Electron Transport in Chloroplasts: Regulation and Alternative Pathways of Electron Transfer.

This work represents an overview of electron transport regulation in chloroplasts as considered in the context of structure-function organization of photosynthetic apparatus in plants. Main focus of the article is on bifurcated oxidation of plastoquinol by the cytochrome b6f complex, which represents the rate-limiting step of electron transfer between photosystems II and I. Electron transport along the chains of non-cyclic, cyclic, and pseudocyclic electron flow, their relationships to generation of the trans-thylakoid difference in electrochemical potentials of protons in chloroplasts, and pH-dependent mechanisms of regulation of the cytochrome b6f complex are considered. Redox reactions with participation of molecular oxygen and ascorbate, alternative mediators of electron transport in chloroplasts, have also been discussed.

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.

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.

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.

PetM Is Essential for the Stabilization and Function of the Cytochrome b6f Complex in Arabidopsis.

The cytochrome b6f (cyt b6f) acts as a common linker of electron transport between photosystems I and II in oxygenic photosynthesis. PetM, one of eight subunits of the cyt b6f complex, is a small hydrophobic subunit at the outside periphery, the functional mechanism of which remains to be elucidated in higher plants. In this work, we found that unlike the PetM mutant in Synechocystis sp. PCC 6803, the Arabidopsis thaliana PetM mutant showed a bleached phenotype with yellowish leaves, block of photosynthetic electron transport and loss of photo-autotrophy, similar to the Arabidopsis PetC mutant. Although PetM is relatively conserved between higher plants and cyanobacteria, Synechocystis PetM could not rescue the PetM-knockout phenotype in Arabidopsis. We provide evidence that the Synechocystis PetM did not stably bind to the Arabidopsis cyt b6f complex. Based on these results, we suggest that PetM is required by Arabidopsis to maintain the function of the cyt b6f complex, likely through its close link with core subunits to form a tight 'fence' that stabilizes the core of the complex.

Catalytic Reactions and Energy Conservation in the Cytochrome bc1 and b6f Complexes of Energy-Transducing Membranes.

This review focuses on key components of respiratory and photosynthetic energy-transduction systems: the cytochrome bc1 and b6f (Cytbc1/b6f) membranous multisubunit homodimeric complexes. These remarkable molecular machines catalyze electron transfer from membranous quinones to water-soluble electron carriers (such as cytochromes c or plastocyanin), coupling electron flow to proton translocation across the energy-transducing membrane and contributing to the generation of a transmembrane electrochemical potential gradient, which powers cellular metabolism in the majority of living organisms. Cytsbc1/b6f share many similarities but also have significant differences. While decades of research have provided extensive knowledge on these enzymes, several important aspects of their molecular mechanisms remain to be elucidated. We summarize a broad range of structural, mechanistic, and physiological aspects required for function of Cytbc1/b6f, combining textbook fundamentals with new intriguing concepts that have emerged from more recent studies. The discussion covers but is not limited to (i) mechanisms of energy-conserving bifurcation of electron pathway and energy-wasting superoxide generation at the quinol oxidation site, (ii) the mechanism by which semiquinone is stabilized at the quinone reduction site, (iii) interactions with substrates and specific inhibitors, (iv) intermonomer electron transfer and the role of a dimeric complex, and (v) higher levels of organization and regulation that involve Cytsbc1/b6f. In addressing these topics, we point out existing uncertainties and controversies, which, as suggested, will drive further research in this field.

Evidence for convergent sensing of multiple abiotic stresses in cyanobacteria.

BACKGROUND: Bacteria routinely utilize two-component signal transduction pathways to sense and alter gene expression in response to environmental cues. While cyanobacteria express numerous two-component systems, these pathways do not regulate all of the genes within many of the identified abiotic stress-induced regulons. METHODS: Electron transport inhibitors combined with western analysis and measurement of chlorophyll a fluorescent yield, using pulse amplitude modulation fluorometry, were used to detect the effect of a diverse range of abiotic stresses on the redox status of the photosynthetic electron transport chain and the accumulation and degradation of the Synechocystis sp. PCC 6803 DEAD box RNA helicase, CrhR. RESULTS: Alterations in CrhR abundance were tightly correlated with the redox poise of the electron transport chain between QA and cytochrome b6f, with reduction favoring CrhR accumulation. CONCLUSIONS: The results provide evidence for an alternative, convergent sensing mechanism mediated through the redox poise of QB/PQH2 that senses multiple, divergent forms of abiotic stress and regulates accumulation of CrhR. The RNA helicase activity of CrhR could then function as a post-translational effector to regulate downstream gene expression. GENERAL SIGNIFICANCE: The potential for a related system in Staphylococcus aureus and higher plant chloroplasts suggest convergent sensing mechanisms may be evolutionarily conserved and occur more widely than anticipated.

