BBA - Bioenergetics (v.1847, #10)

The mitochondrial-targeted antioxidant, MitoQ, increases liver mitochondrial cardiolipin content in obesogenic diet-fed rats by Gilles Fouret; Evanthia Tolika; Jérôme Lecomte; Béatrice Bonafos; Manar Aoun; Michael P. Murphy; Carla Ferreri; Chryssostomos Chatgilialoglu; Eric Dubreucq; Charles Coudray; Christine Feillet-Coudray (1025-1035).
Cardiolipin (CL), a unique mitochondrial phospholipid, plays a key role in several processes of mitochondrial bioenergetics as well as in mitochondrial membrane stability and dynamics. The present study was designed to determine the effect of MitoQ, a mitochondrial-targeted antioxidant, on the content of liver mitochondrial membrane phospholipids, in particular CL, and its fatty acid composition in obesogenic diet-fed rats.To do this, twenty-four 6 week old male Sprague Dawley rats were randomized into three groups of 8 animals and fed for 8 weeks with either a control diet, a high fat diet (HF), or a HF diet with MitoQ (HF + MitoQ). Phospholipid classes and fatty acid composition were assayed by chromatographic methods in liver and liver mitochondria. Mitochondrial bioenergetic function was also evaluated. While MitoQ had no or slight effects on total liver fatty acid composition and phospholipid classes and their fatty acid composition, it had major effects on liver mitochondrial phospholipids and mitochondrial function. Indeed, MitoQ both increased CL synthase gene expression and CL content of liver mitochondria and increased 18:2n-6 (linoleic acid) content of mitochondrial phospholipids by comparison to the HF diet. Moreover, mitochondrial CL content was positively correlated to mitochondrial membrane fluidity, membrane potential and respiration, as well as to ATP synthase activity, while it was negatively correlated to mitochondrial ROS production.These findings suggest that MitoQ may decrease pathogenic alterations to CL content and profiles, thereby preserving mitochondrial function and attenuating the development of some of the features of metabolic syndrome in obesogenic diet-fed rats.
Keywords: Cardiolipin; Fatty acids; Hepatic steatosis; High fat diet; Mitochondria; Phospholipids;

The sarcoplasmic Ca2 +-ATPase (SERCA1a) forms two phosphoenzyme intermediates during Ca2 + pumping. The second intermediate E2P hydrolyzes rapidly, which is essential for the rapid removal of Ca2 + from the cytosol of muscle cells. The present work studies whether a weakening of the scissile P―O bond in the E2P ground state facilitates dephosphorylation. To this end, the experimentally known vibrational spectrum of the E2P phosphate group was calculated with density functional theory (DFT) using structural models at two levels of structural complexity: (i) Models of acetyl phosphate in simple environments and (ii) ~ 150 atom models of the catalytic site. It was found that the enzyme environment distorts the structure of the phosphate group: one of the terminal P―O bonds is shorter in the catalytic site indicating weaker interactions than in water. However, the bond that bridges phosphate and Asp351 is unaffected. This indicates that the scissile P―O bond is not weakened by the enzyme environment of E2P. A second finding was that the catalytic site of the E2P state in aqueous solution appears to adopt a structure as in the crystals with BeF3 , where the ATPase is in a non-reactive conformation. The reactant state of the dephosphorylation reaction differs from the E2P ground state: Glu183 faces Asp351 and positions the attacking water molecule. This state has a 0.04 Å longer, and thus weaker, bridging P―O bond. The reactant state is not detected in our experiments, indicating that its energy is at least 1 kcal/mol higher than that of the E2P ground state.Display Omitted
Keywords: ATPase; SERCA; Phosphate transfer; Density functional theory; Infrared spectroscopy; FTIR spectroscopy;

Echinenone vibrational properties: From solvents to the orange carotenoid protein by Elizabeth Kish; Maria Manuela Mendes Pinto; Diana Kirilovsky; Riccardo Spezia; Bruno Robert (1044-1054).
Orange carotenoid protein (OCP) is a cyanobacterial photoactive protein which binds echinenone as a chromophore; it is involved in photoprotection of these photosynthetic organisms against intense illumination. In its resting state, OCP appears orange (OCPo), and turns into a red form (OCPr) when exposed to blue–green light. Here we have combined resonance Raman spectroscopy and molecular modeling to investigate the mechanisms underlying the electronic absorption properties of the different forms of OCP. Our results show that there are at least two carotenoid configurations in the OCPo, suggesting that it is quite flexible, and that the OCPo to OCPr transition must involve an increase of the apparent conjugation length of the bound echinenone. Resonance Raman indicates that this chromophore must be in an all-trans configuration in OCPo. Density functional theory (DFT) calculations, in agreement with the Raman spectra of both OCP forms, show that the OCPo to OCPr transition must involve either an echinenone s-cis to s-trans isomerization which would affect the position of its conjugated end-chain rings, or a bending of the echinenone rings which would bring them from out of the plane of the C=C conjugated plane in the OCPo form into the C=C plane in the OCPr form.
Keywords: Carotenoid; Raman spectroscopy; Biophysics; Photoprotection; Density functional theory; Orange carotenoid protein (OCP);

Reductive activation of E. coli respiratory nitrate reductase by Pierre Ceccaldi; Julia Rendon; Christophe Léger; René Toci; Bruno Guigliarelli; Axel Magalon; Stéphane Grimaldi; Vincent Fourmond (1055-1063).
Over the past decades, a number of authors have reported the presence of inactive species in as-prepared samples of members of the Mo/W-bisPGD enzyme family. This greatly complicated the spectroscopic studies of these enzymes, since it is impossible to discriminate between active and inactive species on the basis of the spectroscopic signatures alone. Escherichia coli nitrate reductase A (NarGHI) is a member of the Mo/W-bisPGD family that allows anaerobic respiration using nitrate as terminal electron acceptor. Here, using protein film voltammetry on NarGH films, we show that the enzyme is purified in a functionally heterogeneous form that contains between 20 and 40% of inactive species that activate the first time they are reduced. This activation proceeds in two steps: a non-redox reversible reaction followed by an irreversible reduction. By carefully correlating electrochemical and EPR spectroscopic data, we show that neither the two major Mo(V) signals nor those of the two FeS clusters that are the closest to the Mo center are associated with the two inactive species. We also conclusively exclude the possibility that the major “low-pH” and “high-pH” Mo(V) EPR signatures correspond to species in acid–base equilibrium.Display Omitted
Keywords: Protein film voltammetry; EPR spectroscopy; Mo/W bisPGD enzyme family; Respiratory nitrate reductase;

