BBA - Bioenergetics (v.1807, #1)
Reviewer Acknowledgment (iii-iv).
Editorial Board (i).
Bacterial sulfite-oxidizing enzymes by Ulrike Kappler (1-10).
Enzymes belonging to the Sulfite Oxidase (SO) enzyme family are found in virtually all forms of life, and are especially abundant in prokaryotes as shown by analysis of available genome data. Despite this fact, only a limited number of bacterial SO family enzymes has been characterized in detail to date, and these appear to be involved in very different metabolic processes such as energy generation from sulfur compounds, host colonization, sulfite detoxification and organosulfonate degradation. The few characterized bacterial SO family enzymes also show an intriguing range of structural conformations, including monomeric, dimeric and heterodimeric enzymes with varying numbers and types of redox centres. Some of the bacterial enzymes even catalyze novel reactions such as dimethylsulfoxide reduction that previously had been thought not to be catalyzed by SO family enzymes. Classification of the SO family enzymes based on the structure of their Mo domain clearly shows that three distinct groups of enzymes belong to this family, and that almost all SOEs characterized to date are representatives of the same group.The widespread occurrence and obvious structural and functional plasticity of the bacterial SO family enzymes make this an exciting field for further study, in particular the unraveling of the metabolic roles of the three enzyme groups, some of which appear to be associated almost exclusively with pathogenic microorganisms.► Bacterial enzymes make up the majority of enzymes in the sulfite oxidase family. ► Sulfite oxidation is essential for most forms of life. ► Sulfite oxidizing enzymes can serve very different purposes within cellular metabolism. ► At least three basic types of enzymes make up the sulfite oxidase enzyme family. ► Bacterial sulfite oxidizing enzymes are molybdenumenzyme and may contain additional redox centres such as heme groups.
Keywords: Sulfite oxidation; Metalloenzymes; Sulfur oxidizing bacteria; Molybdenum cofactor; Spectroscopy; Phylogenetic analysis;
The formation of the split EPR signal from the S3 state of Photosystem II does not involve primary charge separation by Kajsa G.V. Havelius; Ji-Hu Su; Guangye Han; Fikret Mamedov; Felix M. Ho; Stenbjörn Styring (11-21).
Metalloradical EPR signals have been found in intact Photosystem II at cryogenic temperatures. They reflect the light-driven formation of the tyrosine Z radical (YZ •) in magnetic interaction with the CaMn4 cluster in a particular S state. These so-called split EPR signals, induced at cryogenic temperatures, provide means to study the otherwise transient YZ • and to probe the S states with EPR spectroscopy. In the S0 and S1 states, the respective split signals are induced by illumination of the sample in the visible light range only. In the S3 state the split EPR signal is induced irrespective of illumination wavelength within the entire 415–900 nm range (visible and near-IR region) [Su, J. H., Havelius, K. G. V., Ho, F. M., Han, G., Mamedov, F., and Styring, S. (2007) Biochemistry 46, 10703–10712]. An important question is whether a single mechanism can explain the induction of the Split S3 signal across the entire wavelength range or whether wavelength-dependent mechanisms are required. In this paper we confirm that the YZ • radical formation in the S1 state, reflected in the Split S1 signal, is driven by P680-centered charge separation. The situation in the S3 state is different. In Photosystem II centers with pre-reduced quinone A (QA), where the P680-centered charge separation is blocked, the Split S3 EPR signal could still be induced in the majority of the Photosystem II centers using both visible and NIR (830 nm) light. This shows that P680-centered charge separation is not involved. The amount of oxidized electron donors and reduced electron acceptors (QA −) was well correlated after visible light illumination at cryogenic temperatures in the S1 state. This was not the case in the S3 state, where the Split S3 EPR signal was formed in the majority of the centers in a pathway other than P680-centered charge separation. Instead, we propose that one mechanism exists over the entire wavelength interval to drive the formation of the Split S3 signal. The origin for this, probably involving excitation of one of the Mn ions in the CaMn4 cluster in Photosystem II, is discussed.►Split S3 signal formation in PSII at 5 K is not driven by P680-centered primary charge separation. ►A Mn-centered mechanism for YZ • oxidation operates for both visible and NIR light illuminations in Split S3 signal induction. ►QA − reduction due to P680-centered charge separation can be fully accounted for by donation by secondary donors Chl/Car/Cytb 559 . ►Apart from Mn(III), the involvement of Mn(IV) ions in the excitation of the CaMn4 cluster is also a possibility.
Keywords: Photosystem II; EPR; S3 state; Near-infrared; Split signal;
Analysis of high temperature stress on the dynamics of antenna size and reducing side heterogeneity of Photosystem II in wheat leaves (Triticum aestivum) by Sonal Mathur; Suleyman I. Allakhverdiev; Anjana Jajoo (22-29).
