BBA - Bioenergetics (v.1827, #6)
Editorial Board (i).
Characterization of singlet oxygen production and its involvement in photodamage of Photosystem II in the cyanobacterium Synechocystis PCC 6803 by histidine-mediated chemical trapping by Ateeq Ur Rehman; Krisztián Cser; László Sass; Imre Vass (689-698).
Singlet oxygen production in intact cells of the cynobacterium Synechocystis 6803 was studied using chemical trapping by histidine, which leads to O2 uptake during illumination. The rate of O2 uptake, measured by a standard Clark-type electrode, is enhanced in the presence of D2O, which increases the lifetime of 1O2, and suppressed by the 1O2 quencher NaN3. Due to the limited mobility of 1O2 these data demonstrate that exogenous histidine reaches close vicinity of 1O2 production sites inside the cells. Flash induced chlorophyll fluorescence measurements showed that histidine does not inhibit Photosystem II activity up to 5 mM concentration. By applying the histidine-mediated O2 uptake method we showed that 1O2 production linearly increases with light intensity even above the saturation of photosynthesis. We also studied 1O2 production in site directed mutants in which the Gln residue at the 130th position of the D1 reaction center subunit was changed to either Glu or Leu, which affect the efficiency of nonradiative charge recombination from the primary radical pair (Rappaport et al. 2002, Biochemistry 41: 8518–8527; Cser and Vass 2007, BBA 1767:233–243). We found that the D1-Gln130Glu mutant showed decreased 1O2 production concomitant with decreased rate of photodamage relative to the WT, whereas both 1O2 production and photodamage were enhanced in the D1-Gln130Leu mutant. The data are discussed in the framework of the model of photoinhibition in which 3P680 mediated 1O2 production plays a key role in PSII photodamage, and nonradiative charge recombination of the primary charge separated state provides a photoprotective pathway.
Keywords: Singlet oxygen; Photoinhibition; Photosystem II; Nonradiative charge recombination; Cyanobacteria; Synechocystis PCC 6803;
Conversion of Corynebacterium glutamicum from an aerobic respiring to an aerobic fermenting bacterium by inactivation of the respiratory chain by Abigail Koch-Koerfges; Nina Pfelzer; Laura Platzen; Marco Oldiges; Michael Bott (699-708).
In this study a comparative analysis of three Corynebacterium glutamicum ATCC 13032 respiratory chain mutants lacking either the cytochrome bd branch (ΔcydAB), or the cytochrome bc 1–aa 3 branch (Δqcr), or both branches was performed. The lack of cytochrome bd oxidase was inhibitory only under conditions of oxygen limitation, whereas the absence of a functional cytochrome bc 1–aa 3 supercomplex led to decreases in growth rate, biomass yield, respiration and proton-motive force (pmf) and a strongly increased maintenance coefficient under oxygen excess. These results show that the bc 1–aa 3 supercomplex is of major importance for aerobic respiration. For the first time, a C. glutamicum strain with a completely inactivated aerobic respiratory chain was obtained (ΔcydABΔqcr), named DOOR (devoid of oxygen respiration), which was able to grow aerobically in BHI (brain–heart infusion) glucose complex medium with a 70% reduced biomass yield compared to the wild type. Surprisingly, reasonable aerobic growth was also possible in glucose minimal medium after supplementation with peptone. Under these conditions, the DOOR strain displayed a fermentative type of catabolism with l-lactate as major and acetate and succinate as minor products. The DOOR strain had about 2% of the oxygen consumption rate of the wild type, showing the absence of additional terminal oxidases. The pmf of the DOOR mutant was reduced by about 30% compared to the wild type. Candidates for pmf generation in the DOOR strain are succinate:menaquinone oxidoreductase, which probably can generate pmf in the direction of fumarate reduction, and F1FO-ATP synthase, which can couple ATP hydrolysis to the export of protons.► Importance of cytochrome bc 1–aa 3 complex for Corynebacterium glutamicum is shown. ► Maintenance coefficients of cytochrome bc 1 mutant are strongly increased. ► Growth of a C. glutamicum DOOR mutant incapable of aerobic respiration is shown. ► DOOR mutant performs mixed acid fermentation under aerobic conditions.
