BBA - Bioenergetics (v.1457, #1-2)
Escape probability and trapping mechanism in purple bacteria: revisited by Karen Bernhardt; Hans-Wilhelm Trissl (1-17).
Despite intensive research for decades, the trapping mechanism in the core complex of purple bacteria is still under discussion. In this article, it is attempted to derive a conceptionally simple model that is consistent with all basic experimental observations and that allows definite conclusions on the trapping mechanism. Some experimental data reported in the literature are conflicting or incomplete. Therefore we repeated two already published experiments like the time-resolved fluorescence decay in LH1-only purple bacteria Rhodospirillum rubrum and Rhodopseudomonas viridis chromatophores with open and closed (QA −) reaction centers. Furthermore, we measured fluorescence excitation spectra for both species under the two redox-conditions. These data, all measured at room temperature, were analyzed by a target analysis based on a three-state model (antenna, primary donor, and radical pair). All states were allowed to react reversibly and their decay channels were taken into consideration. This leads to seven rate constants to be determined. It turns out that a unique set of numerical values of these rate constants can be found, when further experimental constraints are met simultaneously, i.e. the ratio of the fluorescence yields in the open and closed (QA −) states F m/F o≈2 and the P+H−-recombination kinetics of 3–6 ns. The model allows to define and to quantify escape probabilities and the transfer equilibrium. We conclude that trapping in LH1-only purple bacteria is largely transfer-to-the-trap-limited. Furthermore, the model predicts properties of the reaction center (RC) in its native LH1-environment. Within the framework of our model, the predicted P+H−-recombination kinetics are nearly indistinguishable for a hypothetically isolated RC and an antenna-RC complex, which is in contrast to published experimental data for physically isolated RCs. Therefore RC preparations may display modified kinetic properties.
Keywords: Fluorescence decay kinetics; Fluorescence excitation spectrum; Fluorescence yield; Modeling; Trapping mechanism;
31P Magnetic resonance spectroscopy study of phosphocreatine recovery kinetics in skeletal muscle: the issue of intersubject variability by M. Roussel; D. Bendahan; J.P. Mattei; Y. Le Fur; P.J. Cozzone (18-26).
We have analyzed by 31P MRS the relationship between kinetic parameters of phosphocreatine (PCr) recovery and end-of-exercise status under conditions of moderate and large acidosis induced by dynamic exercise. Thirteen healthy subjects performed muscular contractions at 0.47 Hz (low frequency, moderate exercise) and 0.85 Hz (high frequency, heavy exercise). The rate constant of PCr resynthesis (k PCr) varied greatly among subjects (variation coefficients: 43 vs. 57% for LF vs. HF exercises) and protocols (k PCr values: 1.3±0.5 min−1 vs. 0.9±0.5 min−1 for LF vs. HF exercises, P<0.03). The large intersubject variability can be captured into a linear relationship between k PCr, the amount of PCr consumed ([PCr2]) and pH reached at the end of exercise (pHend) (k PCr=−3.3+0.7 pHend-0.03 [PCr2]; P=0.0007; r=0.61). This dual relationship illustrates that mitochondrial activity is affected by end-of-exercise metabolic status and allows reliable comparisons between control, diseased and trained muscles. In contrast to k PCr, the initial rate of PCr recovery and the maximum oxidative capacity were always constant whatever the metabolic conditions of end-of-exercise and can then be additionally used in the identification of dysfunctions in the oxidative metabolic pathway.
Keywords: Phosphorus-31 magnetic resonance spectroscopy; Human skeletal muscle; Exercise intensity; Oxidative capacity;
Tyr30 of amicyanin is not critical for electron transfer to cytochrome c-551i: implications for predicting electron transfer pathways by Victor L. Davidson; Limei H. Jones; M.Elizabeth Graichen; Zhenyu Zhu (27-35).