Cryo-EM structure of the spinach cytochrome b6 f†complex at 3.6 Šresolution.

The cytochrome b6 f (cytb6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1-3. Electron transfer within cytb6 f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation2. In higher plants, cytb6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses3. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 f monomer near the PQ oxidation site (Qp) adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis.

Evidence of Simultaneous Spectral Hole Burning Involving Two Tiers of the Protein Energy Landscape in Cytochrome b6f.

Cytochrome b6f, with one chlorophyll molecule per protein monomer, is a simple model system whose studies can help achieve a better understanding of nonphotochemical spectral hole burning (NPHB) and single-complex spectroscopy results obtained in more complicated photosynthetic chlorophyll-protein complexes. We are reporting new data and proposing an alternative explanation for spectral dynamics that was recently observed in cytochrome b6f using NPHB. The relevant distribution of the tunneling parameter λ is a superposition of two components that are nearly degenerate in terms of the resultant NPHB yield and represent two tiers of the energy landscape responsible for NPHB. These two components likely burn competitively; we present the first demonstration of modeling a competitive NPHB process. Similar values of the NPHB yield result from distinctly different combinations of barrier heights, shifts along the generalized coordinate d, and/or masses of the entities involved in conformational changes m, with md2 parameter different by a factor of 2.7. Consequently, in cytochrome b6f, the first (at least) 10 h of fixed-temperature recovery preferentially probe different components of the barrier- and λ-distributions encoded into the spectral holes than thermocycling experiments. Both components most likely represent dynamics of the protein and not of the surrounding buffer/glycerol glass.

Structural and functional contributions of lipids to the stability and activity of the photosynthetic cytochrome b6f lipoprotein complex.

The photosynthetic cytochrome b6f complex, a homodimer containing eight distinct subunits and 26 transmembrane helices per monomer, catalyzes proton-coupled electron transfer across the thylakoid membrane. The 2.5-Å-resolution structure of the complex from the cyanobacterium Nostoc sp. revealed the presence of 23 lipid-binding sites per monomer. Although the crystal structure of the cytochrome b6f from a plant source has not yet been solved, the identities of the lipids present in a plant b6f complex have previously been determined, indicating that the predominant lipid species are monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG), and sulfoquinovosyldiacylglycerol (SQDG). Despite the extensive structural analyses of b6f-lipid interactions, the basis of the stabilization by lipids remains poorly understood. In the present study, we report on the effect of individual lipids on the structural and functional integrity of the b6f complex, purified from Spinacea oleracea It was found that (i) galactolipids (MGDG, DGDG, and SQDG) and phospholipids dilinolenoyl-phosphatidylglycerol (DLPG), 1,2-dioleoylphosphatidylglycerol (DOPG), and 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC) structurally stabilize the complex to varying degrees; (ii) SQDG has a major role in stabilizing the dimeric complex; (iii) the b6f complex is stabilized by incorporation into nanodiscs or bicelles; (iv) removal of bound phospholipid by phospholipase A2 inactivates the cytochrome complex; and (v) activity can be restored significantly by the addition of the anionic lipid PG, which is attributed to stabilization of the quinone portal and the hinge region of the iron-sulfur protein.

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.

Structure-based control of the rate limitation of photosynthetic electron transport.

