BBA - Bioenergetics (v.1553, #3)
Flash-induced turnover of the cytochrome bc 1 complex in chromatophores of Rhodobacter capsulatus: binding of Zn2+ decelerates likewise the oxidation of cytochrome b, the reduction of cytochrome c 1 and the voltage generation by Sergey S. Klishin; Wolfgang Junge; Armen Y. Mulkidjanian (177-182).
The effect of Zn2+ on the rates of electron transfer and of voltage generation in the cytochrome bc 1 complex (bc 1) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn2+ retarded the oxidation of cytochrome b and allowed to monitor (at 561–570 nm) the reduction of its high potential heme b h (in the absence of Zn2+ this reaction was masked by the fast re-oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c 1 reduction (as monitored at 552–570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn2+ <100 μM the reduction of heme b h remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc 1 turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme b h is the main electrogenic reaction in the cytochrome bc 1 complex, (2) imply an allosteric link between the site of heme b h oxidation and the site of cytochrome c 1 reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc 1 complex.
Keywords: Electron transfer; Proton transfer; Coupling; Protonmotive force; Rhodobacter sphaeroides;
Change in electron and spin density upon electron transfer to haem by Mikael P Johansson; Margareta R.A Blomberg; Dage Sundholm; Mårten Wikström (183-187).
Haems are the cofactors of cytochromes and important catalysts of biological electron transfer. They are composed of a planar porphyrin structure with iron coordinated at the centre. It is known from spectroscopy that ferric low-spin haem has one unpaired electron at the iron, and that this spin is paired as the haem receives an electron upon reduction (I. Bertini, C. Luchinat, NMR of Paramagnetic Molecules in Biological Systems, Benjamin/Cummins Publ. Co., Menlo Park, CA, 1986, pp. 165–170; H.M. Goff, in: A.B.P. Lever, H.B. Gray (Eds.), Iron Porphyrins, Part I, Addison-Wesley Publ. Co., Reading, MA, 1983, pp. 237–281; G. Palmer, in: A.B.P. Lever, H.B. Gray (Eds.), Iron Porphyrins, Part II, Addison-Wesley Publ. Co., Reading, MA, 1983, pp. 43–88). Here we show by quantum chemical calculations on a haem a model that upon reduction the spin pairing at the iron is accompanied by effective delocalisation of electrons from the iron towards the periphery of the porphyrin ring, including its substituents. The change of charge of the iron atom is only approx. 0.1 electrons, despite the unit difference in formal oxidation state. Extensive charge delocalisation on reduction is important in order for the haem to be accommodated in the low dielectric of a protein, and may have impact on the distance dependence of the rates of electron transfer. The lost individuality of the electron added to the haem on reduction is another example of the importance of quantum mechanical effects in biological systems.
Keywords: Density functional; Haem charge; Quantum chemistry;
The molecular mechanism of ATP synthesis by F1F0-ATP synthase by Alan E. Senior; Sashi Nadanaciva; Joachim Weber (188-211).
ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F1F0-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis.
Keywords: ATP synthesis; Oxidative phosphorylation; F1F0-ATP synthase; Molecular mechanism; Catalytic site; Subunit rotation;
Density functional calculations modelling tyrosine oxidation in oxygenic photosynthetic electron transfer by Patrick J. O’Malley (212-217).
Hybrid density functional calculations are used to model tyrosine oxidation during electron transfer reactions of photosystem II. The predicted frequency values for the 7a and δCOH modes of the reduced form and the 7a mode of the oxidised radical form are in excellent agreement with experimental data obtained for Mn and Ca depleted systems by Hienerwadel et al. [Biochemistry 36 (1997) 15447] and Berthomieu et al. [Biochemistry 37 (1998) 10547]. The calculations confirm that the two tyrosines YD and YZ are protonated in the reduced form. On oxidation the larger 7a frequency value observed experimentally for YZ • can be best explained by a greater localisation of the protonic charge released on formation of this tyrosyl free radical.
A theoretical approach to the link between oxidoreductions and pyrite formation in the early stage of evolution by Miklós Péter Kalapos (218-222).
