Expression of the plastocyanin gene PETE2 in Camelina sativa improves seed yield and salt tolerance.

Plastocyanin functions as an electron carrier in the photosynthetic electron transport chain, located at the thylakoid membrane. In several species, endogenous plastocyanin levels are correlated with the photosynthetic electron transport rate. Overexpression of plastocyanin genes in Arabidopsis thaliana increases plant size, but this phenomenon has not been observed in crop species. Here, we investigated the effects of heterologous expression of a gene encoding a plastocyanin isoform from Arabidopsis, AtPETE2, in the oil seed crop Camelina sativa under standard growth conditions and under salt stress. AtPETE2 heterologous expression enhanced photosynthetic activity in Camelina, accelerating plant development and improving seed yield under standard growth conditions. Additionally, CsPETE2 from Camelina was induced by salt stress and AtPETE2 expression lines had larger primary roots and more lateral roots than the wild type. AtPETE2 expression lines also had larger seeds and higher total seed yield under long-term salt stress compared with non-transgenic Camelina. Our results demonstrate that increased plastocyanin levels in Camelina can enhance photosynthesis and productivity, as well as tolerance to osmotic and salt stresses. Heterologous expression of plastocyanin may be a useful strategy to mitigate crop stress in saline soils.

The Protein Matrix of Plastocyanin Supports Long-Distance Charge Transport with Photosystem I and the Copper Ion Regulates Its Spatial Span and Conductance.

Charge exchange is the fundamental process that sustains cellular respiration and photosynthesis by shuttling electrons in a cascade of electron transfer (ET) steps between redox cofactors. While intraprotein charge exchange is well characterized in protein complexes bearing multiple redox sites, interprotein processes are less understood due to the lack of suitable experimental approaches and the dynamic nature of the interactions. Proteins constrained between electrodes are known to support electron transport (ETp) through the protein matrix even without redox cofactors, as the charges housed by the redox sites in ET are furnished by the electrodes. However, it is unknown whether protein ETp mechanisms apply to the interprotein medium present under physiological conditions. We study interprotein charge exchange between plant photosystem I (PSI) and its soluble redox partner plastocyanin (Pc) and address the role of the Pc copper center. Using electrochemical scanning tunneling spectroscopy (ECSTS) current-distance and blinking measurements, we quantify the spatial span of charge exchange between individual Pc/PSI pairs and ETp through transient Pc/PSI complexes. Pc devoid of the redox center (Pcapo) can exchange charge with PSI at longer distances than with the copper ion (Pcholo). Conductance bursts associated with Pcapo/PSI complex formation are higher than in Pcholo/PSI. Thus, copper ions are not required for long-distance Pc/PSI ETp but regulate its spatial span and conductance. Our results suggest that the redox center that carries the charge in Pc is not necessary to exchange it in interprotein ET through the aqueous solution and question the canonical view of tight complex binding between redox protein partners.

High-resolution cryo-EM structures of plant cytochrome b6f at work.

Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome b6f (Cyt b6f) is known as one of the key steps of photosynthesis, but the catalytic mechanism in the plastoquinone oxidation site (Qp) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt b6f homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near Qp in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt b6f in one direction, transiently exposing the redox-active ring of quinone during catalysis. Our work proposes an unprecedented one-way traffic model that explains efficient quinol oxidation during photosynthesis and respiration.

RLPredictiOme, a Machine Learning-Derived Method for High-Throughput Prediction of Plant Receptor-like Proteins, Reveals Novel Classes of Transmembrane Receptors.