The 2.5 Å structure of the cytochrome (cyt) b6 f complex provides a basis for control of the rate-limiting electron transfer step of oxygenic photosynthesis associated with the plastoquinol/quinone exchange pathway. Here, a structural change was made at a site containing two proline residues which border the intra-cyt pathway for plastoquinol/quinone exchange. The proline side chains confer a larger aperture for passage of plastoquinol/quinone. Change of these prolines to alanine in the cyanobacterium Synechococcus sp. PCC 7002 results in attenuation of this rate-limiting step, observed by a two-fold reduction in the rate of cell growth, O2 evolution, and plastoquinol-mediated reduction of cyt f. This study demonstrates modification by site-directed mutagenesis of photosynthetic energy transduction based on rational application of information in the atomic structure.

Structure-function of the cytochrome b6f lipoprotein complex: a scientific odyssey and personal perspective.

Structure-function studies of the cytochrome b6f complex, the central hetero-oligomeric membrane protein complex in the electron transport chain of oxygenic photosynthesis, which formed the basis for a high-resolution (2.5 Å) crystallographic solution of the complex, are described. Structure-function differences between the structure of subunits of the bc complexes, b6f, and bc1 from mitochondria and photosynthetic bacteria, which are often assumed to function identically, are discussed. Major differences which suggest that quinone-dependent electron transport pathways can vary in b6f and bc1 complexes are as follows: (a) an additional c-type heme, cn, and bound single copies of chlorophyll a and ß-carotene in the b6f complex; and (b) a cyclic electron transport pathway that encompasses the b6f and PSI reaction center complexes. The importance of including lipid in crystallization of the cytochrome complex, or with any hetero-oligomeric membrane protein complex, is emphasized, and consequences to structure-function of b6f being a lipoprotein complex discussed, including intra-protein dielectric heterogeneity and resultant pathways of trans-membrane electron transport. The role of the b6f complex in trans-membrane signal transduction from reductant generated on the p-side of the electron transport chain to the regulation of light energy to the two photosystems by trans-side phosphorylation of the light-harvesting chlorophyll protein is presented. Regarding structure aspects relevant to plastoquinol-quinone entrance-egress: (i) modification of the p-side channel for plastoquinone access to the iron-sulfur protein would change the rate-limiting step in electron transport; (ii) the narrow niche for entry of plastoquinol into b6f from the PSII reaction center complex would seem to require close proximity between the complexes.

Structure of a PSI-LHCI-cyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions.

Photosynthetic linear electron flow (LEF) produces ATP and NADPH, while cyclic electron flow (CEF) exclusively drives photophosphorylation to supply extra ATP. The fine-tuning of linear and cyclic electron transport levels allows photosynthetic organisms to balance light energy absorption with cellular energy requirements under constantly changing light conditions. As LEF and CEF share many electron transfer components, a key question is how the same individual structural units contribute to these two different functional modes. Here, we report the structural identification of a photosystem I (PSI)-light harvesting complex I (LHCI)-cytochrome (cyt) b6f supercomplex isolated from the unicellular alga Chlamydomonas reinhardtii under anaerobic conditions, which induces CEF. This provides strong evidence for the model that enhanced CEF is induced by the formation of CEF supercomplexes, when stromal electron carriers are reduced, to generate additional ATP. The additional identification of PSI-LHCI-LHCII complexes is consistent with recent findings that both CEF enhancement and state transitions are triggered by similar conditions, but can occur independently from each other. Single molecule fluorescence correlation spectroscopy indicates a physical association between cyt b6f and fluorescent chlorophyll containing PSI-LHCI supercomplexes. Single particle analysis identified top-view projections of the corresponding PSI-LHCI-cyt b6f supercomplex. Based on molecular modeling and mass spectrometry analyses, we propose a model in which dissociation of LHCA2 and LHCA9 from PSI supports the formation of this CEF supercomplex. This is supported by the finding that a Δlhca2 knockout mutant has constitutively enhanced CEF.

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR.

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b6f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast.

The Cytochrome b 6 f Complex: Biophysical Aspects of Its Functioning in Chloroplasts.

