Cytochrome B6f Research Articles

Cryo-EM structures of the Synechocystis sp. PCC 6803 cytochrome b6f complex with and without the regulatory PetP subunit.

In oxygenic photosynthesis, the cytochrome b6f (cytb6f) complex links the linear electron transfer (LET) reactions occurring at photosystems I and II and generates a transmembrane proton gradient via the Q-cycle. In addition to this central role in LET, cytb6f also participates in a range of processes including cyclic electron transfer (CET), state transitions and photosynthetic control. Many of the regulatory roles of cytb6f are facilitated by auxiliary proteins that differ depending upon the species, yet because of their weak and transient nature the structural details of these interactions remain unknown. An apparent key player in the regulatory balance between LET and CET in cyanobacteria is PetP, a ∼10 kDa protein that is also found in red algae but not in green algae and plants. Here, we used cryogenic electron microscopy to determine the structure of the Synechocystis sp. PCC 6803 cytb6f complex in the presence and absence of PetP. Our structures show that PetP interacts with the cytoplasmic side of cytb6f, displacing the C-terminus of the PetG subunit and shielding the C-terminus of cytochrome b6, which binds the heme cn cofactor that is suggested to mediate CET. The structures also highlight key differences in the mode of plastoquinone binding between cyanobacterial and plant cytb6f complexes, which we suggest may reflect the unique combination of photosynthetic and respiratory electron transfer in cyanobacterial thylakoid membranes. The structure of cytb6f from a model cyanobacterial species amenable to genetic engineering will enhance future site-directed mutagenesis studies of structure-function relationships in this crucial ET complex.

Concerning the enigmatic cytochrome b-559 of oxygenic photosynthesis.

Although there is an extensive literature on the properties and possible electron transfer pathways of cytochrome b-559, which is a prominent subunit of the multi-subunit photosystem II complex which functions in oxygenic photosynthesis, there is presently no consensus on the function of b-559 in the photosynthetic electron transport chain. The inability in earlier times to define a redox-linked function of this cytochrome was, to a large extent, a consequence of an absence of biochemical and structure information to complement an extensive array of spectrophotometric studies of the cytochrome in situ. Based on the location of hetero-dimeric b-559 in the photosystem II reaction center complex, derived from crystal crystallographic structure analysis, and the absence of a necessary redox function for the cytochrome in PSII, it is proposed that the main function of cytochrome b-559 is linked to its role as a structure component in the PSII reaction center complex. This function resides in the association of b-559 through its heme histidine residues in the trans-membrane domains of the PsbE and PsbF subunits of the PSII reaction center. These subunits, along with PsbJ, are inferred, from the analysis of structure, to define the intra-membrane portal in the PSII reaction center for plastoquinol (PQH2) export which, through the PSII complex, provides the redox link to the cytochrome b6f complex in the electron transfer chain.

De-etiolation-induced protein 1 (DEIP1) mediates assembly of the cytochrome b6f complex in Arabidopsis

The conversion of light energy to chemical energy by photosynthesis requires the concerted action of large protein complexes in the thylakoid membrane. Recent work has provided fundamental insights into the three-dimensional structure of these complexes, but how they are assembled from hundreds of parts remains poorly understood. Particularly little is known about the biogenesis of the cytochrome b6f complex (Cytb6f), the redox-coupling complex that interconnects the two photosystems. Here we report the identification of a factor that guides the assembly of Cytb6f in thylakoids of chloroplasts. The protein, DE-ETIOLATION-INDUCED PROTEIN 1 (DEIP1), resides in the thylakoid membrane and is essential for photoautotrophic growth. Knock-out mutants show a specific loss of Cytb6f, and are defective in complex assembly. We demonstrate that DEIP1 interacts with the two cytochrome subunits of the complex, PetA and PetB, and mediates the assembly of intermediates in Cytb6f biogenesis. The identification of DEIP1 provides an entry point into the study of the assembly pathway of a crucial complex in photosynthetic electron transfer.