Cell surface receptors play essential roles in perceiving and processing external and internal signals at the cell surface of plants and animals. The receptor-like protein kinases (RLK) and receptor-like proteins (RLPs), two major classes of proteins with membrane receptor configuration, play a crucial role in plant development and disease defense. Although RLPs and RLKs share a similar single-pass transmembrane configuration, RLPs harbor short divergent C-terminal regions instead of the conserved kinase domain of RLKs. This RLP receptor structural design precludes sequence comparison algorithms from being used for high-throughput predictions of the RLP family in plant genomes, as has been extensively performed for RLK superfamily predictions. Here, we developed the RLPredictiOme, implemented with machine learning models in combination with Bayesian inference, capable of predicting RLP subfamilies in plant genomes. The ML models were simultaneously trained using six types of features, along with three stages to distinguish RLPs from non-RLPs (NRLPs), RLPs from RLKs, and classify new subfamilies of RLPs in plants. The ML models achieved high accuracy, precision, sensitivity, and specificity for predicting RLPs with relatively high probability ranging from 0.79 to 0.99. The prediction of the method was assessed with three datasets, two of which contained leucine-rich repeats (LRR)-RLPs from Arabidopsis and rice, and the last one consisted of the complete set of previously described Arabidopsis RLPs. In these validation tests, more than 90% of known RLPs were correctly predicted via RLPredictiOme. In addition to predicting previously characterized RLPs, RLPredictiOme uncovered new RLP subfamilies in the Arabidopsis genome. These include probable lipid transfer (PLT)-RLP, plastocyanin-like-RLP, ring finger-RLP, glycosyl-hydrolase-RLP, and glycerophosphoryldiester phosphodiesterase (GDPD, GDPDL)-RLP subfamilies, yet to be characterized. Compared to the only Arabidopsis GDPDL-RLK, molecular evolution studies confirmed that the ectodomain of GDPDL-RLPs might have undergone a purifying selection with a predominance of synonymous substitutions. Expression analyses revealed that predicted GDPGL-RLPs display a basal expression level and respond to developmental and biotic signals. The results of these biological assays indicate that these subfamily members have maintained functional domains during evolution and may play relevant roles in development and plant defense. Therefore, RLPredictiOme provides a framework for genome-wide surveys of the RLP superfamily as a foundation to rationalize functional studies of surface receptors and their relationships with different biological processes.

Plastoquinol Oxidation: Rate-Limiting Stage in the Electron Transport Chain of Chloroplasts.

This work is devoted to theoretical study of functioning of the cytochrome (Cyt) b6f complex (plastoquinol:plastocyanin oxidoreductase) of the electron transport chain (ETC) in oxygenic photosynthesis. A composition of the chloroplast ETC and molecular mechanisms of functioning of the Cyt b6f complex, which stands between photosystems II and I (PSII and PSI), are briefly reviewed. The Cyt b6f complex oxidizes plastoquinol (PQH2) molecules formed in PSII, and reduces plastocyanin, which serves as an electron donor to PSI. PQH2 oxidation is the rate-limiting step in the chain of electron transfer processes between PSII and PSI. Using the density functional theory (DFT) method, we have analyzed the two-electron (bifurcated) oxidation of PQH2 in the catalytic center Qo of the Cyt b6f complex. Results of DFT calculations are consistent with the fact that the first step of PQH2 oxidation, electron transfer to the Fe2S2 cluster of the iron-sulfur protein (ISP), is an endergonic (energy-accepting) process (ΔE ≈ 15 kJ·mol-1) that can limit turnover of the Cyt b6f complex. The second stage of bifurcated oxidation of PQH2 - electron transfer from semiquinone (PQH•, formed after the first step of PQH2 oxidation) to heme b6L - is the exergonic (energy-donating) process (ΔE < 0). DFT modeling of this stage revealed that semiquinone oxidation should accelerate after the PQH• radical shift towards the heme b6L (an electron acceptor) and the carboxy group of Glu78 (a proton acceptor). The data obtained are discussed within the framework of the Mitchell Q-cycle model describing PQH2 oxidation at the Qo site of the Cyt b6f complex.

Altered coordination in a blue copper protein upon association with redox partner revealed by carbon-deuterium vibrational probes.

Proteins tune the reactivity of metal sites; less understood is the impact of association with a redox partner. We demonstrate the utility of carbon-deuterium labels for selective analysis of delicate metal sites. Introduced into plastocyanin, they reveal substantial strengthening of the key Cu-Cys89 bond upon association with cytochrome f.

Light- and Redox-Dependent Force Spectroscopy Reveals that the Interaction between Plastocyanin and Plant Photosystem I Is Favored when One Partner Is Ready for Electron Transfer.

Photosynthesis is a fundamental process that converts photons into chemical energy, driven by large protein complexes at the thylakoid membranes of plants, cyanobacteria, and algae. In plants, water-soluble plastocyanin (Pc) is responsible for shuttling electrons between cytochrome b6f complex and the photosystem I (PSI) complex in the photosynthetic electron transport chain (PETC). For an efficient turnover, a transient complex must form between PSI and Pc in the PETC, which implies a balance between specificity and binding strength. Here, we studied the binding frequency and the unbinding force between suitably oriented plant PSI and Pc under redox control using single molecule force spectroscopy (SMFS). The binding frequency (observation of binding-unbinding events) between PSI and Pc depends on their respective redox states. The interaction between PSI and Pc is independent of the redox state of PSI when Pc is reduced, and it is disfavored in the dark (reduced P700) when Pc is oxidized. The frequency of interaction between PSI and Pc is higher when at least one of the partners is in a redox state ready for electron transfer (ET), and the post-ET situation (PSIRed-PcOx) leads to lower binding. In addition, we show that the binding of ET-ready PcRed to PSI can be regulated externally by Mg2+ ions in solution.