There are two fundamental axioms of surface metabolism theory: (i) pyrite formation from H2S and FeS is proposed as a source of energy for life, and (ii) archaic reductive citric acid cycle is put into the center of a metabolic network. However, the concept fails to indicate how sulfide oxidation ought to be coupled to processes driven by free energy change occurring during pyrite production, and secondly, how reductive citric acid cycle ought to be supplied with row material(s). Recently, the non-enzymatic methylglyoxalase pathway has been recommended as the anaplerotic route for the reductive citric acid cycle. In this paper a mechanism is proposed by which the oxidation of lactate, the essential step of the anaplerotic path, becomes possible and a coupling system between sulfide oxidation and endergonic reaction(s) is also presented. Oxidoreduction for other redox pairs is discussed too. It is concluded that the So/H2S system may have been the clue to energy production at the early stage of evolution, as hydrogen sulfide produced by the metabolic network may have functioned as a coupling molecule between endergonic and exergonic reactions.
Keywords: Surface metabolism; Pyrite; Oxidoreduction; Common intermediate; Methylglyoxalase pathway;
Metabolic underpinnings of the paradoxical net phosphocreatine resynthesis in contracting rat gastrocnemius muscle by Benoit Giannesini; Marguerite Izquierdo; Patrick J Cozzone; David Bendahan (223-231).
Net phosphocreatine (PCr) resynthesis during muscle contraction is a paradoxical phenomenon because it occurs under conditions of high energy demand. The metabolic underpinnings of this phenomenon were analyzed non-invasively using 31P-magnetic resonance spectroscopy in rat gastrocnemius muscle (n=11) electrically stimulated (7.6 Hz, 6 min duration) in situ under ischemic and normoxic conditions. During ischemic stimulation, [PCr] initially fell to a steady state (9±5% of resting concentration) which was maintained for the last 5 min of stimulation, whereas isometric force production decreased to a non-measurable level beyond 3 min. Throughout normoxic stimulation, [PCr] and force production declined to a steady state after respectively 1 min (5±3% of resting concentration) and 3.25 min (21±8% of initial value) of stimulation. Contrary to the observations under ischemia, a paradoxical net PCr resynthesis was recorded during the last 2 min of normoxic stimulation and was not accompanied by any improvement in force production. These results demonstrate that the paradoxical net PCr resynthesis recorded in contracting muscle relies exclusively on oxidative energy production and could occur in inactivated fibers, similarly to PCr resynthesis during post-exercise recovery.
Keywords: Phosphorus-31 magnetic resonance spectroscopy; Rat skeletal muscle; Bioenergetics; Metabolism; Fatigue; Oxygen;
Effects of cold exposure in vivo and uncouplers and recouplers in vitro on potato tuber mitochondria by V.N. Popov; O.V. Markova; E.N. Mokhova; V.P. Skulachev (232-237).
Effects of cold exposure in vivo and treatment with laurate, carboxyatractylate, atractylate, nucleotides, and BSA in vitro on potato tuber mitochondria have been studied. Cold exposure of tubers for 48–96 h resulted in some uncoupling that could be reversed completely by BSA and partially by ADP, ATP, UDP, carboxyatractylate, and atractylate. UDP was less effective than ADP and ATP, and atractylate was less effective than carboxyatractylate. The recoupling effects of nucleotides were absent when the nucleotides were added after carboxyatractylate. GDP, UDP, and CDP did not recouple mitochondria from either the control or the cold-exposed tubers. This indicates that the cold-induced fatty acid-mediated uncoupling in potato tuber mitochondria is partially due to the operation of the ATP/ADP antiporter. As to the plant uncoupling protein, its contribution to the uncoupling in tuber is negligible or, under the conditions used, somehow desensitized to nucleotides.
Keywords: Uncoupling protein; ADP/ATP antiporter; Adenosine 5′-diphosphate; Uridine 5′-diphosphate; Guanosine 5′-diphosphate; Fatty acid; Carboxyatractylate; Atractylate; Plant mitochondria; Cold adaptation;
3-O-Methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca2+-ATPase by Monica M Freire; Julio A Mignaco; Paulo C de Carvalho-Alves; Hector Barrabin; Helena M Scofano (238-248).
