BBA - Bioenergetics (v.1807, #2)

The structural basis of secondary active transport mechanisms by Lucy R. Forrest; Reinhard Krämer; Christine Ziegler (167-188).
Secondary active transporters couple the free energy of the electrochemical potential of one solute to the transmembrane movement of another. As a basic mechanistic explanation for their transport function the model of alternating access was put forward more than 40 years ago, and has been supported by numerous kinetic, biochemical and biophysical studies. According to this model, the transporter exposes its substrate binding site(s) to one side of the membrane or the other during transport catalysis, requiring a substantial conformational change of the carrier protein. In the light of recent structural data for a number of secondary transport proteins, we analyze the model of alternating access in more detail, and correlate it with specific structural and chemical properties of the transporters, such as their assignment to different functional states in the catalytic cycle of the respective transporter, the definition of substrate binding sites, the type of movement of the central part of the carrier harboring the substrate binding site, as well as the impact of symmetry on fold-specific conformational changes. Besides mediating the transmembrane movement of solutes, the mechanism of secondary carriers inherently involves a mechanistic coupling of substrate flux to the electrochemical potential of co-substrate ions or solutes. Mainly because of limitations in resolution of available transporter structures, this important aspect of secondary transport cannot yet be substantiated by structural data to the same extent as the conformational change aspect. We summarize the concepts of coupling in secondary transport and discuss them in the context of the available evidence for ion binding to specific sites and the impact of the ions on the conformational state of the carrier protein, which together lead to mechanistic models for coupling.► Comparison of structural fold of all major classes of secondary active transporters. ► Correlation of catalytic states of transporters with carrier conformation. ► Structural analysis of substrate and co-substrate binding sites. ► Analysis of the structural basis of coupling in secondary transport.
Keywords: Secondary active transport; Protein structure; Carrier; Coupling; Modeling; Protein conformation;

The purple photosynthetic bacterium Rubrivivax gelatinosus has, at least, four periplasmic electron carriers, i.e., HiPIP, two cytochromes c 8 with low- and high-midpoint potentials, and cytochrome c 4 as electron donors to the photochemical reaction center. The quadruple mutant lacking all four electron carrier proteins showed extremely slow photosynthetic growth. During the long-term cultivation of this mutant under photosynthetic conditions, a suppressor strain recovering the wild-type growth level appeared. In the cells of the suppressor strain, we found significant accumulation of a soluble c-type cytochrome that has not been detected in wild-type cells. This cytochrome c has a redox midpoint potential of about + 280 mV and could function as an electron donor to the photochemical reaction center in vitro. The amino acid sequence of this cytochrome c was 65% identical to that of the high-potential cytochrome c 8 of this bacterium. The gene for this cytochrome c was identified as nirM on the basis of its location in the newly identified nir operon, which includes a gene coding cytochrome cd 1-type nitrite reductase. Phylogenetic analysis and the well-conserved nir operon gene arrangement suggest that the origin of the three cytochromes c 8 in this bacterium is NirM. The two other cytochromes c 8, of high and low potentials, proposed to be generated by gene duplication from NirM, have evolved to function in distinct pathways.►The third cytochrome c8 was found in R. gelatinosus. ►The gene for cytochrome c8 is in the nitrite reductase gene cluster as nirM. ►NirM could donate electron to RC in R. gelatinosus. ►NirM was overexpressed in deletion mutant of soluble electron donors.
Keywords: Cytochrome c 8; Nitrite reduction; Photosynthesis; nir operon; Electron donor;

How the N-terminal extremity of Saccharomyces cerevisiae IF1 interacts with ATP synthase: A kinetic approach by Tiona Andrianaivomananjaona; Martin Moune-Dimala; Sameh Herga; Violaine David; Francis Haraux (197-204).
The N-terminal part of the inhibitory peptide IF1 interacts with the central γ subunit of mitochondrial isolated extrinsic part of ATP synthase in the inhibited complex (J.R. Gledhill, M.G. Montgomery, G.W. Leslie, J.E. Walker, 2007). To explore its role in the different steps of IF1 binding, kinetics of inhibition of the isolated and membrane-bound enzymes were investigated using Saccharomyces cerevisiae IF1 derivatives modified in N-terminal extremity. First, we studied peptides truncated in Nter up to the amino acid immediately preceding Phe17, a well-conserved residue thought to play a key role. These deletions did not affect or even improve the access of IF1 to its target. They decreased the stability of the inhibited complex but much less than previously proposed. We also mutated IF1-Phe17 and found this amino acid not mandatory for the inhibitory effect. The most striking finding came from experiments in which PsaE, a 8 kDa globular-like protein, was attached in Nter of IF1. Unexpectedly, such a modification did not appreciably affect the rate of IF1 binding. Taken together, these data show that IF1-Nter plays no role in the recognition step but contributes to stabilize the inhibited complex. Moreover, the data obtained using chimeric PsaE-IF1 suggest that before binding IF1 presents to the enzyme with its middle part facing a catalytic interface and its Nter extremity folded in the opposite direction.Display Omitted►Kinetic approach reveals the different steps of inhibitory peptide IF1 binding to F0F1-ATPase. ►Yeast IF1 fused in Nter with a 8 kDa globular polypeptide readily inhibits F1-ATPase. ►IF1 mid-part binds to αβ interface before its Nter part wraps around γ subunit. ►Residues 14–15–16 of yeast IF1 help to stabilize the ATPase-IF1 inhibited complex. ►Phenylalanine marking the beginning of IF1 mid-part is not mandatory for inhibition.
Keywords: ATP synthase; IF1; Inhibitory peptide; Mitochondria; Yeast; Binding kinetics;