Keywords: Corynebacterium glutamicum; Cytochrome bd oxidase; Cytochrome bc 1 complex; Cytochrome aa 3 oxidase; Proton-motive force; Maintenance coefficient;
Monogalactosyldiacylglycerol deficiency in tobacco inhibits the cytochrome b6f-mediated intersystem electron transport process and affects the photostability of the photosystem II apparatus by Wang Wu; Wenli Ping; Hanying Wu; Minchun Li; Dan Gu; Yinong Xu (709-722).
Monogalactosyldiacylglycerol (MGDG) is the most abundant lipid component of the thylakoid membrane. Although MGDG is believed to be important in sustaining the structure and function of the photosynthetic membrane, its exact role in photosynthesis in vivo requires further investigation. In this study, the transgenic tobacco plant M18, which has an MGDG deficiency of approximately 53%, and which contains many fewer thylakoid membranes and exhibits retarded growth and a chlorotic phenotype, was used to investigate the role of MGDG. Chlorophyll fluorescence analysis of the M18 line revealed that PSII activity was inhibited when the plants were exposed to light. The inactive linear electron transport found in M18 plants was mainly attributed to a block in the intersystem electron transport process that was revealed by P700 redox kinetics and PSI light response analysis. Immunoblotting and Blue Native SDS-PAGE analysis suggested that a reduction in the accumulation of cytochrome b6f in M18 plants is a direct structural effect of MGDG deficiency, and this is likely to be responsible for the inefficiency observed in intersystem electron transport. Although drastic impairments of PSII subunits were detected in M18 plants grown under normal conditions, further investigations of low-light-grown M18 plants indicated that the impairments are not direct structural effects. Instead, they are likely to result from the cumulative photodamage that occurs due to impaired photostability under long-term exposure to relatively high light levels. The study suggests that MGDG plays important roles in maintaining both the linear electron transport process and the photostability of the PSII apparatus.
Keywords: Monogalactosyldiacylglycerol; Thylakoid membrane; Photosystem II; Cytochrome b6f; Electron transport; Photoinhibition;
Light harvesting complexes of Chromera velia, photosynthetic relative of apicomplexan parasites by Josef Tichy; Zdenko Gardian; David Bina; Peter Konik; Radek Litvin; Miroslava Herbstova; Arnab Pain; Frantisek Vacha (723-729).
The structure and composition of the light harvesting complexes from the unicellular alga Chromera velia were studied by means of optical spectroscopy, biochemical and electron microscopy methods. Two different types of antennae systems were identified. One exhibited a molecular weight (18–19 kDa) similar to FCP (fucoxanthin chlorophyll protein) complexes from diatoms, however, single particle analysis and circular dichroism spectroscopy indicated similarity of this structure to the recently characterized XLH antenna of xanthophytes. In light of these data we denote this antenna complex CLH, for “Chromera Light Harvesting” complex. The other system was identified as the photosystem I with bound Light Harvesting Complexes (PSI–LHCr) related to the red algae LHCI antennae. The result of this study is the finding that C. velia, when grown in natural light conditions, possesses light harvesting antennae typically found in two different, evolutionary distant, groups of photosynthetic organisms.► Antenna complexes of photosynthetic ancestor of apicomplexan parasites were studied. ► A complex of photosystem I with its external antennae (PSI–LHCr) was observed. ► Xanthonema-like antennae rather than fucoxanthin chlorophyll proteins were observed.
Keywords: Chromera velia; FCP; LHCr; Circular dichroism; Photosystem I; Electron microscopy;
A variant conferring cofactor-dependent assembly of Escherichia coli dimethylsulfoxide reductase by Huipo Tang; Richard A. Rothery; Joel H. Weiner (730-737).