Involvement of mitochondrial proteins in calcium signaling and cell death induced by staurosporine in Neurospora crassa by A. Pedro Gonçalves; J. Miguel Cordeiro; João Monteiro; Chiara Lucchi; Paulo Correia-de-Sá; Arnaldo Videira (1064-1074).
Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca2 + influx and mobilization of Ca2 + from internal stores. Here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex I (like the 51 kDa and 14 kDa subunits) or the Ca2 +-binding alternative NADPH dehydrogenase NDE-1 are hypersensitive to staurosporine and incapable of setting up a proper intracellular Ca2 + response. Cells expressing mutant forms of NUO51 that mimic human metabolic diseases also presented Ca2 + signaling deficiencies. Accumulation of reactive oxygen species is increased in cells lacking NDE-1 and seems to be required for Ca2 + oscillations in response to staurosporine. Measurement of the mitochondrial levels of Ca2 + further supported the involvement of these organelles in staurosporine-induced Ca2 + signaling. In summary, our data indicate that staurosporine-induced fungal cell death involves a sophisticated response linking Ca2 + dynamics and bioenergetics.
Keywords: Cell death; Ca2 + dynamics; Mitochondrion; Alternative NAD(P)H dehydrogenase; NDE-1; Complex I;

Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia by Alexander V. Birk; Wesley M. Chao; Shaoyi Liu; Yi Soong; Hazel H. Szeto (1075-1084).
It was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met80 to Met80 sulfoxide (Met-O) in isolated hearts after ischemia–reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met80-Fe ligation of cyt c, forming pentacoordinated heme Fe2 +, which inhibits electron transport (ET) and promotes oxygenase activity.SS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of Met80-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy.Biochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met80-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20.Our results show that ischemia disrupts the Met80-Fe ligation of cyt c resulting in the formation of a globin-like pentacoordinated heme Fe2 + that inhibits ET, and converts cyt c into an oxygenase to cause CL peroxidation and proteolytic degradation of OPA1, resulting in cyt c release.Cyt c heme structure represents a novel target for minimizing ischemic injury. SS-20, which we show to selectively target CL and protect the Met80-Fe ligation, minimizes ischemic injury and promotes ATP recovery.Display Omitted
Keywords: Methionine sulfoxide; cyt c oxygenase; Electron transport; SS-20; OPA1; Mitochondria cristae;

Mitochondrial complex I is a large, membrane-bound enzyme central to energy metabolism, and its dysfunction is implicated in cardiovascular and neurodegenerative diseases. An interesting feature of mammalian complex I is the so-called A/D transition, when the idle enzyme spontaneously converts from the active (A) to the de-active, dormant (D) form. The A/D transition plays an important role in tissue response to ischemia and rate of the conversion can be a crucial factor determining outcome of ischemia/reperfusion. Here, we describe the effects of alkali cations on the rate of the D-to-A transition to define whether A/D conversion may be regulated by sodium.At neutral pH (7–7.5) sodium resulted in a clear increase of rates of activation (D-to-A conversion) while other cations had minor effects. The stimulating effect of sodium in this pH range was not caused by an increase in ionic strength. EIPA, an inhibitor of Na+/H+ antiporters, decreased the rate of D-to-A conversion and sodium partially eliminated this effect of EIPA. At higher pH (> 8.0), acceleration of the D-to-A conversion by sodium was abolished, and all tested cations decreased the rate of activation, probably due to the effect of ionic strength.The implications of this finding for the mechanism of complex I energy transduction and possible physiological importance of sodium stimulation of the D-to-A conversion at pathophysiological conditions in vivo are discussed.
Keywords: Mitochondrial complex I; NADH:ubiquinone oxidoreductase; A/D transition; Conformational change; Sodium; Ischemia/reperfusion;

Oxygen reduction by cytochrome ba 3 oxidase from Thermus thermophilus was studied by stopped-flow and microsecond freeze-hyperquenching analyzed with UV-Vis and EPR spectroscopy. In the initial phase, the low-spin heme b 560 is rapidly and almost completely oxidized (k obs  > 33,000 s− 1) whereas CuA remains nearly fully reduced. The internal equilibrium between CuA and heme b 560 with forward and reverse rate constants of 4621 s− 1 and 3466 s− 1, respectively, indicates a ~ 7.5 mV lower midpoint potential for CuA compared to heme b 560. The formation of the oxidized enzyme is relatively slow (693 s− 1). In contrast to the Paracoccus denitrificans cytochrome aa 3 oxidase, where in the last phase of the oxidative half cycle a radical from the strictly conserved Trp272 is formed, no radical is formed in the cytochrome ba 3 oxidase. Mutation of the Trp229, the cytochrome ba 3 oxidase homologue to the Trp272, did not abolish the activity, again in contrast to the Paracoccus cytochrome aa 3 oxidase. Differences in the proton pumping mechanisms of Type A and Type B oxidases are discussed in view of the proposed role of the strictly conserved tryptophan residue in the mechanism of redox-linked proton pumping in Type A oxidases. In spite of the differences between the Type A and Type B oxidases, we conclude that protonation of the proton-loading site constitutes the major rate-limiting step in both catalytic cycles.
Keywords: Cytochrome c oxidase; Kinetics; Electron transfer; Proton pumping; Catalytic mechanism;