This study demonstrates the effect of high temperature stress on the heterogeneous behavior of PSII in Wheat (Triticum aestivum) leaves. Photosystem II in green plant chloroplasts displays heterogeneity both in the composition of its light harvesting antenna i.e. on the basis of antenna size (α, β and γ centers) and in the ability to reduce the plastoquinone pool i.e. the reducing side of the reaction centers (QB-reducing centers and QB-non-reducing centers). Detached wheat leaves were subjected to high temperature stress of 35 °C, 40 °C and 45 °C. The chlorophyll a (Chl a) fluorescence transient were recorded in vivo with high time resolution and analyzed according to JIP test which can quantify PS II behavior using Plant efficiency analyzer (PEA). Other than PEA, Biolyzer HP-3 software was used to evaluate different types of heterogeneity in wheat leaves. The results revealed that at high temperature, there was a change in the relative amounts of PSII α, β and γ centers. As judged from the complementary area growth curve, it seemed that with increasing temperature the PSIIβ and PSIIγ centers increased at the expense of PSIIα centers. The reducing side heterogeneity was also affected as shown by an increase in the number of QB-non-reducing centers at high temperatures. The reversibility of high temperature induced damage on PSII heterogeneity was also studied. Antenna size heterogeneity was recovered fully up to 40 °C while reducing side heterogeneity showed partial recovery at 40 °C. An irreversible damage to both the types of heterogeneity was observed at 45 °C. The work is a significant contribution to understand the basic mechanism involved in the adaptation of crop plants to stress conditions.►The effect of high temperature on wheat leaves on the two types of PSII heterogeneity (including α, β, γ and QB-reducing and QB-non-reducing type of heterogeneities) simultaneously has never been explored so extensively till yet. ►Both antenna size and reducing side heterogeneity are greatly affected with increasing temperature. It is concluded that certain environmental stimuli like high temperature stress evoke interconversions of α centers into β and γ centers and the active QB-reducing centers into inactive QB-non-reducing centers. ►The plant adapts to high temperature stress by temporary interconversions of PSII heterogeneity up to temperature of 40 °C. ►The changes in energy flux in response to high temp leads to increased dissipation of energy and untrapped photons which may protect the plant from oxidative stress transiently. ►High temperature stress led to incidental changes in PS II heterogeneity which may be one of the adaptive mechanisms to cope with high temperature stress.
Keywords: Photosystem II; Heterogeneity; Stress; High temperature; Wheat (Triticum aestivum);
Excited-state properties of the 16 kDa red carotenoid protein from Arthrospira maxima by Pavel Chábera; Milan Durchan; Patrick M. Shih; Cheryl A. Kerfeld; Tomáš Polívka (30-35).
We have studied spectroscopic properties of the 16 kDa red carotenoid protein (RCP), which is closely related to the orange carotenoid protein (OCP) from cyanobacteria. Both proteins bind the same chromophore, the carotenoid 3′-hydroxyechinenone (hECN), and the major difference between the two proteins is lack of the C-terminal domain in the RCP; this results in exposure of part of the carotenoid. The excited-state lifetime of hECN in the RCP is 5.5 ps, which is markedly longer than in OCP (3.3 ps) but close to 6 ps obtained for hECN in organic solvent. This confirms that the binding of hECN to the C-terminal domain in the OCP changes conformation of hECN, thereby altering its excited-state properties. Hydrogen bonds between the C-terminal domain and the carotenoid are also absent in the RCP. This allows the conformation of hECN in the RCP to be similar to that in solution, which results in comparable excited-state properties of hECN in solution. The red-shift of the RCP absorption spectrum is most likely due to aggregation of RCP induced by hydrophobic nature of hECN that, when exposed to buffer, stimulates formation of assemblies minimizing contact of hECN with water. We suggest that the loss of the C-terminal domain renders the protein amphipathic, containing both hydrophobic (the exposed part of hECN) and hydrophilic (N-terminal domain) regions, and may help the RCP to interact with lipid membranes; exposed hECN can penetrate into the hydrophobic environment of the lipid membrane, possibly to provide additional photoprotection.Display Omitted►Red carotenoid protein lacks the C-terminal domain. ►S1 lifetime of hydroxyechinenone in RCP is similar to that in solution. ►Red-shift of RCP absorption spectrum is due to aggregation.
Keywords: Cyanobacteria; Photoprotection; Carotenoid; Excited-state; Carotenoid-binding protein;
Role of Ca2+ in structure and function of Complex I from Escherichia coli by Marina Verkhovskaya; Juho Knuuti; Mårten Wikström (36-41).