3-O-Methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca2+-ATPase in the absence of Ca2+, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca2+, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca2+ concentration produces a bell-shaped curve with a maximum at 50 μM Ca2+. At optimal Ca2+ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca2+, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca2+ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca2+-ATPases.
Keywords: Ca2+-ATPase; Plasma membrane Ca2+-ATPase; Erythrocyte; Fluorimetric assay; 3-O-Methylfluorescein phosphate;
Control of oxidative phosphorylation by Complex I in rat liver mitochondria: implications for aging by Barbara Ventura; Maria Luisa Genova; Carla Bovina; Gabriella Formiggini; Giorgio Lenaz (249-260).
We compared NAD-dependent state 4 and state 3 respiration, NADH oxidation and Complex I specific activity in liver mitochondria from 4- and 30-month-old rats. All the activities examined were significantly decreased with aging. In both groups of animals, the flux control coefficients measured by rotenone titration indicated that Complex I is largely rate controlling upon NADH aerobic oxidation while, in state 3 respiration, it shares the control with other steps in the pathway. Moreover, we observed a trend wherein flux control coefficients of Complex I became higher with age. This indication was strengthened by examining the rotenone inhibition thresholds showing that Complex I becomes more rate controlling, over all the examined activities, during aging. Our results point out that age-related alterations of the mitochondrial functions are also present in tissues considered less prone to accumulate mitochondrial DNA mutations.
Keywords: Rat liver mitochondria; Aging; Respiratory chain; Flux control; Complex I;
Heart design: free ADP scales with absolute mitochondrial and myofibrillar volumes from mouse to human by Geoffrey P Dobson; Uwe Himmelreich (261-267).
Our aim was to estimate a number of bioenergetic parameters in the beating mouse, rat and guinea pig heart in situ and compare the values to those in hearts of mammals over a 2000-fold range in body mass. For the mouse, rat and guinea pig heart, we report a phosphorylation ratio of 1005±50 (n=16), 460±32 (n=10) and 330±22 (n=5) mM−1 and a free cytosolic [ADP] concentration of 13, 18 and 22 μM, respectively. When each parameter was plotted against body mass, they scaled closely to the quarter power (−0.28, r=0.99 and −0.23, r=0.97). A similar regression slope was found when the inverse of free [ADP] was plotted against absolute mitochondrial (slope=−0.26, r=0.99) and myofibrillar volumes (slope=−0.24, r=0.99). The similar slopes indicate that the ratio of absolute mitochondria and myofibrillar volumes in the healthy mammalian heart is a constant, and independent of body size. In conclusion, our study supports the hypothesis that the mammalian heart has a number of highly conserved thermodynamic and kinetic parameters that obey quarter-power laws linking the phosphorylation ratio, ATP turnover rates, free [ADP] and absolute mitochondrial volumes to body size. The results are discussed in terms of possible mechanisms and potential deviations from these laws in some disease states.
Keywords: Heart; Adenosine 5′-diphosphate; Mitochondria; Scaling; Oxygen consumption;
Interaction of purified NDH-1 from Escherichia coli with ubiquinone analogues by Pamela David; Marc Baumann; Mårten Wikström; Moshe Finel (268-278).
The NADH:ubiquinone oxidoreductase (NDH-1 or Complex I) of Escherichia coli is a smaller version of the mitochondrial enzyme, being composed of 13 protein subunits in comparison to the 43 of bovine heart complex I. The bacterial NDH-1 from an NDH-2-deficient strain was purified using a combination of anion exchange chromatography and sucrose gradient centrifugation. All 13 different subunits were detected in the purified enzyme by either N-terminal sequencing or matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis. In addition, some minor contaminants were observed and identified. The activity of the enzyme was studied and the effects of phospholipid and dodecyl maltoside were characterized. Kinetic analyses were performed for the enzyme in the native membrane as well as for the purified NDH-1, using ubiquinone-1, ubiquinone-2 or decylubiquinone as the electron acceptors. The purified enzyme exhibited between 1.5- and 4-fold increase in the apparent K m for these acceptors. Both ubiquinone-2 and decylubiquinone are good acceptors for this enzyme, while affinity of NDH-1 for ubiquinone-1 is clearly lower than for the other two, particularly in the purified state.