F-type ATP synthases, central energy conversion machines of the cell synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane and, reversely, can also hydrolyze ATP to pump ions across the membrane, depending on cellular conditions such as ATP concentration. To prevent wasteful ATP hydrolysis, mammalian and bacterial ATP synthases possess different regulatory mechanisms. In bacteria, a low ATP concentration induces a conformational change in the ε subunit from the down- to up-states, which inhibits ATP hydrolysis. Moreover, the conformational change of the ε subunit depends on Mg2+ concentration in some bacteria such as Bacillus subtilis, but not in others. This diversity makes the ε subunit a potential target for antibiotics. Here, performing molecular dynamics simulations, we identify the Mg2+ binding site in the ε subunit from B. subtilis as E59 and E86. The free energy analysis shows that the first-sphere bi-dentate coordination of the Mg2+ ion by the two glutamates is the most stable state. In comparison, we also clarify the reason for the absence of Mg2+ dependency in the ε subunit from thermophilic Bacillus PS3, despite the high homology to that from B. subtilis. Sequence alignment suggests that this Mg2+ binding motif is present in the ε subunits of some pathogenic bacteria. In addition we discuss strategies to stabilize an isolated ε subunit carrying the Mg2+ binding motif by site directed mutagenesis, which also can be used to crystallize Mg2+ dependent ε subunits in future.Display Omitted
Keywords: Mg2+ coordination; ATPase inhibition; F-type ATPase; Bacillus subtilis;

Thermodynamic and kinetic characterization of PccH, a key protein in microbial electrosynthesis processes in Geobacter sulfurreducens by Telma C. Santos; André R. de Oliveira; Joana M. Dantas; Carlos A. Salgueiro; Cristina M. Cordas (1113-1118).
The monoheme c-type cytochrome PccH from Geobacter sulfurreducens, involved in the pathway of current-consumption in biofilms, was electrochemically characterized in detail. Cyclic voltammetry was used to determine the kinetics and thermodynamics properties of PccH redox behavior. Entropy, enthalpy and Gibbs free energy changes associated with the redox center transition between the ferric and the ferrous state were determined, indicating an enhanced solvent exposure. The midpoint redox potential is considerably low for a monoheme c-type cytochrome and the heterogeneous electron transfer constant rate reflects a high efficiency of electron transfer process in PccH. The midpoint redox potential dependence on the pH (redox-Bohr effect) was investigated, over the range of 2.5 to 9.1, and is described by the protonation/deprotonation events of two distinct centers in the vicinity of the heme group with pKa values of 2.7 (pKox1 ); 4.1 (pKred1 ) and 5.9 (pKox2 ); 6.4 (pKred2 ). Based on the inspection of PccH structure, these centers were assigned to heme propionic acids P13 and P17, respectively. The observed redox-Bohr effect indicates that PccH is able to thermodynamically couple electron and proton transfer in the G. sulfurreducens physiological pH range.
Keywords: Geobacter; Electrochemistry; Cytochrome; Redox potential; Electron transfer;

Effects of the protonophore carbonyl-cyanide m-chlorophenylhydrazone on intracytoplasmic membrane assembly in Rhodobacter sphaeroides by Kamil Woronowicz; Oluwatobi B. Olubanjo; Daniel Sha; Joseph M. Kay; Robert A. Niederman (1119-1128).
The effect of carbonyl-cyanide m-chlorophenyl-hydrazone (CCCP) on intracytoplasmic membrane (ICM) assembly was examined in the purple bacterium Rhodobacter sphaeroides. CCCP blocks generation of the electrochemical proton gradient required for integral membrane protein insertion. ICM formation was induced for 8 h, followed by a 4-h exposure to CCCP. Measurements of fluorescence induction/relaxation kinetics showed that CCCP caused a diminished quantum yield, a cessation in expansion of the functional absorption cross-section and a 4- to 10-fold slowing in the electron transfer turnover rate. ICM vesicles (chromatophores) and an upper-pigmented band (UPB) containing ICM growth initiation sites, were isolated and subjected to clear-native electrophoresis. Proteomic analysis of the chromatophore gel bands indicated that CCCP produced a 2.7-fold reduction in spectral counts in the preferentially assembled light-harvesting 2 (LH2) antenna, while the RC-LH1 complex, F1FO-ATPase and pyridine nucleotide transhydrogenase decreased by 1.7–1.9-fold. For 35 soluble enzymes, the ratio of 0.99 for treated/control proteins demonstrated that protein synthesis was unaffected by CCCP, suggesting that the membrane complex decline arose from the turnover of unassembled apoproteins. In the UPB fraction, an ~ 2-fold accumulation was observed for the preprotein translocase SecY, the SecA translocation ATPase, SecD and SecF insertion components, and chaperonins DnaJ and DnaK, consistent with the possibility that these factors, which act early in the assembly process, have accumulated in association with nascent polypeptides as stabilized assembly intermediates.Display Omitted
Keywords: Bacterial photosynthesis; Light-harvesting; Reaction center; ATPase; Transhydrogenase; Membrane assembly;

Humic substances (HS) constitute a significant fraction of natural organic matter in terrestrial and aquatic environments and can act as terminal electron acceptors in anaerobic microbial respiration. Geobacter sulfurreducens has a remarkable respiratory versatility and can utilize the HS analog anthraquinone-2,6-disulfonate (AQDS) as a terminal electron acceptor or its reduced form (AH2QDS) as an electron donor. Previous studies set the triheme cytochrome PpcA as a key component for HS respiration in G. sulfurreducens, but the process is far from fully understood. In this work, NMR chemical shift perturbation measurements were used to map the interaction region between PpcA and AH2QDS, and to measure their binding affinity. The results showed that the AH2QDS binds reversibly to the more solvent exposed edge of PpcA heme IV. The NMR and visible spectroscopies coupled to redox measurements were used to determine the thermodynamic parameters of the PpcA:quinol complex. The higher reduction potential of heme IV (− 127 mV) compared to that of AH2QDS (− 184 mV) explains why the electron transfer is more favorable in the case of reduction of the cytochrome by the quinol. The clear evidence obtained for the formation of an electron transfer complex between AH2QDS and PpcA, combined with the fact that the protein also formed a redox complex with AQDS, revealed for the first time the bifunctional behavior of PpcA toward an analog of the HS. Such behavior might confer selective advantage to G. sulfurreducens, which can utilize the HS in any redox state available in the environment for its metabolic needs.
Keywords: Geobacter; Humics; Multiheme cytochromes; NMR; Electron transfer;