Electron paramagnetic resonance study of the electron transfer reactions in photosystem II membrane preparations from Arabidopsis thaliana by Guiying Chen; Yagut Allahverdiyeva; Eva-Mari Aro; Stenbjörn Styring; Fikret Mamedov (205-215).
Arabidopsis thaliana is widely used as a model organism in plant biology as its genome has been sequenced and transformation is known to be efficient. A large number of mutant lines and genomic resources are available for Arabidopsis. All this makes Arabidopsis a useful tool for studies of photosynthetic reactions in higher plants. In this study, photosystem II (PSII) enriched membranes were successfully isolated from thylakoids of Arabidopsis plants and for the first time the electron transfer cofactors in PSII were systematically studied using electron paramagnetic resonance (EPR) spectroscopy. EPR signals from both of the donor and acceptor sides of PSII, as well as from auxiliary electron donors were recorded. From the acceptor side of PSII, EPR signals from QAˉ Fe2+ and Pheˉ QAˉ Fe2+ as well as from the free Pheˉ radical were observed. The multiline EPR signals from the S0- and S2-states of CaMn4O x -cluster in the water oxidation complex were characterized. Moreover, split EPR signals, the interaction signals from YZ • and CaMn4O x -cluster in the S0-, S1-, S2-, and the S3-state were induced by illumination of the PSII membranes at 5 K and characterized. In addition, EPR signals from auxiliary donors YD •, Chl+ and cytochrome b 559 were observed. In total, we were able to detect about 20 different EPR signals covering all electron transfer components in PSII. Use of this spectroscopic platform opens a possibility to study PSII reactions in the library of mutants available in Arabidopsis.►Active Photosystem II membrane preparations from Arabidopsis thaliana were isolated. ►Full range of the Photosystem II redox cofactors was investigated by electron paramagnetic resonance (EPR) spectroscopy. ►EPR signals from all S states of the water oxidizing complex were also detected. ►Spectroscopic platform to study PSII reactions in wild type and mutants from Arabidopsis thaliana is created.
Keywords: Photosystem II; Electron paramagnetic resonance; Arabidopsis thaliana;

Effects of formate binding on the quinone–iron electron acceptor complex of photosystem II by Arezki Sedoud; Lisa Kastner; Nicholas Cox; Sabah El-Alaoui; Diana Kirilovsky; A. William Rutherford (216-226).
EPR was used to study the influence of formate on the electron acceptor side of photosystem II (PSII) from Thermosynechococcus elongatus. Two new EPR signals were found and characterized. The first is assigned to the semiquinone form of QB interacting magnetically with a high spin, non-heme-iron (Fe2+, S = 2) when the native bicarbonate/carbonate ligand is replaced by formate. This assignment is based on several experimental observations, the most important of which were: (i) its presence in the dark in a significant fraction of centers, and (ii) the period-of-two variations in the concentration expected for QB •− when PSII underwent a series of single-electron turnovers. This signal is similar but not identical to the well-know formate-modified EPR signal observed for the QA •−Fe2+ complex (W.F.J. Vermaas and A.W. Rutherford, FEBS Lett. 175 (1984) 243–248). The formate-modified signals from QA •−Fe2+ and QB •−Fe2+ are also similar to native semiquinone–iron signals (QA •−Fe2+/QB •−Fe2+) seen in purple bacterial reaction centers where a glutamate provides the carboxylate ligand to the iron. The second new signal was formed when QA •− was generated in formate-inhibited PSII when the secondary acceptor was reduced by two electrons. While the signal is reminiscent of the formate-modified semiquinone–iron signals, it is broader and its main turning point has a major sub-peak at higher field. This new signal is attributed to the QA •−Fe2+ with formate bound but which is perturbed when QB is fully reduced, most likely as QBH2 (or possibly QBH•− or QB 2•−). Flash experiments on formate-inhibited PSII monitoring these new EPR signals indicate that the outcome of charge separation on the first two flashes is not greatly modified by formate. However on the third flash and subsequent flashes, the modified QA •−Fe2+QBH2 signal is trapped in the EPR experiment and there is a marked decrease in the quantum yield of formation of stable charge pairs. The main effect of formate then appears to be on QBH2 exchange and this agrees with earlier studies using different methods.► A new EPR signal from QB •−Fe is reported when formate is bound to PSII non-heme iron ► A new EPR signal from formate-modified QA •−Fe is reported when QBH2 is also present. ► Flash experiments using EPR indicate that formate blocks PSII at the QA •−FeQBH2 state.
Keywords: Photosystem II; Quinone; Non-heme iron; Formate; Bicarbonate; EPR;