Determining photosynthetic control, a probe for the balance between electron transport and Calvin-Benson cycle activity, with the DUAL-KLAS-NIR.

Photosynthetic Control is defined as the control imposed on photosynthetic electron transport by the lumen-pH-sensitive re-oxidation of plastoquinol (PQH2) by cytochrome b6f. Photosynthetic Control leads at higher actinic light intensities to an electron transport chain with a (relatively) reduced photosystem (PS) II and PQ pool and a (relatively) oxidized PS I. Making Light Curves of more than 33 plant species with the recently introduced DUAL-KLAS-NIR (Chl a fluorescence + the redox states of plastocyanin (PC), P700, and ferredoxin (Fd)) the light intensity-dependent induction of Photosynthetic Control was probed and characterized. It was observed that PC became completely oxidized at light intensities ≤ 400 µmol photons m-2 s-1 (at lower light intensities in shade than in sun leaves). The relationship between qP and P700(red) was used to determine the extent of Photosynthetic Control. Instead of measuring the whole Light Curve, it was shown that a single moderate light intensity can be used to characterize the status of a leaf relative to that of other leaves. It was further found that in some shade-acclimated leaves Fd becomes again more oxidized at high light intensities indicating that electron transfer from the PQ pool to P700 cannot keep up with the outflow of electrons on the acceptor side of PS I. It was observed as well that for NPQ-induction a lower light intensity (less acidified lumen) was needed than for the induction of Photosynthetic Control. The measurements were also used to make a comparison between the parameters qP and qL, a comparison suggesting that qP was the more relevant parameter.

Are plastocyanin and ferredoxin specific electron carriers or generic redox capacitors? Classical and murburn perspectives on two photosynthetic proteins.

In the light reaction of oxygenic photosynthesis, plastocyanin (PC) and ferredoxins (Fd) are small/diffusible redox-active proteins playing key roles in electron transfer/transport phenomena. In the Z-scheme mechanistic purview, they are considered as specific affinity binding-based electron-relay agents, linking the functions of Cytochrome b6f (Cyt. b6f), Photosystem I (PS I) and Fd:NADPH oxidoreductase (FNR). The murburn explanation for photolytic photophosphorylation deems PC/Fd as generic 'redox capacitors', temporally accepting and releasing one-electron equivalents in reaction milieu. Herein, we explore the two theories with respect to structural, distributional and functional aspects of PC/Fd. Amino acid residues located on the surface loci of key patches of PC/Fd vary in electrostatic/contour (topography) signatures. Crystal structures of four different complexes each of Cyt.f-PC and Fd-FNR show little conservation in the contact-surfaces, thereby discrediting 'affinity binding-based electron transfers (ET)' as an evolutionary logic. Further, thermodynamic and kinetic data of wildtype and mutant proteins interactions do not align with Z-scheme. Furthermore, micromolar physiological concentrations of PC and the non-conducive architecture of chloroplasts render the classical model untenable. In the murburn model, as PC is optional, the observation that plants lacking PC survive and grow is justified. Further, the low physiological concentration/distribution of PC in chloroplast lumen/stroma is supported by murburn equilibriums, as higher concentrations would limit electron transfers. Thus, structural evidence, interactive dynamics with redox partners and physiological distribution/role of PC/Fd support the murburn perspective that these proteins serve as generic redox-capacitors in chloroplasts.Communicated by Ramaswamy H. Sarma.

Structure of plant photosystem I-plastocyanin complex reveals strong hydrophobic interactions.

Photosystem I is defined as plastocyanin-ferredoxin oxidoreductase. Taking advantage of genetic engineering, kinetic analyses and cryo-EM, our data provide novel mechanistic insights into binding and electron transfer between PSI and Pc. Structural data at 2.74 Šresolution reveals strong hydrophobic interactions in the plant PSI-Pc ternary complex, leading to exclusion of water molecules from PsaA-PsaB/Pc interface once the PSI-Pc complex forms. Upon oxidation of Pc, a slight tilt of bound oxidized Pc allows water molecules to accommodate the space between Pc and PSI to drive Pc dissociation. Such a scenario is consistent with the six times larger dissociation constant of oxidized as compared with reduced Pc and mechanistically explains how this molecular machine optimized electron transfer for fast turnover.