Crystallographic and solution studies of NAD+- and NADH-bound alkylhydroperoxide reductase subunit F (AhpF) from Escherichia coli provide insight into sequential enzymatic steps by Neelagandan Kamariah; Malathy Sony Subramanian Manimekalai; Wilson Nartey; Frank Eisenhaber; Birgit Eisenhaber; Gerhard Grüber (1139-1152).
Redox homeostasis is significant for the survival of pro- and eukaryotic cells and is crucial for defense against reactive oxygen species like superoxide and hydrogen peroxide. In Escherichia coli, the reduction of peroxides occurs via the redox active disulfide center of the alkyl hydroperoxide reductase C subunit (AhpC), whose reduced state becomes restored by AhpF. The 57 kDa EcAhpF contains an N-terminal domain (NTD), which catalyzes the electron transfer from NADH via an FAD of the C-terminal domain into EcAhpC. The NTD is connected to the C-terminal domain via a linker. Here, the first crystal structure of E. coli AhpF bound with NADH and NAD+ has been determined at 2.5 Å and 2.4 Å resolution, respectively. The NADH-bound form of EcAhpF reveals that the NADH-binding domain is required to alter its conformation to bring a bound NADH to the re-face of the isoalloxazine ring of the flavin, and thereby render the NADH-domain dithiol center accessible to the NTD disulfide center for electron transfer. The NAD+-bound form of EcAhpF shows conformational differences for the nicotinamide end moieties and its interacting residue M467, which is proposed to represent an intermediate product-release conformation. In addition, the structural alterations in EcAhpF due to NADH- and NAD+-binding in solution are shown by small angle X-ray scattering studies. The EcAhpF is revealed to adopt many intermediate conformations in solution to facilitate the electron transfer from the substrate NADH to the C-terminal domain, and subsequently to the NTD of EcAhpF for the final step of AhpC reduction.Display Omitted
Keywords: Bioenergetics; Reactive oxygen species; Oxidative stress; Alkylhydroperoxide reductase; Redox homeostasis; Structural biology;

Carotenoids are essential for the assembly of cyanobacterial photosynthetic complexes by Tünde N. Tóth; Volha Chukhutsina; Ildikó Domonkos; Jana Knoppová; Josef Komenda; Mihály Kis; Zsófia Lénárt; Győző Garab; László Kovács; Zoltán Gombos; Herbert van Amerongen (1153-1165).
In photosynthetic organisms, carotenoids (carotenes and xanthophylls) are important for light harvesting, photoprotection and structural stability of a variety of pigment–protein complexes. Here, we investigated the consequences of altered carotenoid composition for the functional organization of photosynthetic complexes in wild-type and various mutant strains of the cyanobacterium Synechocystis sp. PCC 6803.Although it is generally accepted that xanthophylls do not play a role in cyanobacterial photosynthesis in low-light conditions, we have found that the absence of xanthophylls leads to reduced oligomerization of photosystems I and II. This is remarkable because these complexes do not bind xanthophylls. Oligomerization is even more disturbed in crtH mutant cells, which show limited carotenoid synthesis; in these cells also the phycobilisomes are distorted despite the fact that these extramembranous light-harvesting complexes do not contain carotenoids. The number of phycocyanin rods connected to the phycobilisome core is strongly reduced leading to high amounts of unattached phycocyanin units. In the absence of carotenoids the overall organization of the thylakoid membranes is disturbed: Photosystem II is not formed, photosystem I hardly oligomerizes and the assembly of phycobilisomes remains incomplete. These data underline the importance of carotenoids in the structural and functional organization of the cyanobacterial photosynthetic machinery.
Keywords: Carotenoid deficiency; Cyanobacterial photosynthesis; Phycobilisome; Photosynthetic complexes; Time-resolved fluorescence;

Role of vacuolar-type proton ATPase in signal transduction by Ge-Hong Sun-Wada; Yoh Wada (1166-1172).
The vacuolar H+-ATPase (V-ATPase) was first identified as an electrogenic proton pump that acidifies the lumen of intra- and extracellular compartments. The acidic pH generated by V-ATPase is important for a wide range of cellular processes as well as acidification-independent processes such as secretion and membrane fusion. In addition to these housekeeping functions, recent studies implicate V-ATPase in the direct regulation and function of signaling pathways. In this review, we describe recent findings on the functions of V-ATPase in growth regulation and tissue physiology.
Keywords: V-ATPase; Wnt; mTOR; Notch; Signaling pathway;

One of the remaining mysteries regarding the respiratory enzyme cytochrome c oxidase is how proton pumping can occur in all reduction steps in spite of the low reduction potentials observed in equilibrium titration experiments for two of the active site cofactors, Cu B (II) and Fe a3(III). It has been speculated that, at least the copper cofactor can acquire two different states, one metastable activated state occurring during enzyme turnover, and one relaxed state with lower energy, reached only when the supply of electrons stops. The activated state should have a transiently increased Cu B (II) reduction potential, allowing proton pumping. The relaxed state should have a lower reduction potential, as measured in the titration experiments. However, the structures of these two states are not known. Quantum mechanical calculations show that the proton coupled reduction potential for Cu B is inherently high in the active site as it appears after reaction with oxygen, which explains the observed proton pumping. It is suggested here that, when the flow of electrons ceases, a relaxed resting state is formed by the uptake of one extra proton, on top of the charge compensating protons delivered in each reduction step. The extra proton in the active site decreases the proton coupled reduction potential for Cu B by almost half a volt, leading to agreement with titration experiments. Furthermore, the structure for the resting state with an extra proton is found to have a hydroxo-bridge between Cu B (II) and Fe a3(III), yielding a magnetic coupling that can explain the experimentally observed EPR silence.Display Omitted
Keywords: Cytochrome c oxidase; Density functional theory; Reduction potentials; Magnetic coupling; Oxidized state;