Paradoxes in judging the inhibition of photosynthetic electron transfer chain using P700 oxidation and dark re-reduction analyses

• P700 redox kinetics analyses to identify functions of photosynthetic inhibitors • DNP-INT is unable to block electron flow between photosystem II and photosystem I • Leakage of electrons from Cytochrome b6f to plastocyanine cause reduction of oxidized P700 • P700 redox kinetics ensures inhibition of electron flow to PSI and alternate electron flow from cytochrome b6f and PQ-pool

Gene expression and organization of thylakoid protein complexes in the PSII-less mutant of Synechocystis sp. PCC 6803.

Photosystems I and II (PSI and PSII) are the integral components of the photosynthetic electron transport chain that utilize light to provide chemical energy for CO2 fixation. In this study, we investigated how the deficiency of PSII affects the gene expression, accumulation, and organization of thylakoid protein complexes as well as physiological characteristics of Synechocystis sp. PCC 6803 by combining biochemical, biophysical, and transcriptomic approaches. RNA‐seq analysis showed upregulated expression of genes encoding the PSII core proteins, and downregulation of genes associated with interaction between light‐harvesting phycobilisomes and PSI. Two‐dimensional separation of thylakoid protein complexes confirmed the lack of PSII complexes, yet unassembled PSII subunits were detected. The content of PsaB representing PSI was lower, while the content of cytochrome b6f complexes was higher in the PSII‐less strain as compared with control (CS). Application of oxygraph measurements revealed higher rates of dark respiration and lower PSI activity in the mutant. The latter likely resulted from the detected decrease in the accumulation of PSI, PSI monomerization, increased proportion of energetically decoupled phycobilisomes in PSII‐less cultures, and low abundance of phycocyanin. Merging the functional consequences of PSII depletion with differential protein and transcript accumulation in the mutant, in comparison to CS, identified signal transduction from the photosynthetic apparatus to the genome level.

Ser/Thr Protein Kinase SpkI Affects Photosynthetic Efficiency in Synechocystis sp. PCC 6803 upon Salt Stress.

High salinity is a common environmental factor that limits productivity and growth for photosynthetic organisms. Here, we identified a mutant defected in gene sll1770, which encodes a Ser/Thr protein kinase SpkI, with a significantly low maximal quantum yield of PSII under high salt condition in Synechocystis sp. PCC 6803. Physiological characterization demonstrated that the ΔspkI mutant had normal growth and photosynthesis under control condition. And a significantly higher NPQ capacity was also observed in ΔspkI when grown under control condition. However, when grown under high salt condition, ΔspkI exhibited apparently slower growth as well as decreased net photosynthesis and PSII activity. Western blot analysis demonstrated that the amount of major photosynthetic proteins declined sharply in ΔspkI when cells grown under high salt condition. Redox kinetics measurement suggested that the activities of PSI and cytochrome b6f complex were modified in ΔspkI under high salt condition, which resulted in a more reduced PQ pool in ΔspkI. Chlorophyll fluorescence traces suggested that the OA− reoxidation and state transition was also impaired in ΔspkI under high salt condition. Above all, we propose that Ser/Thr protein kinase SpkI plays a role in maintaining high-effective photosynthesis during high-salt acclimation process in Synechocystis.

Quinone binding sites of cyt bc complexes analysed by X-ray crystallography and cryogenic electron microscopy.