A protease-mediated mechanism regulates the cytochrome c6/plastocyanin switch in Synechocystis sp. PCC 6803.

After the Great Oxidation Event (GOE), iron availability was greatly decreased, and photosynthetic organisms evolved several alternative proteins and mechanisms. One of these proteins, plastocyanin, is a type I blue-copper protein that can replace cytochrome c6 as a soluble electron carrier between cytochrome b6f and photosystem I. In most cyanobacteria, expression of these two alternative proteins is regulated by copper availability, but the regulatory system remains unknown. Herein, we provide evidence that the regulatory system is composed of a BlaI/CopY-family transcription factor (PetR) and a BlaR-membrane protease (PetP). PetR represses petE (plastocyanin) expression and activates petJ (cytochrome c6), while PetP controls PetR levels in vivo. Using whole-cell extracts, we demonstrated that PetR degradation requires both PetP and copper. Transcriptomic analysis revealed that the PetRP system regulates only four genes (petE, petJ, slr0601, and slr0602), highlighting its specificity. Furthermore, the presence of petE and petRP in early branching cyanobacteria indicates that acquisition of these genes could represent an early adaptation to decreased iron bioavailability following the GOE.

The heterologous expression of a plastocyanin in the diatom Phaeodactylum tricornutum improves cell growth under iron-deficient conditions.

We have investigated if the heterologous expression of a functional green alga plastocyanin in the diatom Phaeodactylum tricornutum can improve photosynthetic activity and cell growth. Previous in vitro assays showed that a single-mutant of the plastocyanin from the green algae Chlamydomonas reinhardtii is effective in reducing P. tricornutum photosystem I. In this study, in vivo assays with P. tricornutum strains expressing this plastocyanin indicate that even the relatively low intracellular concentrations of holo-plastocyanin detected (≈4 µM) are enough to promote an increased growth (up to 60%) under iron-deficient conditions as compared with the WT strain, measured as higher cell densities, content in pigments and active photosystem I, global photosynthetic rates per cell, and even cell volume. In addition, the presence of plastocyanin as an additional photosynthetic electron carrier seems to decrease the over-reduction of the plastoquinone pool. Consequently, it promotes an improvement in the maximum quantum yield of both photosystem II and I, together with a decrease in the acceptor side photoinhibition of photosystem II-also associated to a reduced oxidative stress-a decrease in the peroxidation of membrane lipids in the choroplast, and a lower degree of limitation on the donor side of photosystem I. Thus the heterologous plastocyanin appears to act as a functional electron carrier, alternative to the native cytochrome c6 , under iron-limiting conditions.

Plastocyanin is the long-range electron carrier between photosystem II and photosystem I in plants.

In photosynthetic electron transport, large multiprotein complexes are connected by small diffusible electron carriers, the mobility of which is challenged by macromolecular crowding. For thylakoid membranes of higher plants, a long-standing question has been which of the two mobile electron carriers, plastoquinone or plastocyanin, mediates electron transport from stacked grana thylakoids where photosystem II (PSII) is localized to distant unstacked regions of the thylakoids that harbor PSI. Here, we confirm that plastocyanin is the long-range electron carrier by employing mutants with different grana diameters. Furthermore, our results explain why higher plants have a narrow range of grana diameters since a larger diffusion distance for plastocyanin would jeopardize the efficiency of electron transport. In the light of recent findings that the lumen of thylakoids, which forms the diffusion space of plastocyanin, undergoes dynamic swelling/shrinkage, this study demonstrates that plastocyanin diffusion is a crucial regulatory element of plant photosynthetic electron transport.

Identification of copper-regulated proteins in an oceanic diatom, Thalassiosira oceanica 1005.