We have investigated the final steps of complex iron–sulfur molybdoenzyme (CISM) maturation using Escherichia coli DMSO reductase (DmsABC) as a model system. The catalytic subunit of this enzyme, DmsA, contains an iron–sulfur cluster (FS0) and a molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD). We have identified a variant of DmsA (Cys59Ser) that renders enzyme maturation sensitive to molybdenum cofactor availability. DmsA-Cys59 is a ligand to the FS0 [4Fe–4S] cluster. In the presence of trace amounts of molybdate, the Cys59Ser variant assembles normally to the cytoplasmic membrane and supports respiratory growth on DMSO, although the ground state of FS0 as determined by EPR is converted from high-spin (S = 3/2) to low-spin (S = 1/2). In the presence of the molybdenum antagonist tungstate, wild-type DmsABC lacks Mo-bisPGD, but is translocated via the Tat translocon and assembles on the periplasmic side of the membrane as an apoenzyme. The Cys59Ser variant cannot overcome the dual insults of amino acid substitution plus lack of Mo-bisPGD, leading to degradation of the DmsABC subunits. This indicates that the cofactor can serve as a chemical chaperone to mitigate the destabilizing effects of alteration of the FS0 cluster. These results provide insights into the role of the Mo–bisPGD–protein interaction in stabilizing the tertiary structure of DmsA during enzyme maturation.► DmsABC maturation depends on [Fe–S] cluster (FS0) assembly and cofactor availability. ► The molybdenum cofactor can function as a chemical chaperone. ► ApoDmsAB is translocated to the periplasm by the Tat translocon. ► Serine is an acceptable ligand for the FS0 cluster, but alters its EPR properties. ► The dual insults of replacing cystine with serine and lack of cofactor cause enzyme degradation.
Keywords: Iron–sulfur molybdoenzyme; Molybdo-bis(pyranopterin guanine dinucleotide) cofactor; Nitrate reductase; Tat translocon;
An NMR comparison of the light-harvesting complex II (LHCII) in active and photoprotective states reveals subtle changes in the chlorophyll a ground-state electronic structures by Anjali Pandit; Michael Reus; Tomas Morosinotto; Roberto Bassi; Alfred R. Holzwarth; Huub J.M. de Groot (738-744).
To protect the photosynthetic apparatus against photo-damage in high sunlight, the photosynthetic antenna of oxygenic organisms can switch from a light-harvesting to a photoprotective mode through the process of non-photochemical quenching (NPQ). There is growing evidence that light-harvesting proteins of photosystem II participate in photoprotection by a built-in capacity to switch their conformation between light-harvesting and energy-dissipating states. Here we applied high-resolution Magic-Angle Spinning Nuclear Magnetic Resonance on uniformly 13C-enriched major light-harvesting complex II (LHCII) of Chlamydomonas reinhardtii in active or quenched states. Our results reveal that the switch into a dissipative state is accompanied by subtle changes in the chlorophyll (Chl) a ground-state electronic structures that affect their NMR responses, particularly for the macrocycle 13C4, 13C5 and 13C6 carbon atoms. Inspection of the LHCII X-ray structures shows that of the Chl molecules in the terminal emitter domain, where excited-state energy accumulates prior to further transfer or dissipation, the C4, 5 and 6 atoms are in closest proximity to lutein; supporting quenching mechanisms that involve altered Chl–lutein interactions in the dissipative state. In addition the observed changes could represent altered interactions between Chla and neoxanthin, which alters its configuration under NPQ conditions. The Chls appear to have increased dynamics in unquenched, detergent-solubilized LHCII. Our work demonstrates that solid-state Nuclear Magnetic Resonance is applicable to investigate high-resolution structural details of light-harvesting proteins in varied functional conditions, and represents a valuable tool to address their molecular plasticity associated with photoprotection.
Keywords: Non-photochemical quenching; Photosynthetic light-harvesting; Major light-harvesting complex II; Conformational switch;
Relative importance of driving force and electrostatic interactions in the reduction of multihaem cytochromes by small molecules by Pedro O. Quintas; Andreia P. Cepeda; Nuno Borges; Teresa Catarino; David L. Turner (745-750).