Cytochrome (cyt) bc1, bcc and b6f complexes, collectively referred to as cyt bc complexes, are homologous isoprenoid quinol oxidising enzymes present in diverse phylogenetic lineages. Cyt bc1 and bcc complexes are constituents of the electron transport chain (ETC) of cellular respiration, and cyt b6f complex is a component of the photosynthetic ETC. Cyt bc complexes share in general the same Mitchellian Q cycle mechanism, with which they accomplish proton translocation and thus contribute to the generation of proton motive force which drives ATP synthesis. They therefore require a quinol oxidation (Qo) and a quinone reduction (Qi) site. Yet, cyt bc complexes evolved to adapt to specific electrochemical properties of different quinone species and exhibit structural diversity. This review summarises structural information on native quinones and quinone-like inhibitors bound in cyt bc complexes resolved by X-ray crystallography and cryo-EM structures. Although the Qi site architecture of cyt bc1 complex and cyt bcc complex differs considerably, quinone molecules were resolved at the respective Qi sites in very similar distance to haem bH. In contrast, more diverse positions of native quinone molecules were resolved at Qo sites, suggesting multiple quinone binding positions or captured snapshots of trajectories toward the catalytic site. A wide spectrum of inhibitors resolved at Qo or Qi site covers fungicides, antimalarial and antituberculosis medications and drug candidates. The impact of these structures for characterising the Q cycle mechanism, as well as their relevance for the development of medications and agrochemicals are discussed.

Differentially expressed genes in Mythimna separata under chlorantraniliprole exposure and functional identification

The oriental armyworm, Mythimna separata has led to great economic losses of corn in China. The main method to control this pest relies on chemical insecticides. Plenty of genes in armyworm can be activated in response to insecticides. In this study, the critical genes involved in detoxification of chlorantraniliprole in armyworm were investigated using RNA-seq (transcriptome sequencing), digital gene expression profiles, and functional identification by RNA interference (RNAi) technology. The de novo assembly of the transcriptomes was achieved based on Illumina short-read sequencing technology in M. separata. There were 70,845 unigenes produced and 35,327 (49.86%) annotated. 748 unigenes were significantly up-expressed and 741 were significantly down-expressed in chlorantraniliprole treatment group compared to the control group. Silencing of cytochrome B6-F (CYB6), and long chain fatty acid CoA ligase 4 (CoA) of the top 18 highly expressed genes selected in chlorantraniliprole treatment group, resulted in more significant sensibility of M. separata to chlorantraniliprole, growth retardation and antifeedant phenomenon, which indicated that both CYB6 and CoA were most possibly responsible for chlorantraniliprole metabolism/detoxication.

Non-rolling flag leaves use an effective mechanism to reduce water loss and light-induced damage under drought stress.

The study reports on four different types of flag leaf rolling under soil drought in relation to the level of cell wall-bound phenolics. The flag leaf colonization by aphids, as a possible bioindicator of the accumulation of cell wall-bound phenolics, was also estimated. The proteins of the photosynthetic apparatus that form its core and are crucial for maintaining its stability (D1/PsbA protein), limit destructive effects of light (PsbS, a protein binding carotenoids in the antennas) and participate in efficient electron transport between photosystems II (PSII) and PSI (Rieske iron-sulfur protein of the cytochrome b6f complex) were evaluated in two types of flag leaf rolling. Additionally, biochemical and physiological reactions to drought stress in rolling and non-rolling flag leaves were compared. The study identified four types of genome-related types of flag leaf rolling. The biochemical basis for these differences was a different number of phenolic molecules incorporated into polycarbohydrate structures of the cell wall. In an extreme case of non-rolling dehydrated flag leaves, they were found to accumulate high amounts of cell wall-bound phenolics that limited cell water loss and protected the photosynthetic apparatus against excessive light. PSII was also additionally protected against excess light by the accumulation of photosynthetic apparatus proteins that ensured stable and efficient transport of excitation energy beyond PSII and its dissipation as far-red fluorescence and heat. Our analysis revealed a new type of flag leaf rolling brought about by an interaction between wheat and rye genomes, and resulting in biochemical specialization of flexible, rolling and rigid, non-rolling parts of the flag leaf. The study confirmed limited aphid colonization of the flag leaves with enhanced content of cell wall-bound phenolics. Non-rolling leaves developed effective adaptation mechanisms to reduce both water loss and photoinhibitory damage to the photosynthetic apparatus under drought stress.

Low Light Increases the Abundance of Light Reaction Proteins: Proteomics Analysis of Maize (Zea mays L.) Grown at High Planting Density.