The dependence of E. coli Complex I activity on cation chelators such as EDTA, EGTA, NTA and o-phenanthroline was studied in bacterial membranes, purified solubilized enzyme and Complex I reconstituted into liposomes. Purified Complex I was strongly inhibited by EDTA with an I50 of approximately 2.5 μM. The effect of Mg2+ and Ca2+ on EGTA inhibition of purified Complex I activity indicated that Ca2+ is tightly bound to the enzyme and essential for the activity. Low sensitivity to o-phenanthroline argues against the occupation of this cation binding site by Fe2+ or Zn2+. The sensitivity of Complex I to EDTA/EGTA strongly depends on the presence of monovalent cations in the medium, and on whether the complex is native, membrane-bound, or purified. The data is discussed in terms of a possible loss either of an additional 14th, subunit of E. coli Complex I, analogous to Nqo15 in the T. thermophilus enzyme, or another component of the native membrane that affects the affinity and/or accessibility of the Ca2+ binding site.►E. coli Complex I activity is strongly inhibited by divalent cation chelators. ►The extent of the inhibition is dependent on whether Complex I is membrane-bound or purified. ►The chelator's effect can be completely prevented by an equimolar amount of Ca2+ but not Mg2+. ►Complex I is suggested to contain tightly bound Ca2+ and the loss of this bound Ca2+ results in irreversible damage of the enzyme.
Keywords: Complex I; Cation binding site; Ca2+-binding site;
Ubiquinol (QH2) functions as a negative regulator of purine nucleotide inhibition of Acanthamoeba castellanii mitochondrial uncoupling protein by Andrzej Woyda-Ploszczyca; Wieslawa Jarmuszkiewicz (42-52).
We compared the influence of different adenine and guanine nucleotides on the free fatty acid-induced uncoupling protein (UCP) activity in non-phosphorylating Acanthamoeba castellanii mitochondria when the membranous ubiquinone (Q) redox state was varied. The purine nucleotides exhibit an inhibitory effect in the following descending order: GTP > ATP > GDP > ADP ≫ GMP > AMP. The efficiency of guanine and adenine nucleotides to inhibit UCP-sustained uncoupling in A. castellanii mitochondria depends on the Q redox state. Inhibition by purine nucleotides can be increased with decreasing Q reduction level (thereby ubiquinol, QH2 concentration) even with nucleoside monophosphates that are very weak inhibitors at the initial respiration. On the other hand, the inhibition can be alleviated with increasing Q reduction level (thereby QH2 concentration). The most important finding was that ubiquinol (QH2) but not oxidised Q functions as a negative regulator of UCP inhibition by purine nucleotides. For a given concentration of QH2, the linoleic acid-induced GTP-inhibited H+ leak was the same for two types of A. castellanii mitochondria that differ in the endogenous Q content. When availability of the inhibitor (GTP) or the negative inhibition modulator (QH2) was changed, a competitive influence on the UCP activity was observed. QH2 decreases the affinity of UCP for GTP and, vice versa, GTP decreases the affinity of UCP for QH2. These results describe the kinetic mechanism of regulation of UCP affinity for purine nucleotides by endogenous QH2 in the mitochondria of a unicellular eukaryote.Display Omitted► Purine nucleotides inhibit A. castellanii UCP in a Q redox state-dependent way. ► QH2 functions as a negative regulator of UCP inhibition by purine nucleotides. ► UQ2 and purine nucleotides interact in a competitive way on UCP activity.
Keywords: Acanthamoeba castellanii; Mitochondria; Purine nucleotides; Uncoupling protein; Quinone redox state; Ubiquinol;
The simulation of interquinone charge transfer in a bacterial photoreaction center highlights the central role of a hydrogen-bonded non-heme iron complex by Fabian Burggraf; Thorsten Koslowski (53-58).
We consider electron transfer between the quinones QA and QB, one of the final steps in the photoinduced charge separation in the photoreaction center of Rhodobacter sphaeroides. The system is described by a model with atomic resolution using classical force fields and a carefully parameterized tight-binding Hamiltonian. The rates estimated for direct interquinone charge transfer hopping involving a non-heme iron complex bridging the quinones and superexchange based on the geometry of the photochemically inactive dark state are orders of magnitude smaller than those obtained experimentally. Only if the iron complex is attached to both quinones via hydrogen bonds – as characteristic of the charge transfer active light state – the computed rate for superexchange involving the histidine ligands of the complex will become comparable to the experimental value of k CT = 105 s − 1.►Biological interquinone charge transfer is modelled by computer simulations. ►Direct charge transfer, hopping and dark state superexchange can be ruled out. ►Only light state superexchange is compatible with experimental measurements.
Keywords: Electron transfer; Theory; Simulation; Photosynthesis; Bacterial reaction center;
The transmembrane domain 6 of vacuolar H+-pyrophosphatase mediates protein targeting and proton transport by Yih-Jiuan Pan; Chien-Hsien Lee; Shen-Hsing Hsu; Yun-Tzu Huang; Ching-Hung Lee; Tseng-Huang Liu; Yen-Wei Chen; Shih-Ming Lin; Rong-Long Pan (59-67).