The interprotein electron transfer (ET) reactions of the cupredoxin amicyanin, which mediates ET from the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase to cytochrome c-551i have been extensively studied. However, it was not possible to perform certain key experiments in that native system. This study examines the ET reaction from reduced amicyanin to an alternative electron acceptor, the diheme protein MauG. It was possible to vary the ΔG° for this ET reaction by simply changing pH to determine the dependence of k ET on ΔG°. A P94A mutation of amicyanin significantly altered its oxidation–reduction midpoint potential value. It was not possible to study the ET from reduced P94A amicyanin to cytochrome c-551i in the native system because that reaction was kinetically coupled. However, the reaction from reduced P94A amicyanin to MauG was a true ET reaction and it was possible to determine values of reorganization energy (λ) and electronic coupling for the reactions of this variant as well as native amicyanin. Comparison of the λ values associated with the ET reactions between amicyanin and the TTQ of methylamine dehydrogenase, the diheme center of MauG and the single heme of cytochrome c-551i, provides insight into the factors that dictate the λ values for the respective reactions. These results demonstrate how study of ET reactions with alternative redox partner proteins can complement and enhance our understanding of the reactions with the natural redox partners, and further our understanding of mechanisms of protein ET reactions.Display Omitted
Keywords: Metalloprotein; Reorganization energy; Cupredoxin; Heme; Cofactor; Redox protein;

The structures and environments of the protein-bound peridinins (Pers) and chlorophylls (Chls) a/c 2 in the membrane-intrinsic major light-harvesting complex of the dinoflagellate Amphidinium carterae (LHC Amph ) are characterised using resonance Raman (RR) spectroscopy with 11 excitation wavelengths, at 77 K. The excitation-dependent variation in the C=C stretching mode (ν1) suggests the presence of three Pers with conjugation lengths over 8 double bonds (dBs), and one diadinoxanthin, between 413.7 and 528.7 nm. Two Per red species are revealed on excitation at 550 and 560 nm. These Per red species exhibit anomalously low ν1 values, together with notable resonant enhancement of lactone ring-breathing and -deformation modes. To discern protein-specific effects, the RR spectra are compared to that of Per in polar (acetonitrile), polarisable (toluene) and polar-protic (ethanol) solvents. Resonantly enhanced lactone, ring-breathing (942 cm− 1) and ring-deformation (~ 650 cm− 1), modes are identified both in solution, and in the protein, and discussed in the context of the mixing of the S1 and S2 states, and formation of the intramolecular charge-transfer (ICT) state. In the Chl-absorbing region, two sets of Chl c 2's, and (at least) six Chl a's can be differentiated. With a pigment ratio of 5–6 (Chl a):2 (Chl c 2):5–6 (Per):1 Ddx determined from the fit to the RT absorption and 77 K RR spectra, sequence comparison of LHC Amp to LHCII, and the diatom LHC, fucoxanthin–chlorophyll-a/c–protein (FCP), a template for the conserved pigment binding sites is proposed, to fill the paucity of structural information in the absence of a crystal structure for LHC Amph .
Keywords: light-harvesting complex; Structure; Pigment organisation; Chlorophyll c2; Peridinin lactone ring modes; Sequence analysis;

Physicochemical nature of interfaces controlling ferredoxin NADP+ reductase activity through its interprotein interactions with ferredoxin by Misaki Kinoshita; Ju yaen Kim; Satoshi Kume; Yukiko Sakakibara; Toshihiko Sugiki; Chojiro Kojima; Genji Kurisu; Takahisa Ikegami; Toshiharu Hase; Yoko Kimata-Ariga; Young-Ho Lee (1200-1211).
Although acidic residues of ferredoxin (Fd) are known to be essential for activities of various Fd-dependent enzymes, including ferredoxin NADP+ reductase (FNR) and sulfite reductase (SiR), through electrostatic interactions with basic residues of partner enzymes, non-electrostatic contributions such as hydrophobic forces remain largely unknown. We herein demonstrated that intermolecular hydrophobic and charge–charge interactions between Fd and enzymes were both critical for enzymatic activity. Systematic site-directed mutagenesis, which altered physicochemical properties of residues on the interfaces of Fd for FNR /SiR, revealed various changes in activities of both enzymes. The replacement of serine 43 of Fd to a hydrophobic residue (S43W) and charged residue (S43D) increased and decreased FNR activity, respectively, while S43W showed significantly lower SiR activity without affecting SiR activity by S43D, suggesting that hydrophobic and electrostatic interprotein forces affected FNR activity. Enzyme kinetics revealed that changes in FNR activity by mutating Fd correlated with K m, but not with k cat or activation energy, indicating that interprotein interactions determined FNR activity. Calorimetry-based binding thermodynamics between Fd and FNR showed different binding modes of FNR to wild-type, S43W, or S43D, which were controlled by enthalpy and entropy, as shown by the driving force plot. Residue-based NMR spectroscopy of 15N FNR with Fds also revealed distinct binding modes of each complex based on different directions of NMR peak shifts with similar overall chemical shift differences. We proposed that subtle adjustments in both hydrophobic and electrostatic forces were critical for enzymatic activity, and these results may be applicable to protein-based electron transfer systems.
Keywords: Electrostatic interaction; Electron transfer; Enzyme activity; Binding thermodynamics; Driving force plot; Hydrophobic interaction;

Solution structure of the NDH-1 complex subunit CupS from Thermosynechococcus elongatus by Annika Korste; Hannes Wulfhorst; Takahisa Ikegami; Marc M. Nowaczyk; Raphael Stoll (1212-1219).
The cyanobacterial multi-subunit membrane protein complex NDH-1 is both structurally and functionally related to Complex I of eubacteria and mitochondria. In addition to functions in respiration and cyclic electron transfer around photosystem I (PSI), the cyanobacterial NDH-1 complex is involved in a unique mechanism for inorganic carbon concentration. Although the crystal structures of the similar respiratory Complex I from Thermus thermophilus and Escherichia coli are known, atomic structural information is not available for the cyanobacterial NDH-1 complex yet. In particular, the structures of those subunits that are not homologous to Complex I will help to understand their distinct functions. The 15.7 kDa protein CupS is a small soluble subunit of the complex variant NDH-1MS, which is thought to play a role in CO2 conversion.Here, we present the NMR structure of CupS from Thermosynechococcus elongatus, which is the very first structure of a specific cyanobacterial NDH-1 complex subunit. CupS shares a structural similarity with members of the Fasciclin protein superfamily. The structural comparison to Fasciclin type proteins based on known NMR structures and protein sequences of human TGFBIp, MPB70 from Mycobacterium bovis, and Fdp from Rhodobacter sphaeroides, together with a virtual docking model of CupS and NdhF3, provide first insight into the specific binding of CupS to the NDH-1MS complex at atomic resolution.
Keywords: Bioenergetics/electron transfer complex; CupS; Cyanobacteria; Membrane proteins; NDH-1 complex; NMR protein structure;