Copper (Cu) is an essential cofactor of photosynthetic and respiratory redox proteins in phytoplankton and a scarce resource in parts of the open sea. Although its importance for growth is well recognized, the molecular mechanisms by which phytoplankton respond and acclimate to Cu deficiency are not well known. In this study, we identified the dominant Cu-regulated proteins and measured key physiological traits of Thalassiosira oceanica (CCMP 1005) under Cu-limiting and sufficient conditions. Growth limitation of T. oceanica occurred at environmentally relevant Cu concentrations (1 nM) as a result of decreased photosynthetic efficiency (ΦPSII). In Cu-limited cells, levels of plastocyanin decreased by 3-fold compared to Cu-replete cells and rates of maximum photosynthetic electron transport were reduced. Proteins associated with light harvesting complexes also declined in response to Cu limitation, presumably to adjust to reduced photosynthetic electron flow and to avoid photodamage to the photosystems. Key enzymes involved in carbon and nitrogen assimilation were down-regulated in low-Cu cells, as were steady state rates of C and N uptake. Relatively fewer proteins were up-regulated by Cu limitation, but among them were two enzymes involved in fatty acid oxidation (FAO). The increase in FAO may be a sign of increased turnover of cellular lipids caused by damage from oxidative stress. A putative transcription factor containing three, repetitive methionine motifs (MpgMgggM; MpgMggM) increased significantly in Cu-limited cells. The collective results provide a general description of how plastocyanin-dependent diatoms adjust metabolism to cope with chronic Cu deficiency.

Near-infrared in vivo measurements of photosystem I and its lumenal electron donors with a recently developed spectrophotometer.

In photosynthesis research, non-invasive in vivo spectroscopic analyses have been used as a practical tool for studying photosynthetic electron transport. Klas-NIR spectrophotometer has been recently developed by Klughammer and Schreiber (Photosynth Res 128:195-214, 2016) for in vivo measurements of redox changes of P700, plastocyanin (Pcy) and ferredoxin (Fd). Here we show examples using the Klas-NIR spectrophotometer for the evaluation of the redox states and quantities of these components in plant leaves and cyanobacterial suspensions. The redox poise under light of the electron transport components is different in leaves from higher plants compared with cyanobacteria. During a short illumination with an actinic light, P700, Pcy, and Fd are kept reduced in barley leaves but are oxidized in cyanobacteria. During far-red light illumination, P700 and Pcy are mostly oxidized in the leaves but are partially kept reduced in cyanobacteria. In the cyanobacterium, Thermosynechococcus elongatus, which has no Pcy but uses cytochrome c6 (cyt c6) as the electron donor to photosystem I, a cyt c6 signal was detected in vivo. To show the potential of Klas-NIR spectrophotometer for studying different developmental stages of a leaf, we performed measurements on fully mature and early senescing barley leaves. Pcy content in leaves decreased during senescence at an early stage. The Pcy loss was quantitatively analyzed using Klas-NIR spectrophotometer, giving absolute ratios of Pcy to PSI of 2.5 and 1.6 in younger and older leaves, respectively. For quantification of the signals in vivo, in vitro data (Sétif et al. in Photosynth Res142:307-319, 2019) obtained with Klas-NIR spectrophotometer were used.

Probing the electric field across thylakoid membranes in cyanobacteria.

In plants, algae, and some photosynthetic bacteria, the ElectroChromic Shift (ECS) of photosynthetic pigments, which senses the electric field across photosynthetic membranes, is widely used to quantify the activity of the photosynthetic chain. In cyanobacteria, ECS signals have never been used for physiological studies, although they can provide a unique tool to study the architecture and function of the respiratory and photosynthetic electron transfer chains, entangled in the thylakoid membranes. Here, we identified bona fide ECS signals, likely corresponding to carotenoid band shifts, in the model cyanobacteria Synechococcus elongatus PCC7942 and Synechocystis sp. PCC6803. These band shifts, most likely originating from pigments located in photosystem I, have highly similar spectra in the 2 species and can be best measured as the difference between the absorption changes at 500 to 505 nm and the ones at 480 to 485 nm. These signals respond linearly to the electric field and display the basic kinetic features of ECS as characterized in other organisms. We demonstrate that these probes are an ideal tool to study photosynthetic physiology in vivo, e.g., the fraction of PSI centers that are prebound by plastocyanin/cytochrome c6 in darkness (about 60% in both cyanobacteria, in our experiments), the conductivity of the thylakoid membrane (largely reflecting the activity of the ATP synthase), or the steady-state rates of the photosynthetic electron transport pathways.

Near-infrared in vitro measurements of photosystem I cofactors and electron-transfer partners with a recently developed spectrophotometer.