Vacuolar H+-pyrophosphatase (V-PPase; EC 220.127.116.11) plays a significant role in the maintenance of the pH in cytoplasm and vacuoles via proton translocation from the cytosol to the vacuolar lumen at the expense of PPi hydrolysis. The topology of V-PPase as predicted by TopPred II suggests that the catalytic site is putatively located in loop e and exposed to the cytosol. The adjacent transmembrane domain 6 (TM6) is highly conserved and believed to participate in the catalytic function and conformational stability of V-PPase. In this study, alanine-scanning mutagenesis along TM6 of the mung bean V-PPase was carried out to identify its structural and functional role. Mutants Y299A, A306S and L317A exhibited gross impairment in both PPi hydrolysis and proton translocation. Meanwhile, mutations at L307 and N318 completely abolished the targeting of the enzyme, causing broad cytosolic localization and implicating a possible role of these residues in protein translocation. The location of these amino acid residues was on the same side of the helix wheel, suggesting their involvement in maintaining the stability of enzyme conformation. G297A, E301A and A305S mutants showed declines in proton translocation but not in PPi hydrolysis, consequently resulting in decreases in the coupling efficiency. These amino acid residues cluster at one face of the helix wheel, indicating their direct/indirect participation in proton translocation. Taken together, these data indicate that TM6 is crucial to vacuolar H+-pyrophosphatase, probably mediating protein targeting, proton transport, and the maintenance of enzyme structure.►TM6 is crucial to V-PPase, probably mediating protein targeting, proton transport, and the maintenance of enzyme structure. ►The E301 is the critical residue in proton pumping of V-PPase. ►TM6 of V-PPase may contain signal for expression and targeting.
Keywords: Proton translocation; Tonoplast; Vacuole; Vacuolar H+-pyrophosphatase; Site-directed mutagenesis;
Biochemical and biophysical characterization of succinate: Quinone reductase from Thermus thermophilus by Olga Kolaj-Robin; Sarah R. O'Kane; Wolfgang Nitschke; Christophe Léger; Frauke Baymann; Tewfik Soulimane (68-79).
Enzymes serving as respiratory complex II belong to the succinate:quinone oxidoreductases superfamily that comprises succinate:quinone reductases (SQRs) and quinol:fumarate reductases. The SQR from the extreme thermophile Thermus thermophilus has been isolated, identified and purified to homogeneity. It consists of four polypeptides with apparent molecular masses of 64, 27, 14 and 15 kDa, corresponding to SdhA (flavoprotein), SdhB (iron–sulfur protein), SdhC and SdhD (membrane anchor proteins), respectively. The existence of [2Fe–2S], [4Fe–4S] and [3Fe–4S] iron–sulfur clusters within the purified protein was confirmed by electron paramagnetic resonance spectroscopy which also revealed a previously unnoticed influence of the substrate on the signal corresponding to the [2Fe–2S] cluster. The enzyme contains two heme b cofactors of reduction midpoint potentials of −20 mV and −160 mV for b H and b L, respectively. Circular dichroism and blue-native polyacrylamide gel electrophoresis revealed that the enzyme forms a trimer with a predominantly helical fold. The optimum temperature for succinate dehydrogenase activity is 70 °C, which is in agreement with the optimum growth temperature of T. thermophilus. Inhibition studies confirmed sensitivity of the enzyme to the classical inhibitors of the active site, as there are sodium malonate, sodium diethyl oxaloacetate and 3-nitropropionic acid. Activity measurements in the presence of the semiquinone analog, nonyl-4-hydroxyquinoline-N-oxide (NQNO) showed that the membrane part of the enzyme is functionally connected to the active site. Steady-state kinetic measurements showed that the enzyme displays standard Michaelis–Menten kinetics at a low temperature (30 °C) with a K M for succinate of 0.21 mM but exhibits deviation from it at a higher temperature (70 °C). This is the first example of complex II with such a kinetic behavior suggesting positive cooperativity with k' of 0.39 mM and Hill coefficient of 2.105. While the crystal structures of several SQORs are already available, no crystal structure of type A SQOR has been elucidated to date. Here we present for the first time a detailed biophysical and biochemical study of type A SQOR—a significant step towards understanding its structure–function relationship.►Succinate unusually influences the EPR spectrum of complex II. ►T. thermophilus complex II exhibits positive cooperativity at high temperature. ►Trimeric complex II from T. thermophilus belongs to type A SQORs.