Functional characterization and organ distribution of three mitochondrial ATP–Mg/Pi carriers in Arabidopsis thaliana by Magnus Monné; Daniela Valeria Miniero; Toshihiro Obata; Lucia Daddabbo; Luigi Palmieri; Angelo Vozza; M. Cristina Nicolardi; Alisdair R. Fernie; Ferdinando Palmieri (1220-1230).
The Arabidopsis thaliana genome contains 58 membrane proteins belonging to the mitochondrial carrier family. Three members of this family, here named AtAPC1, AtAPC2, and AtAPC3, exhibit high structural similarities to the human mitochondrial ATP–Mg2 +/phosphate carriers. Under normal physiological conditions the AtAPC1 gene was expressed at least five times more than the other two AtAPC genes in flower, leaf, stem, root and seedlings. However, in stress conditions the expression levels of AtAPC1 and AtAPC3 change. Direct transport assays with recombinant and reconstituted AtAPC1, AtAPC2 and AtAPC3 showed that they transport phosphate, AMP, ADP, ATP, adenosine 5′-phosphosulfate and, to a lesser extent, other nucleotides. AtAPC2 and AtAPC3 also had the ability to transport sulfate and thiosulfate. All three AtAPCs catalyzed a counter-exchange transport that was saturable and inhibited by pyridoxal-5′-phosphate. The transport activities of AtAPCs were also inhibited by the addition of EDTA or EGTA and stimulated by the addition of Ca2 +. Given that phosphate and sulfate can be recycled via their own specific carriers, these findings indicate that AtAPCs can catalyze net transfer of adenine nucleotides across the inner mitochondrial membrane in exchange for phosphate (or sulfate), and that this transport is regulated both at the transcriptional level and by Ca2 +.
Keywords: Arabidopsis; ATP–Mg/phosphate carrier; Membrane transport; Mitochondria; Mitochondrial carrier; Mitochondrial transporter;

The two transmembrane helices of CcoP are sufficient for assembly of the cbb 3-type heme-copper oxygen reductase from Vibrio cholerae by Young O. Ahn; Hyun Ju Lee; Daniel Kaluka; Syun-Ru Yeh; Denis L. Rousseau; Pia Ädelroth; Robert B. Gennis (1231-1239).
The C-family (cbb 3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb 3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb 3 from Vibrio cholerae (CcoNOQPX) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQPX and O2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c 4, since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the KC-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.
Keywords: Oxygen reductase; Membrane protein assembly; Bioenergetics; Vibrio cholerae; cbb 3;

Structure and properties of the catalytic site of nitric oxide reductase at ambient temperature by Vangelis Daskalakis; Takehiro Ohta; Teizo Kitagawa; Constantinos Varotsis (1240-1244).
Nitric oxide reductase (Nor) is the third of the four enzymes of bacterial denitrification responsible for the catalytic formation of laughing gas (N2O). Here we report the detection of the hyponitrite (HO–N = N–O) species (νN–N  = 1332 cm− 1) in the heme b 3 Fe–FeB dinuclear center of Nor from Paracoccus denitrificans. We have also applied density functional theory (DFT) to characterize the bimetallic-bridging hyponitrite species in the reduction of NO to N2O by Nor and compare the present results with those recently reported for the N–N bond formation in the ba 3 and caa 3 oxidoreductases from Thermus thermophilus.Display Omitted
Keywords: Heme proteins; Oxidoreductases; UV Raman spectroscopy; Hyponitrite; Density functional theory;

Calcium-induced conformational changes in the regulatory domain of the human mitochondrial ATP-Mg/Pi carrier by Steven P.D. Harborne; Jonathan J. Ruprecht; Edmund R.S. Kunji (1245-1253).
The mitochondrial ATP-Mg/Pi carrier imports adenine nucleotides from the cytosol into the mitochondrial matrix and exports phosphate. The carrier is regulated by the concentration of cytosolic calcium, altering the size of the adenine nucleotide pool in the mitochondrial matrix in response to energetic demands. The protein consists of three domains; (i) the N-terminal regulatory domain, which is formed of two pairs of fused calcium-binding EF-hands, (ii) the C-terminal mitochondrial carrier domain, which is involved in transport, and (iii) a linker region with an amphipathic α-helix of unknown function. The mechanism by which calcium binding to the regulatory domain modulates substrate transport in the carrier domain has not been resolved. Here, we present two new crystal structures of the regulatory domain of the human isoform 1. Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric. Therefore, the ATP-Mg/Pi carrier must have a different mechanism of calcium regulation than the architecturally related aspartate/glutamate carrier, which is dimeric. The structure showed that an amphipathic α-helix is bound to the regulatory domain in a hydrophobic cleft of EF-hand 3/4. Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane.Display Omitted
Keywords: Calcium regulation mechanism; EF-hand conformational change; SCaMC; Adenine nucleotide translocase; Regulation of adenine nucleotides;

A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF target gene expression by Alexander V. Zhdanov; Irina A. Okkelman; Fergus W.J. Collins; Silvia Melgar; Dmitri B. Papkovsky (1254-1266).
Abnormal accumulation of oncometabolite fumarate and succinate is associated with inhibition of mitochondrial function and carcinogenesis. By competing with α-ketoglutarate, oncometabolites also activate hypoxia inducible factors (HIFs), which makes oncometabolite mimetics broadly utilised in hypoxia research. We found that dimethyloxalylglycine (DMOG), a synthetic analogue of α-ketoglutarate, commonly used to induce HIF signalling, inhibits O2 consumption in cancer cell lines HCT116 and PC12, well before activation of HIF pathways. A rapid suppression of cellular respiration was accompanied by a decrease in histone H4 lysine 16 acetylation and not abolished by double knockdown of HIF-1α and HIF-2α. In agreement with this, production of NADH and state 3 respiration in isolated mitochondria were down-regulated by the de-esterified DMOG derivative, N-oxalylglycine. Exploring the roles of DMOG as a putative inhibitor of glutamine/α-ketoglutarate metabolic axis, we found that the observed suppression of OxPhos and compensatory activation of glycolytic ATP flux make cancer cells vulnerable to combined treatment with DMOG and inhibitors of glycolysis. On the other hand, DMOG treatment impairs deep cell deoxygenation in 3D tissue culture models, demonstrating a potential to relieve functional stress imposed by deep hypoxia on tumour, ischemic or inflamed tissues. Indeed, using a murine model of colitis, we found that O2 availability in the inflamed colon tissue rapidly increased after application of DMOG, which could contribute to the known therapeutic effect of this compound. Overall, our results provide new insights into the relationship between mitochondrial function, O2 availability, metabolic reprogramming and associated diseases.
Keywords: DMOG; Mitochondrial respiration; Energy stress; Tissue O2 imaging; Oncometabolite; Inflammation;

