the diversification of some of the known groups of anoxygenic photosynthetic bacteria. Based on a structural and functional rationale we hypothesize that this early Archean photosystem was capable of water oxidation and had already evolved some level of protection against the formation of reactive oxygen species, which would place primordial forms of oxygenic photosynthesis at a very early stage in the evolutionary history of life.

evolutionary biology↗