Keywords: Complex II; Succinate:quinone oxidoreductase; Succinate dehydrogenase; Cooperativity; Thermus thermophilus;
The identity of the transient proton loading site of the proton-pumping mechanism of cytochrome c oxidase by Ville R.I. Kaila; Vivek Sharma; Mårten Wikström (80-84).
Cellular respiration is driven by cytochrome c oxidase (CcO), which reduces oxygen to water and couples the released energy to proton pumping across the mitochondrial or bacterial membrane. Proton pumping in CcO involves proton transfer from the negatively charged side of the membrane to a transient proton-loading or pump site (PLS), before it is ejected to the opposite side. Although many details of the reaction mechanism are known, the exact location of the PLS has remained elusive. We report here results from combined classical molecular dynamics simulations and continuum electrostatic calculations, which show that the hydrogen-bonded system around the A-propionate of heme a 3 dissociates reversibly upon reduction of heme a. The dissociation increases the pK a value of the propionate to a value above ~ 9, making it accessible for redox-state dependent protonation. The redox state of heme a is of key importance in controlling proton leaks by polarizing the PLS both statically and dynamically. These findings suggest that the propionate region of heme a 3 fulfills the criteria of the pump site in the proton translocation mechanism of CcO.►Redox-state dependent dissociation of the A-propionate of heme a 3 is observed. ►The pK a of the A-propionate increases above ~ 9 upon the dissociation. ►The propionate is suggested to fulfill the criteria of the transient proton-loading site.
Keywords: Proton pump; Heme-copper oxidase; MD simulation; Proton-coupled electron transfer; Continuum electrostatics;
The specificity of proton-translocating transhydrogenase for nicotinamide nucleotides by Lucinda Huxley; Philip G. Quirk; Nick P.J. Cotton; Scott A. White; J. Baz Jackson (85-94).
In its forward direction, transhydrogenase couples the reduction of NADP+ by NADH to the outward translocation of protons across the membrane of bacteria and animal mitochondria. The enzyme has three components: dI and dIII protrude from the membrane and dII spans the membrane. Hydride transfer takes place between nucleotides bound to dI and dIII. Studies on the kinetics of a lag phase at the onset of a “cyclic reaction” catalysed by complexes of the dI and dIII components of transhydrogenase from Rhodospirillum rubrum, and on the kinetics of fluorescence changes associated with nucleotide binding, reveal two features. Firstly, the binding of NADP+ and NADPH to dIII is extremely slow, and is probably limited by the conversion of the occluded to the open state of the complex. Secondly, dIII can also bind NAD+ and NADH. Extrapolating to the intact enzyme this binding to the “wrong” site could lead to slip: proton translocation without change in the nucleotide redox state, which would have important consequences for bacterial and mitochondrial metabolism.►Transhydrogenase is utilised for NADP+ reduction in bacteria and mitochodria. ►Subcomplexes of transhydrogenase bind NADP+ and NADPH very slowly. ►Nucleotide binding is restricted by conversion of the occluded to the open state. ►Unexpectedly, NAD+ and NADH can bind into the NADP(H)-binding site. ►The metabolic significance of “wrong”-site binding is discussed.
Keywords: Transhydrogenase; Membrane protein; Proton translocation; Tryptophan fluorescence; Nucleotide binding;
Experimental evidence that the membrane-spanning helix of PufX adopts a bent conformation that facilitates dimerisation of the Rhodobacter sphaeroides RC–LH1 complex through N-terminal interactions by Emma C. Ratcliffe; Richard B. Tunnicliffe; Irene W. Ng; Peter G. Adams; Pu Qian; Katherine Holden-Dye; Michael R. Jones; Michael P. Williamson; C. Neil Hunter (95-107).
The PufX polypeptide is an integral component of some photosynthetic bacterial reaction center-light harvesting 1 (RC–LH1) core complexes. Many aspects of the structure of PufX are unresolved, including the conformation of its long membrane-spanning helix and whether C-terminal processing occurs. In the present report, NMR data recorded on the Rhodobacter sphaeroides PufX in a detergent micelle confirmed previous conclusions derived from equivalent data obtained in organic solvent, that the α-helix of PufX adopts a bent conformation that would allow the entire helix to reside in the membrane interior or at its surface. In support of this, it was found through the use of site-directed mutagenesis that increasing the size of a conserved glycine on the inside of the bend in the helix was not tolerated. Possible consequences of this bent helical structure were explored using a series of N-terminal deletions. The N-terminal sequence ADKTIFNDHLN on the cytoplasmic face of the membrane was found to be critical for the formation of dimers of the RC–LH1 complex. It was further shown that the C-terminus of PufX is processed at an early stage in the development of the photosynthetic membrane. A model in which two bent PufX polypeptides stabilise a dimeric RC–LH1 complex is presented, and it is proposed that the N-terminus of PufX from one half of the dimer engages in electrostatic interactions with charged residues on the cytoplasmic surface of the LH1α and β polypeptides on the other half of the dimer.Display Omitted►NMR on the Rba. sphaeroides PufX polypeptide in a micelle reveals a bent structure. ►The bend allows the entire PufX helix to reside in the membrane or at its surface. ►Gly29 on the inside of the bend is sufficiently small to allow the bend to form. ►The N-terminal sequence ADKTIFNDHLN is critical for the formation of RC–LH1 dimers. ►The C-terminus of PufX is processed at an early stage in membrane development.