An easily reversible structural change underlies mechanisms enabling desert crust cyanobacteria to survive desiccation by Leeat Bar-Eyal; Ido Eisenberg; Adam Faust; Hagai Raanan; Reinat Nevo; Fabrice Rappaport; Anja Krieger-Liszkay; Pierre Sétif; Adrien Thurotte; Ziv Reich; Aaron Kaplan; Itzhak Ohad; Yossi Paltiel; Nir Keren (1267-1273).
Biological desert sand crusts are the foundation of desert ecosystems, stabilizing the sands and allowing colonization by higher order organisms. The first colonizers of the desert sands are cyanobacteria. Facing the harsh conditions of the desert, these organisms must withstand frequent desiccation–hydration cycles, combined with high light intensities. Here, we characterize structural and functional modifications to the photosynthetic apparatus that enable a cyanobacterium, Leptolyngbya sp., to thrive under these conditions.Using multiple in vivo spectroscopic and imaging techniques, we identified two complementary mechanisms for dissipating absorbed energy in the desiccated state. The first mechanism involves the reorganization of the phycobilisome antenna system, increasing excitonic coupling between antenna components. This provides better energy dissipation in the antenna rather than directed exciton transfer to the reaction center. The second mechanism is driven by constriction of the thylakoid lumen which limits diffusion of plastocyanin to P700. The accumulation of P700 + not only prevents light-induced charge separation but also efficiently quenches excitation energy.These protection mechanisms employ existing components of the photosynthetic apparatus, forming two distinct functional modes. Small changes in the structure of the thylakoid membranes are sufficient for quenching of all absorbed energy in the desiccated state, protecting the photosynthetic apparatus from photoinhibitory damage. These changes can be easily reversed upon rehydration, returning the system to its high photosynthetic quantum efficiency.
Keywords: Photosynthesis; Cyanobacteria; Desiccation tolerance; Desert;

Energy transfer dynamics in dimeric photosystem II (PSII) complexes isolated from four diatoms, Chaetoceros gracilis, Cyclotella meneghiniana, Thalassiosira pseudonana, and Phaeodactylum tricornutum, are examined. Time-resolved fluorescence measurements were conducted in the range of 0–80 ns. Delayed fluorescence spectra showed a clear difference between PSII monomer and PSII dimer isolated from the four diatoms. The difference can be interpreted as reflecting suppressed energy transfer between PSII monomers in the PSII dimer for efficient energy trapping at the reaction center. The observation was especially prominent in C. gracilis and T. pseudonana. The pathways seem to be suppressed under a low pH condition in isolated PSII complexes from C. gracilis, and excitation energy may be quenched with fucoxanthin chlorophyll a/c-binding protein (FCP) that was closely associated with PSII in C. gracilis. The energy transfer between PSII monomers in the PSII dimer may play a role in excitation energy regulation in diatoms.
Keywords: PSII dimer; Energy transfer; Quench; Time-resolved fluorescence; Diatom;

The photo-induced oxidation of TyrZ and TyrD by P680•+, that involves both electron and proton transfer (PCET), has been studied in oxygen-evolving photosystem II from Thermosynechococcus elongatus. We used time-resolved absorption spectroscopy to measure the kinetics of P680•+ reduction by tyrosine after the first flash given to dark-adapted PS II as a function of temperature and pH. The half-life of TyrZ oxidation by P680•+ increases from 20 ns at 300 K to about 4 μs at 150 K. Analyzing the temperature dependence of the rate, one obtains a reorganization energy of about 770 meV. Between 260 K and 150 K, the reduction of P680•+ by TyrZ is increasingly replaced by charge recombination between P680•+ and QA •−. We propose that the driving force for TyrZ oxidation by P680•+ decreases upon lowering the temperature. TyrZ oxidation cannot be excluded in a minority of PS II complexes at cryogenic temperatures.TyrD oxidation by P680•+ with a half-life of about 30 ns was observed at high pH. The pH dependence of the yield of TyrD oxidation can be described by a single protonable group with a pK of approximately 8.4. The rate of TyrD oxidation by P680•+ is virtually identical upon substitution of solvent exchangeable protons with deuterons indicating that the rate is limited by electron transfer. The rate is independent of temperature between 5 K and 250 K. It is concluded that TyrD donates the electron to P680•+ via PD2.Display Omitted
Keywords: Photosystem II; PCET; Proton-coupled-electron transfer; Water oxidation; TyrZ and TyrD; P680; Transient absorption spectroscopy; EPR;

Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels by Patrycja Kaczara; Roberto Motterlini; Gerald M. Rosen; Bartlomiej Augustynek; Piotr Bednarczyk; Adam Szewczyk; Roberta Foresti; Stefan Chlopicki (1297-1309).
Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10–100 μM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.Display Omitted
Keywords: Carbon monoxide; CO-RM; Endothelium; Respiration; Oxidative phosphorylation; Glycolysis; Mitochondrial BKCa channels;