Keywords: Complex I; NADH dehydrogenase; Matrix assisted laser desorption/ionization time of flight; Ubiquinone-2; Respiratory chain; Phospholipid;
Interference of an apcA insertion with complementary chromatic adaptation in the diazotrophic Synechocystis sp. strain BO 8402 by Olaf Neuschaefer-Rube; Peter Böger; Anneliese Ernst (279-295).
Complementary chromatic adaptation was studied in two unicellular diazotrophic Synechocystis-type cyanobacteria, strains BO 8402 and BO 9201. Strain BO 8402 was isolated from Lake Constance as a mutant lacking phycobilisomes due to an insertion sequence element in the gene apcA, encoding α-allophycocyanin. Strain BO 9201 recovered the ability to assemble functional phycobilisomes after a spontaneous excision of the insertion sequence element in apcA. Simultaneously, the strain became able to perform group II complementary chromatic adaptation by regulating the synthesis of phycoerythrin. The two strains had identical phycoerythrin operons, cpeBA, and similar-sized transcripts were formed upon induction by green light. However, in strain BO 8402 the cpeBA transcript level was approx. 20-fold lower than in strain BO 9201. Because strain BO 8402 cannot synthesize allophycocyanin and phycocyanin is sequestered in paracrystalline inclusion bodies, non-assembled phycoerythrin may accumulate inside the cells. It was examined whether non-assembled phycoerythrin or other effects caused by the absence of phycobilisomes, such as a permanently oxidized redox status of the photosynthetic electron transport chain or a distorted ratio of C and N assimilation mediated the repression of cpeBA transcription in strain BO 8402. No such links could be established. We therefore concluded that in these diazotrophic Synechocystis-type cyanobacteria the green light-induced transcription of the cpe operon directly required a functional apc operon.
Keywords: Nitrogen metabolism; Phycoerythrin; Complementary chromatic adaptation; Internal signaling; Cyanobacterium; Synechocystis;
pH dependent inactivation of solubilized F1F0 ATP synthase by dicyclohexylcarbodiimide: pK a of detergent unmasked aspartyl-61 in Escherichia coli subunit c by Francis Valiyaveetil; Joe Hermolin; Robert H Fillingame (296-301).
The pH dependence of the reaction of dicyclohexylcarbodiimide with the essential aspartyl-61 residue in subunit c of Escherichia coli ATP synthase was compared in membranes and in a detergent dispersed preparation of the enzyme. The rate of reaction was estimated by measuring the inactivation of ATPase activity. The reaction with the detergent dispersed form of the enzyme proved to be pH sensitive with the essential aspartyl group titrating with a pK a=8. However, when measured with E. coli membranes, the reaction proved to be pH insensitive. The results suggest that the reacting aspartyl-61 residues are shielded from the bulk aqueous solvent when in the membrane, but then become aqueous-accessible following detergent solubilization.
Keywords: ATP synthase; Subunit c; Essential carboxyl; Dicyclohexylcarbodiimide; pH dependence; ATPase inhibition; Proton transport;
The rate of charge recombination in Photosystem II by Rik de Wijn; Hans J. van Gorkom (302-308).