Keywords: Bacterial photosynthesis; PufX, light harvesting; Reaction center; Membrane protein; Photosynthetic membrane;
Short circuiting a sulfite oxidising enzyme with direct electrochemistry: Active site substitutions and their effect on catalysis and electron transfer by Trevor D. Rapson; Ulrike Kappler; Graeme R. Hanson; Paul V. Bernhardt (108-118).
Sulfite dehydrogenase (SDH) from Starkeya novella is a heterodimeric enzyme comprising a Mo active site and a heme c electron relay, which mediates electron transfer from the Mo cofactor to cytochrome c following sulfite oxidation. Studies on the wild type enzyme (SDHWT) and its variants have identified key amino acids at the active site, specifically Arg-55 and His-57. We report the MoVI/V, MoV/IV and FeIII/II (heme) redox potentials of the variants SDHR55K, SDHR55M, SDHR55Q and SDHH57A in comparison with those of SDHWT. For SDHR55M, SDHR55Q and SDHH57A the heme potentials are lowered from ca. 240 mV in SDHWT to ca. 200 mV, while the heme potential in SDHR55K remains unchanged and the Mo redox potentials are not affected significantly in any of these variants. Protein film voltammetry reveals a pH dependence of the electrochemical catalytic half-wave potential (E cat ) of −59 mV/pH in SDHWT and SDHR55K which tracks the pH dependence of the MoVI/V redox potential. By contrast, the catalytic potentials for SDHR55M and SDHH57A are pH-independent and follow the potential of the heme cofactor. These results highlight a switch in the pathway of electron exchange as a function of applied potential that is revealed by protein film voltammetry where an actuation of rate limiting intramolecular electron transfer (IET, Mo to heme) at high potential attenuates the catalytic current relative to faster, direct electron transfer (Mo to electrode) at lower potential. The same change in electron transfer pathway is linked to an unusual peak-shaped profile of the ideally sigmoidal steady state voltammogram in SDHWT alone, which has been associated with a potential dependent change in the orientation of the enzyme on the electrode surface. All other variants show purely sigmoidal voltammetry due to their inherently slower turnover numbers which are always lower than IET rates.Display Omitted► Arg-55 and His-57 play a crucial role in SDH catalysis. ► Loss of positive charge in SDHR55M lowers heme potential by 40 mV. ► SDHWT electrochemical catalysis bypasses higher potential heme cofactor. ► Unusual waveforms indicate potential dependent electron transfer pathway.
Keywords: Molybdenum; Enzyme; Voltammetry;
Probing the quinone binding site of Photosystem II from Thermosynechococcus elongatus containing either PsbA1 or PsbA3 as the D1 protein through the binding characteristics of herbicides by Alain Boussac; Miwa Sugiura; Fabrice Rappaport (119-129).
The main cofactors involved in Photosystem II (PSII) oxygen evolution activity are borne by two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is predominantly encoded by either the psbA 1 or the psbA 3 gene, the expression of which depends on the environmental conditions. In this work, the QB site properties in PsbA1-PSII and PsbA3-PSII were probed through the binding properties of DCMU, a urea-type herbicide, and bromoxynil, a phenolic-type herbicide. This was done by using helium temperature EPR spectroscopy and by monitoring the time-resolved changes of the redox state of QA by absorption spectroscopy in PSII purified from a His6-tagged WT strain expressing PsbA1 or from a His6-tagged strain in which both the psbA 1 and psbA 2 genes have been deleted and which therefore only express PsbA3. It is shown that, in both PsbA1-PSII and PsbA3-PSII, bromoxynil does not bind to PSII when QB is in its semiquinone state which indicates a much lower affinity for PSII when QA is in its semiquinone state than when it is in its oxidized state. This is consistent with the midpoint potential of QA •−/QA being more negative in the presence of bromoxynil than in its absence [Krieger-Liszkay and Rutherford, Biochemistry 37 (1998) 17339–17344]. The addition in the dark of DCMU, but not that of bromoxynil, to PSII with a secondary electron acceptor in the QB •− state induces the oxidation of the non-heme iron in a fraction of PsbA3-PSII but not in PsbA1-PSII. These results are explained as follows: i) bromoxynil has a lower affinity for PSII with the non-heme iron oxidized than DCMU therefore, ii) the midpoint potential of the FeII/FeIII couple is lower with DCMU bound than with bromoxynil bound in PsbA3-PSII; and iii) the midpoint potential of the FeII/FeIII couple is higher in PsbA1-PSII than in PsbA3-PSII. The observation of DCMU-induced oxidation of the non-heme iron leads us to propose that Q2, an electron acceptor identified by Joliot and Joliot [FEBS Lett. 134 (1981) 155–158], is the non-heme iron.►Probing the quinone binding site of Photosystem II from Thermosynechococcus elongatus containing either PsbA1 or PsbA3 as the D1 protein through the binding characteristics of DCMU and bromoxynil. ► The electron acceptor Q2 is very likely the oxidized non-heme iron. ► The midpoint potential of the FeII/FeIII couple is higher in PsbA1-PSII than in PsbA3-PSII.