A kinetic-LED-array-spectrophotometer (Klas) was recently developed for measuring in vivo redox changes of P700, plastocyanin (PCy), and ferredoxin (Fd) in the near-infrared (NIR). This spectrophotometer is used in the present work for in vitro light-induced measurements with various combinations of photosystem I (PSI) from tobacco and two different cyanobacteria, spinach plastocyanin, cyanobacterial cytochrome c6 (cyt. c6), and Fd. It is shown that cyt. c6 oxidation contributes to the NIR absorption changes. The reduction of (FAFB), the terminal electron acceptor of PSI, was also observed and the shape of the (FAFB) NIR difference spectrum is similar to that of Fd. The NIR difference spectra of the electron-transfer cofactors were compared between different organisms and to those previously measured in vivo, whereas the relative absorption coefficients of all cofactors were determined by using single PSI turnover conditions. Thus, the (840 nm minus 965 nm) extinction coefficients of the light-induced species (oxidized minus reduced for PC and cyt. c6, reduced minus oxidized for (FAFB), and Fd) were determined with values of 0.207 ± 0.004, - 0.033 ± 0.006, - 0.036 ± 0.008, and - 0.021 ± 0.005 for PCy, cyt. c6, (FAFB) (single reduction), and Fd, respectively, by taking a reference value of + 1 for P700+. The fact that the NIR P700 coefficient is larger than that of PCy and much larger than that of other contributing species, combined with the observed variability in the NIR P700 spectral shape, emphasizes that deconvolution of NIR signals into different components requires a very precise determination of the P700 spectrum.

Copper and iron metabolism in Ostreococcus tauri - the role of phytotransferrin, plastocyanin and a chloroplast copper-transporting ATPase.

Iron and copper are essential elements for practically all living organisms. Their metabolism is frequently interconnected, and while copper is relatively abundant in the ocean, iron is often a limiting factor for the growth of many marine microorganisms. In the present study, we aimed to elucidate the metabolisms of copper and iron and the connection of both in the marine picoalga Ostreococcus tauri. We show that O. tauri adjusts its copper economy in response to copper deficiency by downregulation of the expression of plastocyanin in favor of cytochrome c oxidase without significant changes in growth and physiology. Copper deprivation leads to increased expression of copper transporting ATPase and proteins involved in tetrapyrrole synthesis, most likely to ensure higher turnover of chlorophyll and/or heme. Elucidation of the effect of copper on the incorporation of iron into O. tauri proteins led us to identify the major iron uptake mediating protein, Ot-Fea1, whose expression and binding of iron is copper dependent. Based on our investigation of the incorporation of iron into Ot-Fea1 and ferritin, we hypothesize that O. tauri possesses another Fea1-independent iron uptake system.

Single-molecule study of redox control involved in establishing the spinach plastocyanin-cytochrome b6f electron transfer complex.

Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral cytochrome b6f (cytb6f) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytb6f complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytb6f-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120 µs). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytb6f are in opposite redox states, so complementary charges on the cytb6f and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytb6f molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the 'Z-scheme' of photosynthetic ET.

Comparative analysis of plastocyanin-cytochrome f complex formation in higher plants, green algae and cyanobacteria.

Mechanisms of the complex formation between plastocyanin and cytochrome f in higher plants (Spinacia oleracea and Brassica rapa), green microalgae Chlamydomonas reinhardtii and two species of cyanobacteria (Phormidium laminosum and Nostoc sp.) were investigated using combined Brownian and molecular dynamics simulations and hierarchical cluster analysis. In higher plants and green algae, electrostatic interactions force plastocyanin molecule close to the heme of cytochrome f. In the subsequent rotation of plastocyanin molecule around the point of electrostatic contact in the vicinity of cytochrome f, copper (Cu) atom approaches cytochrome heme forming a stable configuration where cytochrome f molecule behaves as a rather rigid body without conformational changes. In Nostoc plastocyanin molecule approaches cytochrome f in a different orientation (head-on) where the stabilization of the plastocyanin-cytochrome f complex is accompanied by the conformational changes of the G188E189D190 loop that stabilizes the whole complex. In cyanobacterium P. laminosum, electrostatic preorientation of the approaching molecules was not detected, thus indicating that random motions rather than long-range electrostatic interactions are responsible for the proper mutual orientation. We demonstrated that despite the structural similarity of the investigated electron transport proteins in different photosynthetic organisms, the complexity of molecular mechanisms of the complex formation increases in the following sequence: non-heterocystous cyanobacteria - heterocystous cyanobacteria - green algae - flowering plants.