Multihaem cytochromes are essential to the energetics of organisms capable of bioremediation and energy production. The haems in several of these cytochromes have been discriminated thermodynamically and their individual rates of reduction by small electron donors were characterized. The kinetic characterization of individual haems used the Marcus theory of electron transfer and assumed that the rates of reduction of each haem by sodium dithionite depend only on the driving force, while electrostatic interactions were neglected. To determine the relative importance of these factors in controlling the rates, we studied the effect of ionic strength on the redox potential and the rate of reduction by dithionite of native Methylophilus methylotrophus cytochrome c″ and three mutants at different pH values. We found that the main factor determining the rate is the driving force and that Marcus theory describes this satisfactorily. This validates the method of the simultaneous fitting of kinetic and thermodynamic data in multihaem cytochromes and opens the way for further investigation into the mechanisms of these proteins.► The reduction of cytochrome c″ by dithionite was studied at various ionic strengths. ► The electron transfer involves a positively charged region close to the haem edge. ► Surface charge was changed by mutation and pH but had little effect on the rates. ► Using Marcus theory shows that the driving force is the main factor determining rates. ► This validates kinetic and thermodynamic characterizations of multihaem cytochromes.
Keywords: Electron transfer; Multihaem cytochromes; Driving force; Electrostatics; Ionic strength; Reduction kinetics;
Catalytically-relevant electron transfer between two hemes b L in the hybrid cytochrome bc 1-like complex containing a fusion of Rhodobacter sphaeroides and capsulatus cytochromes b by Monika Czapla; Ewelina Cieluch; Arkadiusz Borek; Marcin Sarewicz; Artur Osyczka (751-760).
To address mechanistic questions about the functioning of dimeric cytochrome bc 1 new genetic approaches have recently been developed. They were specifically designed to enable construction of asymmetrically-mutated variants suitable for functional studies. One approach exploited a fusion of two cytochromes b that replaced the separate subunits in the dimer. The fusion protein, built from two copies of the same cytochrome b of purple bacterium Rhodobacter capsulatus, served as a template to create a series of asymmetrically-mutated cytochrome bc 1-like complexes (B–B) which, through kinetic studies, disclosed several important principles of dimer engineering. Here, we report on construction of another fusion protein complex that adds a new tool to investigate dimeric function of the enzyme through the asymmetrically mutated forms of the protein. This complex (BS–B) contains a hybrid protein that combines two different cytochromes b: one coming from R. capsulatus and the other — from a closely related species, R. sphaeroides. With this new fusion we addressed a still controversial issue of electron transfer between the two hemes b L in the core of dimer. Kinetic data obtained with a series of BS–B variants provided new evidence confirming the previously reported observations that electron transfer between those two hemes occurs on a millisecond timescale, thus is a catalytically-relevant event. Both types of the fusion complexes (B–B and BS–B) consistently implicate that the heme-b L–b L bridge forms an electronic connection available for inter-monomer electron transfer in cytochrome bc 1.► We created a hybrid fusion of cytochromes b from R. capsulatus and R. sphaeroides. ► Hybrid fusion replaces two cytochrome b subunits in a dimer of cytochrome bc 1. ► Complex with hybrid fusion confirms fast electron transfer between two hemes b L.
Keywords: Cytochrome bc 1; Asymmetric mutagenesis; Fusion hybrid membrane protein; Rhodobacter capsulatus; Rhodobacter sphaeroides; Electron transfer;
Key role of water in proton transfer at the Qo-site of the cytochrome bc 1 complex predicted by atomistic molecular dynamics simulations by Pekka A. Postila; Karol Kaszuba; Marcin Sarewicz; Artur Osyczka; Ilpo Vattulainen; Tomasz Róg (761-768).