Maize (Zea mays L.) is usually planted at high density, so most of its leaves grow in low light. Certain morphological and physiological traits improve leaf photosynthetic capacity under low light, but how light absorption, transmission, and transport respond at the proteomic level remains unclear. Here, we used tandem mass tag (TMT) quantitative proteomics to investigate maize photosynthesis-related proteins under low light due to dense planting, finding increased levels of proteins related to photosystem II (PSII), PSI, and cytochrome b6f. These increases likely promote intersystem electron transport and increased PSI end electron acceptor abundance. OJIP transient curves revealed increases in some fluorescence parameters under low light: quantum yield for electron transport (φEo), probability that an electron moves beyond the primary acceptor QA− (ψo), efficiency/probability of electron transfer from intersystem electron carriers to reduction end electron acceptors at the PSI acceptor side (δRo), quantum yield for reduction of end electron acceptors at the PSI acceptor side (φRo), and overall performance up to the PSI end electron acceptors (PItotal). Thus, densely planted maize shows elevated light utilization through increased electron transport efficiency, which promotes coordination between PSII and PSI, as reflected by higher apparent quantum efficiency (AQE), lower light compensation point (LCP), and lower dark respiration rate (Rd).

Evidence for a robust photosystem II in the photosynthetic amoeba Paulinella.

Paulinella represents the only known case of an independent primary plastid endosymbiosis, outside Archaeplastida, that occurred c. 120 (million years ago) Ma. These photoautotrophs grow very slowly in replete culture medium with a doubling time of 6-7 d at optimal low light, and are highly sensitive to photodamage under moderate light levels. We used genomic and biophysical methods to investigate the extreme slow growth rate and light sensitivity of Paulinella, which are key to photosymbiont integration. All photosystem II (PSII) genes except psb28-2 and all cytochrome b6 f complex genes except petM and petL are present in Paulinella micropora KR01 (hereafter, KR01). Biophysical measurements of the water oxidation complex, variable chlorophyll fluorescence, and photosynthesis-irradiance curves show no obvious evidence of PSII impairment. Analysis of photoacclimation under high-light suggests that although KR01 can perform charge separation, it lacks photoprotection mechanisms present in cyanobacteria. We hypothesize that Paulinella species are restricted to low light environments because they are deficient in mitigating the formation of reactive oxygen species formed within the photosystems under peak solar intensities. The finding that many photoprotection genes have been lost or transferred to the host-genome during endosymbiont genome reduction, and may lack light-regulation, is consistent with this hypothesis.

Chloroplast translational regulation uncovers nonessential photosynthesis genes as key players in plant cold acclimation.

Plants evolved efficient multifaceted acclimation strategies to cope with low temperatures. Chloroplasts respond to temperature stimuli and participate in temperature sensing and acclimation. However, very little is known about the involvement of chloroplast genes and their expression in plant chilling tolerance. Here we systematically investigated cold acclimation in tobacco seedlings over 2 days of exposure to low temperatures by examining responses in chloroplast genome copy number, transcript accumulation and translation, photosynthesis, cell physiology, and metabolism. Our time-resolved genome-wide investigation of chloroplast gene expression revealed substantial cold-induced translational regulation at both the initiation and elongation levels, in the virtual absence of changes at the transcript level. These cold-triggered dynamics in chloroplast translation are widely distinct from previously described high light-induced effects. Analysis of the gene set responding significantly to the cold stimulus suggested nonessential plastid-encoded subunits of photosynthetic protein complexes as novel players in plant cold acclimation. Functional characterization of one of these cold-responsive chloroplast genes by reverse genetics demonstrated that the encoded protein, the small cytochrome b6f complex subunit PetL, crucially contributes to photosynthetic cold acclimation. Together, our results uncover an important, previously underappreciated role of chloroplast translational regulation in plant cold acclimation.

True oxygen reduction capacity during photosynthetic electron transfer in thylakoids and intact leaves.