Far-red light-regulated efficient energy transfer from phycobilisomes to photosystem I in the red microalga Galdieria sulphuraria and photosystems-related heterogeneity of phycobilisome population by Igor N. Stadnichuk; Alexander A. Bulychev; Evgeni P. Lukashev; Mariya P. Sinetova; Mikhail S. Khristin; Matthew P. Johnson; Alexander V. Ruban (227-235).
Phycobilisomes (PBS) are the major photosynthetic antenna complexes in cyanobacteria and red algae. In the red microalga Galdieria sulphuraria, action spectra measured separately for photosynthetic activities of photosystem I (PSI) and photosystem II (PSII) demonstrate that PBS fraction attributed to PSI is more sensitive to stress conditions and upon nitrogen starvation disappears from the cell earlier than the fraction of PBS coupled to PSII. Preillumination of the cells by actinic far-red light primarily absorbed by PSI caused an increase in the amplitude of the PBS low-temperature fluorescence emission that was accompanied by the decrease in PBS region of the PSI 77 K fluorescence excitation spectrum. Under the same conditions, fluorescence excitation spectrum of PSII remained unchanged. The amplitude of P700 photooxidation in PBS-absorbed light at physiological temperature was found to match the fluorescence changes observed at 77 K. The far-red light adaptations were reversible within 2–5 min. It is suggested that the short-term fluorescence alterations observed in far-red light are triggered by the redox state of P700 and correspond to the temporal detachment of the PBS antenna from the core complexes of PSI. Furthermore, the absence of any change in the 77 K fluorescence excitation cross-section of PSII suggests that light energy transfer from PBS to PSI in G. sulphuraria is direct and does not occur through PSII. Finally, a novel photoprotective role of PBS in red algae is discussed.► Red alga Galdieria has individual fraction of phycobilisomes docked to photosystem I. ► In far-red light phycobilisomes are reversibly detached from photosystem I-complexes. ► Under the same conditions, phycobilisomes coupled to photosystem II stay invariable. ► A novel photoadaptation process corresponds to regulation of cyclic electron transport.
Keywords: Galdieria; Fluorescence; Photosystem I (II); Phycobilisome(s);

Protection by α-tocopherol of the repair of photosystem II during photoinhibition in Synechocystis sp. PCC 6803 by Shuhei Inoue; Kayoko Ejima; Eri Iwai; Hidenori Hayashi; Jens Appel; Esa Tyystjärvi; Norio Murata; Yoshitaka Nishiyama (236-241).
α-Tocopherol is a lipophilic antioxidant that is an efficient scavenger of singlet oxygen. We investigated the role of α-tocopherol in the protection of photosystem II (PSII) from photoinhibition using a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that is deficient in the biosynthesis of α-tocopherol. The activity of PSII in mutant cells was more sensitive to inactivation by strong light than that in wild-type cells, indicating that lack of α-tocopherol enhances the extent of photoinhibition. However, the rate of photodamage to PSII, as measured in the presence of chloramphenicol, which blocks the repair of PSII, did not differ between the two lines of cells. By contrast, the repair of PSII from photodamage was suppressed in mutant cells. Addition of α-tocopherol to cultures of mutant cells returned the extent of photoinhibition to that in wild-type cells, without any effect on photodamage. The synthesis de novo of various proteins, including the D1 protein that plays a central role in the repair of PSII, was suppressed in mutant cells under strong light. These observations suggest that α-tocopherol promotes the repair of photodamaged PSII by protecting the synthesis de novo of the proteins that are required for recovery from inhibition by singlet oxygen.► α-Tocopherol protects PSII from photoinhibition. ► α-Tocopherol promotes the repair of photodamaged PSII. ► α-Tocopherol protects protein synthesis that is required for repair. ► Singlet oxygen acts by inhibiting the repair of PSII via suppression of protein synthesis.
Keywords: α-Tocopherol; Photosystem II; Photoinhibition; Protein synthesis; Repair; Singlet oxygen;