Cytochrome (cyt) bc 1 complex, which is an integral part of the respiratory chain and related energy-conserving systems, has two quinone-binding cavities (Qo- and Qi-sites), where the substrate participates in electron and proton transfer. Due to its complexity, many of the mechanistic details of the cyt bc 1 function have remained unclear especially regarding the substrate binding at the Qo-site. In this work we address this issue by performing extensive atomistic molecular dynamics simulations with the cyt bc 1 complex of Rhodobacter capsulatus embedded in a lipid bilayer. Based on the simulations we are able to show the atom-level binding modes of two substrate forms: quinol (QH2) and quinone (Q). The QH2 binding at the Qo-site involves a coordinated water arrangement that produces an exceptionally close and stable interaction between the cyt b and iron sulfur protein subunits. In this arrangement water molecules are positioned suitably in relation to the hydroxyls of the QH2 ring to act as the primary acceptors of protons detaching from the oxidized substrate. In contrast, water does not have a similar role in the Q binding at the Qo-site. Moreover, the coordinated water molecule is also a prime candidate to act as a structural element, gating for short-circuit suppression at the Qo-site.Display Omitted► Mechanism of proton transfer at Qo-site of cyt bc1 is proposed. ► Water molecules seem to play a crucial role in the binding of the quinol. ► The solvent is clearly not involved in the quinone binding after the oxidation. ► Predicted binding modes suggest that water is the primary acceptors of protons. ► The water coordination assures close iron sulfur protein-cyt b association.
Keywords: Cytochrome bc 1; Molecular dynamics simulation; Quinol/quinone; Electron transfer; Proton transfer; Short-circuit suppression;
Atomistic simulations indicate cardiolipin to have an integral role in the structure of the cytochrome bc 1 complex by Sanja Pöyry; Oana Cramariuc; Pekka A. Postila; Karol Kaszuba; Marcin Sarewicz; Artur Osyczka; Ilpo Vattulainen; Tomasz Róg (769-778).
The reaction mechanism of the cytochrome (cyt) bc 1 complex relies on proton and electron transfer to/from the substrate quinone/quinol, which in turn generate a proton gradient across the mitochondrial membrane. Cardiolipin (CL) have been suggested to play an important role in cyt bc 1 function by both ensuring the structural integrity of the protein complex and also by taking part in the proton uptake. Yet, the atom-scale understanding of these highly charged four-tail lipids in the cyt bc 1 function has remained quite unclear. We consider this issue through atomistic molecular dynamics simulations that are applied to the entire cyt bc 1 dimer of the purple photosynthetic bacterium Rhodobacter capsulatus embedded in a lipid bilayer. We find CLs to spontaneously diffuse to the dimer interface to the immediate vicinity of the higher potential heme b groups of the complex's catalytic Qi-sites. This observation is in full agreement with crystallographic studies of the complex, and supports the view that CLs are key players in the proton uptake. The simulation results also allow us to present a refined picture for the dimer arrangement in the cyt bc 1 complex, the novelty of our work being the description of the role of the surrounding lipid environment: in addition to the specific CL–protein interactions, we observe the protein domains on the positive side of the membrane to settle against the lipids. Altogether, the simulations discussed in this article provide novel views into the dynamics of cyt bc 1 with lipids, complementing previous experimental findings.
Keywords: Cardiolipin; Cytochrome bc 1; Membrane protein; Molecular dynamics simulation; Proton transfer;
Photochemical trapping heterogeneity as a function of wavelength, in plant photosystem I (PSI–LHCI) by Robert C. Jennings; Giuseppe Zucchelli; Stefano Santabarbara (779-785).
In the present paper the marked changes in photochemical trapping time over the absorption/fluorescence band of isolated PSI–LHCI are studied by means of time resolved fluorescence decay measurements. For emission at 680–690 nm the effective trapping time is close to 17–18 ps, and represents the effective trapping time from the bulk antenna. At wavelengths above 700 nm the effective trapping time increases in a monotonic way, over the entire emission band, to attain values in the range of 70–80 ps near 760 nm. This is argued to be caused by “uphill” energy transfer from the low energy states to the core antenna and reaction centre. These data, together with the steady state emission spectrum, permit calculation of the overall trapping time for maize PSI–LHCI, which is estimated to be approximately 40 ps. The wavelength dependence of the trapping time indicates, that in PSI–LHCI there exists at least one red form which emits at lower energies than the 735 nm state. These data indicate that Photosystem I is about 55% diffusion limited.