The effects of inborn oxidative phosphorylation (OXPHOS) complex deficiencies or possible each-step activation (ESA) dysfunction on the bioenergetic system in working intact skeletal muscle are studied using a computer model of OXPHOS published previously. The curves representing the dependencies of V ˙ O 2 and metabolite concentrations on single complex activity, entire OXPHOS activity or ESA intensity exhibit a characteristic threshold at some OXPHOS complex activity/ESA intensity. This threshold for V ˙ O 2 of single complex activities is significantly lower in intact muscle during moderate and heavy work, than in isolated mitochondria in state 3. Metabolite concentrations and pH in working muscle start to change significantly at much higher OXPHOS complex activities/ESA intensities than V ˙ O 2 . The effect of entire OXPHOS deficiency or ESA dysfunction is potentially much stronger than the effect of a single complex deficiency. Implications of these findings for the genesis of mitochondrial myopathies are discussed. It is concluded that V ˙ O 2 in state 3 and its dependence on complex activity in isolated mitochondria is not a universal quantitative determinant of the effect of mitochondrial dysfunctions in vivo. Moderate and severe mitochondria dysfunctions are defined: the former affect significantly only metabolite concentrations and pH, while the latter also decrease significantly V ˙ O 2 in intact skeletal muscle during work. The dysfunction-caused decrease in V ˙ O 2 /oxidative ATP synthesis flux, disturbance of metabolite homeostasis, elevated ROS production and anaerobic glycolysis recruitment can account for such mitochondrial myopathy symptoms as muscle weakness, exercise intolerance (exertional fatigue) and lactic acidosis.
Keywords: Mitochondrial diseases; mtDNA mutations; Oxidative phosphorylation; Inborn enzyme deficiencies; Computer model;

Proton pumping complex I increases growth yield in Candida utilis by Nicole Avéret; Marie-Lise Jobin; Anne Devin; Michel Rigoulet (1320-1326).
In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and cellular maintenance processes. A crucial parameter for growth evaluation is its yield, i.e. the efficiency of the transformation processes. The yeast Candida utilis is of peculiar interest since its mitochondria exhibit a complex I that is proposed to pump protons but also an external NADH dehydrogenase that do not pump protons. Here, we show that in C. utilis cells grown on non-fermentable media, growth yield is 30% higher as compared to that of Saccharomyces cerevisiae that do not exhibit a complex I. Moreover, ADP/O determination in C. utilis shows that electrons coming from internal NADH dehydrogenase go through proton pumping complex I, whereas electrons coming from external NADH dehydrogenases do not go through proton pumping complex I. Furthermore, we show that electron competition strictly depends on extra-mitochondrial NADH concentration, i.e. the higher the extra-mitochondrial NADH concentration, the higher the competition process with a right way for electrons coming from external NADH dehydrogenases. Such a complex regulation in C. utilis allows an increase in growth yield when cytosolic NADH is not plentiful but still favors the cytosolic NADH re-oxidation at high NADH, favoring biomass generation metabolic pathways.
Keywords: Yeast; Mitochondria; Candida utilis; Saccharomyces cerevisiae; Growth yield; Oxidative phosphorylation;

Interaction of the PsbH subunit with a chlorophyll bound to histidine 114 of CP47 is responsible for the red 77 K fluorescence of Photosystem II by Sandrine E. D'Haene; Roman Sobotka; Lenka Bučinská; Jan P. Dekker; Josef Komenda (1327-1334).
A characteristic feature of the active Photosystem II (PSII) complex is a red-shifted low temperature fluorescence emission at about 693 nm. The origin of this emission has been attributed to a monomeric ‘red’ chlorophyll molecule located in the CP47 subunit. However, the identity and function of this chlorophyll remain uncertain. In our previous work, we could not detect the red PSII emission in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking PsbH, a small transmembrane subunit bound to CP47. However, it has not been clear whether the PsbH is structurally essential for the red emission or the observed effect of mutation has been indirectly caused by compromised PSII stability and function. In the present work we performed a detailed spectroscopic characterization of PSII in cells of a mutant lacking PsbH and Photosystem I and we also characterized PSII core complexes isolated from this mutant. In addition, we purified and characterized the CP47 assembly modules containing and lacking PsbH. The results clearly confirm an essential role of PsbH in the origin of the PSII red emission and also demonstrate that PsbH stabilizes the binding of one β-carotene molecule in PSII. Crystal structures of the cyanobacterial PSII show that PsbH directly interacts with a single monomeric chlorophyll ligated by the histidine 114 residue of CP47 and we conclude that this peripheral chlorophyll hydrogen-bonded to PsbH is responsible for the red fluorescence state of CP47. Given the proximity of β-carotene this state could participate in the dissipation of excessive light energy.
Keywords: PSII; PsbH; CP47; Synechocystis sp. PCC 6803; Red chlorophyll; Energy dissipation;

Detailed insight into the ultrafast photoconversion of the cyanobacteriochrome Slr1393 from Synechocystis sp. by Chavdar Slavov; Xiuling Xu; Kai-hong Zhao; Wolfgang Gärtner; Josef Wachtveitl (1335-1344).
The initial light-induced processes of the photochromic, phycocyanobilin-binding GAF domain of Slr1393 from Synechocystis sp. PCC6803 have been studied by ultrafast transient absorption spectroscopy. We use lifetime density analysis as a model-independent method for the evolution of the experimental data, which gives a comprehensive overview of the excitation wavelength dependence of the photoconversion kinetics. The method is particularly suitable for this highly complex and not purely exponential kinetics. In contrast to previously studied cyanobacteriochromes (CBCRs), here both the red- and the green-absorbing forms show significantly slower reaction dynamics, which proceed also via ground state intermediates. The photoconversion of the green-absorbing form is faster than that of the red state, which allowed a clear detection of the primary photoproduct Lumi-G. Strong coherent oscillations of the recorded transient absorption due to wavepacket motion on the excited state potential energy surface were observed and analyzed for both (red and green) forms of Slr1393g3. The vibrational modes responsible for the coherent oscillations could play a role in the dynamics of the initially heterogeneous excited state (ES) population and direct the system towards the minima on the potential energy surface that determine the ES decay pathway. Furthermore, the coherent oscillations appear to be a common feature of bilin-based photoreceptors and thus deserve further attention. The investigated CBCR exhibits an extraordinary high level of heterogeneity due to the remarkable flexibility of the phycocyanobilin and the protein binding pocket. These properties should allow spectrally tuned response to the light stimuli and thus have significant biological implications.Display Omitted
Keywords: Photoreceptors; Cyanobacteriochrome; Photoisomerization; Ultrafast spectroscopy; Transient absorption; Lifetime density analysis;