This chapter presents an overview of structural properties of the cytochrome (Cyt) b 6 f complex and its functioning in chloroplasts. The Cyt b 6 f complex stands at the crossroad of photosynthetic electron transport pathways, providing connectivity between Photosystem (PSI) and Photosysten II (PSII) and pumping protons across the membrane into the thylakoid lumen. After a brief review of the chloroplast electron transport chain, the consideration is focused on the structural organization of the Cyt b 6 f complex and its interaction with plastoquinol (PQH2, reduced form of plastoquinone), a mediator of electron transfer from PSII to the Cyt b 6 f complex. The processes of PQH2 oxidation by the Cyt b 6 f complex have been considered within the framework of the Mitchell's Q-cycle. The overall rate of the intersystem electron transport is determined by PQH2 turnover at the quinone-binding site Qo of the Cyt b 6 f complex. The rate of PQH2 oxidation is controlled by the intrathylakoid pHin, which value determines the protonation/deprotonation events in the Qo-center. Two other regulatory mechanisms associated with the Cyt b 6 f complex are briefly overviewed: (i) redistribution of electron fluxes between alternative (linear and cyclic) pathways, and (ii) "state transitions" related to redistribution of solar energy between PSI and PSII.

Natively oxidized amino acid residues in the spinach cytochrome b 6 f complex.

The cytochrome b 6 f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b 6 f is 10-20 fold higher than that observed for the analogous respiratory cytochrome bc1 complex. The types of ROS produced (O2•-, 1O2, and, possibly, H2O2) and the site(s) of ROS production within the b 6 f complex have been the subject of some debate. Proposed sources of ROS have included the heme b p , PQ p•- (possible sources for O2•-), the Rieske iron-sulfur cluster (possible source of O2•- and/or 1O2), Chl a (possible source of 1O2), and heme c n (possible source of O2•- and/or H2O2). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b 6 f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b 6 f complex.

Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid.

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc1 complex (cytbc1) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc1 complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc1 complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc1 to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc1 complexes retain their native dimeric structure and co-purify with 56±6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b6f complex (cytb6f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc1/cytb6f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q0 and Qi sites.

Probing Energy Landscapes of Cytochrome b6f with Spectral Hole Burning: Effects of Deuterated Solvent and Detergent.

In non-photochemical spectral hole burning (NPHB) and spectral hole recovery experiments, cytochrome b6f protein exhibits behavior that is almost independent of the deuteration of the buffer/glycerol glassy matrix containing the protein, apart from some differences in heat dissipation. On the other hand, strong dependence of the hole burning properties on sample preparation procedures was observed and attributed to a large increase of the electron-phonon coupling and shortening of the excited-state lifetime occurring when n-dodecyl ß-d-maltoside (DM) is used as a detergent instead of n-octyl ß-d-glucopyranoside (OGP). The data was analyzed assuming that the tunneling parameter distribution or barrier distribution probed by NPHB and encoded into the spectral holes contains contributions from two nonidentical components with accidentally degenerate excited state λ-distributions. Both components likely reflect protein dynamics, although with some small probability one of them (with larger md2) may still represent the dynamics involving specifically the -OH groups of the water/glycerol solvent. Single proton tunneling in the water/glycerol solvent environment or in the protein can be safely excluded as the origin of observed NPHB and hole recovery dynamics. The intensity dependence of the hole growth kinetics in deuterated samples likely reflects differences in heat dissipation between protonated and deuterated samples. These differences are most probably due to the higher interface thermal resistivity between (still protonated) protein and deuterated water/glycerol outside environment.

Ultrafast dynamics of the photo-excited hemes b and cn in the cytochrome b6f complex.

The dynamics of hemes b and cn within the cytochrome b6f complex are investigated by means of ultrafast broad-band transient absorption spectroscopy. On the one hand, the data reveal that, subsequent to visible light excitation, part of the b hemes undergoes pulse-limited photo-oxidation, with the liberated electron supposedly being transferred to one of the adjacent aromatic amino acids. Photo-oxidation is followed by charge recombination in about 8.2 ps. Subsequent to charge recombination, heme b is promoted to a vibrationally excited ground state that relaxes in about 4.6 ps. On the other hand, heme cn undergoes ultrafast ground state recovery in about 140 fs. Interestingly, the data also show that, in contrast to previous beliefs, Chl a is involved in the photochemistry of hemes. Indeed, subsequent to heme excitation, Chl a bleaches and recovers to its ground state in 90 fs and 650 fs, respectively. Chl a bleaching allegedly corresponds to the formation of a short lived Chl a anion. Beyond the previously suggested structural role, this study provides unique evidence that Chl a is directly involved in the photochemistry of the hemes.