Keywords: Photosystem I; Light Harvesting Complex I; Fluorescence decay; Trapping rate heterogeneity; Red spectral forms;
On the analysis of non-photochemical chlorophyll fluorescence quenching curves by Alfred R. Holzwarth; Dagmar Lenk; Peter Jahns (786-792).
Non-photochemical quenching (NPQ) protects photosynthetic organisms against photodamage by high light. One of the key measuring parameters for characterizing NPQ is the high-light induced decrease in chlorophyll fluorescence. The originally measured data are maximal fluorescence (Fm′) signals as a function of actinic illumination time (Fm′(t)). Usually these original data are converted into the so-called Stern–Volmer quenching function, NPQSV(t), which is then analyzed and interpreted in terms of various NPQ mechanisms and kinetics. However, the interpretation of this analysis essentially depends on the assumption that NPQ follows indeed a Stern–Volmer relationship. Here, we question this commonly assumed relationship, which surprisingly has never been proven. We demonstrate by simulation of quenching data that particularly the conversion of time-dependent quenching curves like Fm′(t) into NPQSV(t) is (mathematically) not “innocent” in terms of its effects. It distorts the kinetic quenching information contained in the originally measured function Fm′(t), leading to a severe (often sigmoidal) distortion of the time-dependence of quenching and has negative impact on the ability to uncover the underlying quenching mechanisms and their contribution to the quenching kinetics. We conclude that the commonly applied analysis of time-dependent NPQ in NPQSV(t) space should be reconsidered. First, there exists no sound theoretical basis for this common practice. Second, there occurs no loss of information whatsoever when analyzing and interpreting the originally measured Fm′(t) data directly. Consequently, the analysis of Fm′(t) data has a much higher potential to provide correct mechanistic answers when trying to correlate quenching data with other biochemical information related to quenching.
Keywords: Energy dissipation; Fluorescence quenching; Photoprotection; Photosynthesis; Kinetic analysis; NPQ-analysis;
Deletion of β-strands 9 and 10 converts VDAC1 voltage-dependence in an asymmetrical process by Simona Reina; Andrea Magrì; Marco Lolicato; Francesca Guarino; Agata Impellizzeri; Elke Maier; Roland Benz; Matteo Ceccarelli; Vito De Pinto; Angela Messina (793-805).
Voltage-dependent anion selective channel isoform1 maintains the permeability of the outer mitochondrial membrane. Its voltage-gating properties are relevant in bioenergetic metabolism and apoptosis. The N-terminal domain is suspected to be involved in voltage-gating, due to its peculiar localization. However this issue is still controversial. In this work we exchanged or deleted the β-strands that take contact with the N-terminal domain. The exchange of the whole hVDAC1 β-barrel with the homologous hVDAC3 β-barrel produces a chimeric protein that, in reconstituted systems, loses completely voltage-dependence. hVDAC3 β-barrel has most residues in common with hVDAC1, including V143 and L150 considered anchor points for the N-terminus. hVDAC1 mutants completely lacking either the β-strand 9 or both β-strands 9 and 10 were expressed, refolded and reconstituted in artificial bilayers. The mutants formed smaller pores. Molecular dynamics simulations of the mutant structure supported its ability to form smaller pores. The mutant lacking both β-strands 9 and 10 showed a new voltage-dependence feature resulting in a fully asymmetric behavior. These data indicate that a network of β-strands in the pore-walls, and not single residues, are required for voltage-gating in addition to the N-terminus.
Keywords: Mutant protein; Voltage dependent anion channel; Voltage gating process; Molecular dynamics simulation;
Corrigendum to “Turnstiles and bifurcators: The disequilibrium converting engines that put metabolism on the road” [Biochim. Biophys. Acta 1827 (2013) 62–78] by Elbert Branscomb; Michael J. Russell (806).