Loss by recombination of the charge separated state P680 +QA − limits the performance of Photosystem II (PS II) as a photochemical energy converter. Time constants reported in literature for this process are mostly either near 0.17 ms or near 1.4 ms. The shorter time is found in plant PS II when reduction of P680 + by the secondary electron donor Tyrosine Z cannot occur because YZ is already oxidized. The 1.4 ms recombination is seen in YZ-less mutants of the cyanobacterium Synechocystis. However, the rate of P680 +QA − recombination that actually competes with the stabilization of the charge separation has not been previously reported. We have measured the kinetics of the flash-induced fluorescence yield changes in the microsecond time domain in Tris-washed spinach chloroplasts. In this way the kinetics and yield of P680 + reduction by YZ were obtained, and the rate of the competing P680 +QA − recombination could be evaluated. The recombination time was less than 0.5 ms; the best-fitting time constant was 0.1 ms. The presence of YZ ox slightly decreased the efficiency of excitation trapping but did not seem to accelerate P680 +QA − recombination. The two P680 +QA − lifetimes in the literature probably reflect a significant difference between plant and cyanobacterial PS II.
Keywords: Photosystem II; Charge recombination; P680; Tyrosine Z; Fluorescence yield;
Xanthophyll biosynthetic mutants of Arabidopsis thaliana: altered nonphotochemical quenching of chlorophyll fluorescence is due to changes in Photosystem II antenna size and stability by Heiko Lokstein; Li Tian; Jürgen E.W. Polle; Dean DellaPenna (309-319).
Xanthophylls (oxygen derivatives of carotenes) are essential components of the plant photosynthetic apparatus. Lutein, the most abundant xanthophyll, is attached primarily to the bulk antenna complex, light-harvesting complex (LHC) II. We have used mutations in Arabidopsis thaliana that selectively eliminate (and substitute) specific xanthophylls in order to study their function(s) in vivo. These include two lutein-deficient mutants, lut1 and lut2, the epoxy xanthophyll-deficient aba1 mutant and the lut2aba1 double mutant. Photosystem stoichiometry, antenna sizes and xanthophyll cycle activity have been related to alterations in nonphotochemical quenching of chlorophyll fluorescence (NPQ). Nondenaturing polyacrylamide gel electrophoresis indicates reduced stability of trimeric LHC II in the absence of lutein (and/or epoxy xanthophylls). Photosystem (antenna) size and stoichiometry is altered in all mutants relative to wild type (WT). Maximal ΔpH-dependent NPQ (qE) is reduced in the following order: WT>aba1>lut1≈lut2>lut2aba1, paralleling reduction in Photosystem (PS) II antenna size. Finally, light-activation of NPQ shows that zeaxanthin and antheraxanthin present constitutively in lut mutants are not qE active, and hence, the same can be inferred of the lutein they replace. Thus, a direct involvement of lutein in the mechanism of qE is unlikely. Rather, altered NPQ in xanthophyll biosynthetic mutants is explained by disturbed macro-organization of LHC II and reduced PS II-antenna size in the absence of the optimal, wild-type xanthophyll composition. These data suggest the evolutionary conservation of lutein content in plants was selected for due to its unique ability to optimize antenna structure, stability and macro-organization for efficient regulation of light-harvesting under natural environmental conditions.
Keywords: Antenna size; Carotenoid; Lutein; Nonphotochemical quenching of chlorophyll fluorescence; Photoprotection; Xanthophyll; Arabidopsis thaliana;
Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to QB in Rhodobacter sphaeroides reaction centers by Alberto Mezzetti; Eliane Nabedryk; Jacques Breton; Melvin Y. Okamura; Mark L. Paddock; Giovanni Giacometti; Winfried Leibl (320-330).
Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction QA −QB→QAQB − (k AB (1)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k AB (1), attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to QB [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4–29 ms following a flash showed peaks at 1670 (−), 1601 (−) and 1467 (+) cm−1, characteristic of QA reduction. The time evolution of the spectra shows reoxidation of QA − and concomitant reduction of QB with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of QB − occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm−1 [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of QB in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from QA − to QB and the identification of Glu-L212 as the main proton acceptor in the state QAQB −.
Keywords: Proton uptake; Bacterial photosynthesis; Proton-coupled electron transfer; Site-directed mutation; Rhodobacter sphaeroides;
Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins by Mauro Degli Esposti (331-340).
Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure–function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.
Keywords: Mitochondria; Apoptosis; Bid; Bcl-2; Nix; Protein sequence similarity; Lipid transfer; Cytochrome c;
Author index (341-342).
Cumulative contents (343-344).