Reactive oxygen species (ROS) are generated in electron transport processes of living organisms in oxygenic environments. Chloroplasts are plant bioenergetics hubs where imbalances between photosynthetic inputs and outputs drive ROS generation upon changing environmental conditions. Plants have harnessed various site-specific thylakoid membrane ROS products into environmental sensory signals. Our current understanding of ROS production in thylakoids suggests that oxygen (O2) reduction takes place at numerous components of the photosynthetic electron transfer chain (PETC). To refine models of site-specific O2 reduction capacity of various PETC components in isolated thylakoids of Arabidopsis thaliana, we quantified the stoichiometry of oxygen production and consumption reactions associated with hydrogen peroxide (H2O2) accumulation using membrane inlet mass spectrometry and specific inhibitors. Combined with P700 spectroscopy and electron paramagnetic resonance spin trapping, we demonstrate that electron flow to photosystem I (PSI) is essential for H2O2 accumulation during the photosynthetic linear electron transport process. Further leaf disc measurements provided clues that H2O2 from PETC has a potential of increasing mitochondrial respiration and CO2 release. Based on gas exchange analyses in control, site-specific inhibitor-, methyl viologen-, and catalase-treated thylakoids, we provide compelling evidence of no contribution of plastoquinone pool or cytochrome b6f to chloroplastic H2O2 accumulation. The putative production of H2O2 in any PETC location other than PSI is rapidly quenched and therefore cannot function in H2O2 translocation to another cellular location or in signaling.

Electrochemically Driven Photosynthetic Electron Transport in Cyanobacteria Lacking Photosystem II.

Light-activated photosystem II (PSII) carries out the critical step of splitting water in photosynthesis. However, PSII is susceptible to light-induced damage. Here, results are presented from a novel microbial electro-photosynthetic system (MEPS) that uses redox mediators in conjunction with an electrode to drive electron transport in live Synechocystis (ΔpsbB) cells lacking PSII. MEPS-generated, light-dependent current increased with light intensity up to 2050 μmol photons m-2 s-1, which yielded a delivery rate of 113 μmol electrons h-1 mg-chl-1 and an average current density of 150 A m-2 s-1 mg-chl-1. P700+ re-reduction kinetics demonstrated that initial rates exceeded wildtype PSII-driven electron delivery. The electron delivery occurs ahead of the cytochrome b6f complex to enable both NADPH and ATP production. This work demonstrates an electrochemical system that can drive photosynthetic electron transport, provides a platform for photosynthetic foundational studies, and has the potential for improving photosynthetic performance at high light intensities.

Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex

Ferredoxin-NADP+ reductase (FNR) was previously inferred to bind to the cytochrome b6f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b6f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b6f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b6f complex in detergent solution nor with the complex reconstituted in liposomes.

Electron transfer via cytochrome b6f complex displays sensitivity to antimycin A upon STT7 kinase activation.

The cytochrome b6f complex (b6f) has been initially considered as the ferredoxin-plastoquinone reductase (FQR) during cyclic electron flow (CEF) with photosystem I that is inhibited by antimycin A (AA). The binding of AA to the b6f Qi-site is aggravated by heme-ci, which challenged the FQR function of b6f during CEF. Alternative models suggest that PROTON GRADIENT REGULATION5 (PGR5) is involved in a b6f-independent, AA-sensitive FQR. Here, we show in Chlamydomonas reinhardtii that the b6f is conditionally inhibited by AA in vivo and that the inhibition did not require PGR5. Instead, activation of the STT7 kinase upon anaerobic treatment induced the AA sensitivity of b6f which was absent from stt7-1. However, a lock in State 2 due to persisting phosphorylation in the phosphatase double mutant pph1;pbcp did not increase AA sensitivity of electron transfer. The latter required a redox poise, supporting the view that state transitions and CEF are not coercively coupled. This suggests that the b6f-interacting kinase is required for structure-function modulation of the Qi-site under CEF favoring conditions. We propose that PGR5 and STT7 independently sustain AA-sensitive FQR activity of the b6f. Accordingly, PGR5-mediated electron injection into an STT7-modulated Qi-site drives a Mitchellian Q cycle in CEF conditions.