Keywords: Photosystem II; D1 protein; PsbA protein; Herbicide; EPR; Absorption change;
Quantitative evaluation of the intrinsic uncoupling modulated by ADP and P i in the reconstituted ATP synthase of Escherichia coli by Manuela D'Alessandro; Paola Turina; B. Andrea Melandri (130-143).
The ATP synthase from Escherichia coli was isolated and reconstituted into liposomes. The ATP hydrolysis by these proteoliposomes was coupled to proton pumping, and the ensuing inner volume acidification was measured by the fluorescent probe 9-amino-6-chloro-2-methoxyacridine (ACMA). The ACMA response was calibrated by acid–base transitions, and converted into internal pH values. The rates of internal acidification and of ATP hydrolysis were measured in parallel, as a function of P i or ADP concentration. Increasing P i monotonically inhibited the hydrolysis rate with a half-maximal effect at 510 μM, whereas it stimulated the acidification rate up to 100–200 μM, inhibiting it only at higher concentrations. The ADP concentration in the assay, due both to contaminant ADP in ATP and to the hydrolysis reaction, was progressively decreased by means of increasing pyruvate kinase activities. Decreasing ADP stimulated the hydrolysis rate, whereas it inhibited the internal acidification rate. The quantitative analysis showed that the relative number of translocated protons per hydrolyzed ATP, i.e. the relative coupling ratio, depended on the concentrations of P i and ADP with apparent K d values of 220 μM and 27 nM respectively. At the smallest ADP concentrations reached, and in the absence of P i, the coupling ratio dropped down to 15% relative to the value observed at the highest ADP and P i concentrations tested. In addition, the data indicate the presence of two ADP and P i binding sites, of which only the highest affinity one is related to changes in the coupling ratio.►The ATP synthase from E. coli can work at different coupling efficiencies under physiological conditions. ►The physiological ligands ADP and P i can modulate the degree of coupling efficiency. ►On the ATP synthase from E. coli there are al least two binding affinity sites which during hydrolysis can bind both ADP and P i, the highest affinity of which is the one involved in modulating the coupling efficiency. ►The binding of P i to these two sites requires bound ADP.
Keywords: E. coli; ATP synthase; H+/ATP; Coupling ratio; Intrinsic uncoupling; Proteoliposomes;
Expression and processing of the TMEM70 protein by Kateřina Hejzlarová; Markéta Tesařová; Alena Vrbacká-Čížková; Marek Vrbacký; Hana Hartmannová; Vilma Kaplanová; Lenka Nosková; Hana Kratochvílová; Jana Buzková; Vendula Havlíčková; Jiří Zeman; Stanislav Kmoch; Josef Houštěk (144-149).
TMEM70 protein represents a novel ancillary factor of mammalian ATP synthase. We have investigated import and processing of this factor in human cells using GFP- and FLAG-tagged forms of TMEM70 and specific antibodies. TMEM70 is synthesized as a 29 kDa precursor protein that is processed to a 21 kDa mature form. Immunocytochemical detection of TMEM70 showed mitochondrial colocalization with MitoTracker Red and ATP synthase. Western blot of subcellular fractions revealed the highest signal of TMEM70 in isolated mitochondria and mitochondrial location was confirmed by mass spectrometry analysis. Based on analysis of submitochondrial fractions, TMEM70 appears to be located in the inner mitochondrial membrane, in accordance with predicated transmembrane regions in the central part of the TMEM70 sequence. Two-dimensional electrophoretic analysis did not show direct interaction of TMEM70 with assembled ATP synthase but indicated the presence of dimeric form of TMEM70. No TMEM70 protein could be found in cells and isolated mitochondria from patients with ATP synthase deficiency due to TMEM70 c.317-2A>G mutation thus confirming that TMEM70 biosynthesis is prevented in these patients.►TMEM70 ancillary factor of ATP synthase is a mitochondrial protein. ►29 kDa precursor is processed into 21 kDa form upon import. ►TMEM70 is membrane bound and exerts low level of expression. ►Protein is absent in patients with TMEM70 c.317-2A>G mutation.
Keywords: Mitochondria; ATP synthase; TMEM70; Biogenesis;
VDAC3 has differing mitochondrial functions in two types of striated muscles by Keltoum Anflous-Pharayra; Nha Lee; Dawna L. Armstrong; William J. Craigen (150-156).
Voltage-dependent anion channel (VDAC) is an abundant mitochondrial outer membrane protein. In mammals, three VDAC isoforms have been characterized. We have previously reported alterations in the function of mitochondria when assessed in situ in different muscle types in VDAC1 deficient mice (Anflous et al., 2001). In the present report we extend the study to VDAC3 deficient muscles and measure the respiratory enzyme activity in both VDAC1 and VDAC3 deficient muscles. While in the heart the absence of VDAC3 causes a decrease in the apparent affinity of in situ mitochondria for ADP, in the gastrocnemius, a mixed glycolytic/oxidative muscle, the affinity of in situ mitochondria for ADP remains unchanged. The absence of VDAC1 causes multiple defects in respiratory complex activities in both types of muscle. However, in VDAC3 deficient mice the defect is restricted to the heart and only to complex IV. These functional alterations correlate with structural aberrations of mitochondria. These results demonstrate that, unlike VDAC1, there is muscle-type specificity for VDAC3 function and therefore in vivo these two isoforms may fulfill different physiologic functions.► VDAC3 is involved in the transport of ADP across the mitochondrial outer membrane in the cardiac muscle (oxidative muscle) but not in the gastrocnemius muscle (mixed muscle). ► The absence of VDAC3 is associated with altered ultrastructure of mitochondria in the heart but not in the gastrocnemius. ► While the absence of VDAC1 is associated with multiple respiratory chain defects in the heart and the gastrocnemius, the absence of VDAC3 is associated with only complex IV deficiency that is restricted to the heart. ► Unlike VDAC1, VDAC3 fulfills different functions in two types of striated muscles.
Keywords: Mitochondrial outer membrane; VDAC; ADP; Mitochondrial inner membrane;
Structural model and spectroscopic characteristics of the FMO antenna protein from the aerobic chlorophototroph, Candidatus Chloracidobacterium thermophilum by Jianzhong Wen; Yusuke Tsukatani; Weidong Cui; Hao Zhang; Michael L. Gross; Donald A. Bryant; Robert E. Blankenship (157-164).
The Fenna–Matthews–Olson protein (FMO) binds seven or eight bacteriochlorophyll a (BChl a) molecules and is an important model antenna system for understanding pigment-protein interactions and mechanistic aspects of photosynthetic light harvesting. FMO proteins of green sulfur bacteria (Chlorobiales) have been extensively studied using a wide range of spectroscopic and theoretical approaches because of their stability, the spectral resolution of their pigments, their water-soluble nature, and the availability of high-resolution structural data. We obtained new structural and spectroscopic insights by studying the FMO protein from the recently discovered, aerobic phototrophic acidobacterium, Candidatus Chloracidobacterium thermophilum. Native C. thermophilum FMO is a trimer according to both analytical gel filtration and native-electrospray mass spectrometry. Furthermore, the mass of intact FMO trimer is consistent with the presence of 21–24 BChl a in each. Homology modeling of the C. thermophilum FMO was performed by using the structure of the FMO protein from Chlorobaculum tepidum as a template. C. thermophilum FMO differs from C. tepidum FMO in two distinct regions: the baseplate, CsmA-binding region and a region that is proposed to bind the reaction center subunit, PscA. C. thermophilum FMO has two fluorescence emission peaks at room temperature but only one at 77 K. Temperature-dependent fluorescence spectroscopy showed that the two room-temperature emission peaks result from two excited-state BChl a populations that have identical fluorescence lifetimes. Modeling of the data suggests that the two populations contain 1–2 BChl and 5–6 BChl a molecules and that thermal equilibrium effects modulate the relative population of the two emitting states.Display Omitted►Subunit and pigment stoichiometry of C. thermophilum FMO was determined. ►Structural model of C. thermophilum FMO by homology modeling. ►Identify CsmA and RC binding sites on the FMO antenna protein. ►Temperature-dependent fluorescence emission and lifetime of C thermophilum FMO.
Keywords: Acidobacteria; Type-1 reaction center; FMO protein; Baseplate; Native-electrospray mass spectrometry; Homology modeling;
Corrigendum to “Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers” [Biochim. Biophys. Acta 1797 (2010)1696 –1704] by A. Orzechowska; M. Lipińska; J. Fiedor; A. Chumakov; M. Zając; T. Ślęzak; K. Matlak; K. Strzałka; J. Korecki; L. Fiedor; K. Burda (165-166).