Structure function of the Cytochrome C and Cytochrome b6f complex genes in Moringa peregrina

Abstract To satisfy the needs of a growing global population, it is vital to develop viable replacement crops. Moringa is a tropical and subtropical plant that grows in multiple regions in the world. The Moringaceae family includes the genus M. peregrina, it has been discovered to have a variety of medical purposes; therefore M. peregrina must be grown in a variety of locations around Egypt. The purpose of this study is to determine the RNA sequences of tow genes and their phylogenetic relationships of 2780 sequence alignments collected from NCBI as well as M. peregrina from 255 genera across the novel gene cyt C, and 1955 sequence alignments as well as M. peregrina from 177 genera accessions numbers in NCBI (MZ005963 and MZ005966, respectively). Furthermore, cyt C (879 bp) whilst, cyt b6f complex (468bp). The present study included the investigation of allele frequency, partial haploid, haploid SNPs, SNPs transitions, and Transversions M. peregrina. The phylogenetic analysis was utilized to evaluate M. peregrina genetic variation and association with other plant species, according to our findings. Using sequence alignment from NCBI as well as Moringa, we were able to identify multiple cytochrome genes according to sequence alignment. The sequence alignment of the cytochrome C and cytochrome b6f complex genes reveals a high level of genetic variety and diversity among Moringa and other species of plants.

Plant Photochemistry, Reactive Oxygen Species, and Photoprotection

Light energy, absorbed as photons by chlorophylls and other pigment molecules consisting of light-harvesting complexes (LHCs), is transferred to the reaction centres (RCs), where, through charge separation, electrons flow from photosystem II (PSII) through cytochrome b6f and diffusible electron carriers to photosystem I (PSI) [...]

Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex

Ferredoxin-NADP+ reductase (FNR) was previously inferred to bind to the cytochrome b6f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b6f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b6f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b6f complex in detergent solution nor with the complex reconstituted in liposomes. A previous inference of a small amplitude but defined FNR-b6f interaction is explained by FNR interaction with micelles of the undecyl β-D maltoside (UDM) detergent micelles used to purify b6f. Circular dichroism, employed to analyze the effect of detergent on the FNR structure, did not reveal significant changes in secondary or tertiary structures of FNR domains in the presence of UDM detergent. However, thermodynamic analysis implied a significant decrease in an interaction between the N-terminal FAD-binding and C-terminal NADP+-binding domains of FNR caused by detergent. The enthalpy, ΔHo, and the entropy, ΔSo, associated with FNR unfolding decreased four-fold in the presence of 1 mM UDM at pH 6.5. In addition to the conclusion regarding the absence of a binding interaction of significant amplitude between FNR and the b6f complex, these studies provide a precedent for consideration of significant background protein-detergent interactions in ITC analyses involving integral membrane proteins.

Divergent physiological and molecular responses of light‐ and iron‐limited Southern Ocean phytoplankton

AbstractIt has recently been shown that Southern Ocean phytoplankton species have evolved to optimize their light‐harvesting potential without increasing the high iron‐requiring proteins used for photosynthesis. We measured molecular and physiological responses of phytoplankton cultures under a combination of iron and light conditions. While iron‐replete cultures mostly increased biovolume, photochemical efficiency (Fv/Fm) and the relative abundance of photosystem II (PSII) and Cytochrome b6f protein compared to iron‐limited cultures, light also regulated cellular chlorophyll a content and played a role in controlling PSII protein abundance. Investment of protein resources into the carbon fixing enzyme Ribulose 1,5‐bisphosphate carboxylase oxygenase (Rubisco) was species‐specific, but increased growth rates correlated with increased investment into Rubisco for all species. Our results suggest that Proboscia inermis uses a divergent molecular strategy to compete for nutrients, light, and CO2 in the Southern Ocean.