A Pathways analysis of the methylamine dehydrogenase–amicyanin–cytochrome c-551i protein electron transfer (ET) complex predicts two sets of ET pathways of comparable efficiency from the type I copper of amicyanin to the heme of cytochrome c-551i. In one pathway, the electron exits copper via the Cys92 copper ligand, and in the other, it exits via the Met98 copper ligand. If the Pathways algorithm is modified to include contributions from the anisotropy of metal–ligand coupling, independent of differences in copper–ligand bond length, then the pathways via Cys92 are predicted to be at least 100-fold more strongly coupled than the pathways via any of the other copper ligands. All of the favored pathways via Cys92 include a through-space jump from Cys92 to the side chain of Tyr30. To determine whether or not the pathways via Cys92 are preferentially used for ET, Tyr30 was changed to other amino acid residues by site-directed mutagenesis. Some mutant proteins were very unstable suggesting a role for Tyr30 in stabilizing the protein structure. Y30F and Y30I mutant amicyanins could be isolated and analyzed. For the Y30I mutant, the modified Pathways analysis which favors ET via Cys92 predicts a decrease in ET rate of at least two orders of magnitude, whereas the standard Pathways analysis predicts no change in ET rate since ET via Met98 is not affected. Experimentally, the ET rates of the Y30I and Y30F mutants were indistinguishable from that of wild-type amicyanin. Likely explanations for these observations are discussed as are their implications for predicting pathways for ET reactions of metalloproteins.
Keywords: Copper protein; Metalloprotein; Methylamine dehydrogenase; Kinetics;
Mutations in the tether region of the iron–sulfur protein affect the activity and assembly of the cytochrome bc 1 complex of yeast mitochondria by Victor H Obungu; Yudong Wang; Suzelle M Amyot; Christian B Gocke; Diana S Beattie (36-44).
Resolution of the crystal structure of the mitochondrial cytochrome bc 1 complex has indicated that the extra-membranous extrinsic domain of the iron–sulfur protein containing the 2Fe2S cluster is connected by a tether to the transmembrane helix that anchors the iron–sulfur protein to the complex. To investigate the role of this tether in the cytochrome bc 1 complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants ΔVLA(88–90) and ΔAMA(90–92) and an insertion mutant I87AAA88 in the iron–sulfur protein of the yeast, Saccharomyces cerevisiae. In cells grown at 30°C, enzymatic activities of the bc 1 complex were reduced 22–56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, ΔVLA(88–90) and ΔAMA(90–92), while activity of the insertion mutant was reduced 90%. No loss of cytochromes b or c–c 1, detected spectrally, or the iron–sulfur protein, determined by quantitative immunoblotting, was observed in these mutants with the exception of the mutants of Ala-92 in which the loss of activity paralleled a loss in the amount of the iron–sulfur protein. EPR spectroscopy revealed no changes in the iron–sulfur cluster of mutants A86L, A90I, A92R or the deletion mutant ΔVLA(88–90). Greater losses of both protein and activity were observed in all of the mutants of Ala-92 as well as in A90F grown at 37°C. suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron–sulfur protein and its assembly into the bc 1 complex. By contrast, no significant loss of iron–sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30°C or 37°C despite the 50–70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc 1 complex.
Keywords: Iron-sulphur protein; Cytochrome bc1; Yeast; Mitochondria;
Growth of the yeast Saccharomyces cerevisiae on a non-fermentable substrate: control of energetic yield by the amount of mitochondria by Laurent Dejean; Bertrand Beauvoit; Bernard Guérin; Michel Rigoulet (45-56).
The purpose of this study was to investigate the long-term control of ATP synthesis during the course of Saccharomyces cerevisiae batch grown on lactate, a purely respiratory substrate. For this, we used a respirometric and on-line calorimetric approach to analyse the energetic balances and the control of energetic metabolism during growth. Enthalpic growth yields assessed by enthalpy balance (taking account of substrate consumption, by-product accumulation, biomass formation and heat dissipation) remained constant during the entire exponential growth. Moreover, at the same time, a parallel decrease in basal respiratory rate and enthalpy flux occurred. It is shown that the decrease in respiration corresponds to a decrease in the amount of mitochondria per cell but not to a change of steady state of oxidative phosphorylation. Taking into account the part of energy used for maintenance, it can be concluded that mitochondria by themselves are the major heat dissipative system in a fully aerobic metabolism, and that the decrease in the amount of mitochondria when growth rate decreases leads to an enthalpic growth yield constant.
Keywords: Yeast; Microcalorimetry; Enthalpic growth yield; Mitochondria; Oxidative phosphorylation;
Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria by N.Irene Kavanagh; Edward K. Ainscow; Martin D. Brand (57-70).
Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem but had no effect on the proton leak kinetics. The response of mitochondria respiring on 2-oxoglutarate at two physiological concentrations of free calcium was quantified using control and regulation analysis. The partial integrated response coefficients showed that direct stimulation of substrate oxidation contributed 86% of the effect of calcium on state 3 oxygen consumption, and direct activation of the phosphorylation reactions caused 37% of the increase in phosphorylation flux. Calcium directly activated phosphorylation more strongly than substrate oxidation (78% compared to 45%) to achieve homeostasis of mitochondrial membrane potential during large increases in flux.
Keywords: Calcium; Oxidative phosphorylation; Top-down elasticity analysis; Control analysis; Regulation analysis; Muscle mitochondria;
Fast energy transfer between BChl d and BChl c in chlorosomes of the green sulfur bacterium Chlorobium limicola by Dorte B. Steensgaard; Cornelis A. van Walree; Hjalmar Permentier; Lluis Bañeras; Carles M. Borrego; Jesus Garcia-Gil; Thijs J. Aartsma; Jan Amesz; Alfred R. Holzwarth (71-80).
We have studied energy transfer in chlorosomes of Chlorobium limicola UdG6040 containing a mixture of about 50% bacteriochlorophyll (BChl) c and BChl d each. BChl d-depleted chlorosomes were obtained by acid treatment. The energy transfer between the different pigment pools was studied using both steady-state and time-resolved fluorescence spectroscopy at room temperature and low temperature. The steady-state emission of the intact chlorosome originated mainly from BChl c, as judged by comparison of fluorescence emission spectra of intact and BChl d-depleted chlorosomes. This indicated that efficient energy transfer from BChl d to BChl c takes place. At room temperature BChl c/d to BChl a excitation energy transfer (EET) was characterized by two components of 27 and 74 ps. At low temperature we could also observe EET from BChl d to BChl c with a time constant of ∼4 ps. Kinetic modeling of the low temperature data indicated heterogeneous fluorescence kinetics and suggested the presence of an additional BChl c pool, E790, which is more or less decoupled from the baseplate BChl a. This E790 pool is either a low-lying exciton state of BChl c which acts as a trap at low temperature or alternatively represents the red edge of a broad inhomogeneous absorption band of BChl c. We present a refined model for the organization of the spatially separated pigment pools in chlorosomes of Cb. limicola UdG6040 in which BChl d is situated distal and BChl c proximal with respect to the baseplate.
Keywords: Bacteriochlorophyll; Energy transfer; Fluorescence kinetics; Fluorescence spectroscopy; Green bacterium; Photosynthesis; Single-photon timing;
A covalent tandem dimer of the mitochondrial ADP/ATP carrier is functional in vivo by Véronique Trézéguet; Agnès Le Saux; Claudine David; Céline Gourdet; Christelle Fiore; Anne-Christine Dianoux; Gérard Brandolin; Guy J.-M. Lauquin (81-93).
The adenine nucleotide carrier, or Ancp, is an integral protein of the inner mitochondrial membrane. It is established that the inactive Ancp bound to one of its inhibitors (CATR or BA) is a dimer, but different contradictory models were proposed over the past years to describe the organization of the active Ancp. In order to decide in favor of a single model, it is necessary to establish the orientations of the N- and C-termini and thus the parity of the Ancp transmembrane segments (TMS). According to this, we have constructed a gene encoding a covalent tandem dimer of the Saccharomyces cerevisiae Anc2p and we demonstrate that it is stable and active in vivo as well as in vitro. The properties of the isolated dimer are strongly similar to those of the native Anc2p, as seen from nucleotide exchange and inhibitor binding experiments. We can therefore conclude that the native Anc2p has an even number of TMS and that the N- and C-terminal regions are exposed to the same cellular compartment. Furthermore, our results support the idea of a minimal dimeric functional organization of the Ancp in the mitochondrial membrane and we can suggest that TMS 1 of one monomer and TMS 6 of the other monomer in the native dimer are very close to each other.
Keywords: ADP/ATP carrier; Topography; Subunit stoichiometry; Covalent tandem dimer; Saccharomyces cerevisiae;
Effects of fluorescent pseudo-ATP and ATP-metal analogs on secondary structure of Na+/K+-ATPase by Fabio Tanfani; Holger Linnertz; Tomas Obsil; Rita Krumscheid; Petra Urbanova; Otakar Jelinek; Laura Mazzanti; Enrico Bertoli; Wilhelm Schoner; Evzen Amler (94-102).
The secondary structure of Na+/K+-ATPase after modification of the ATP-binding sites was analyzed. Consistently with recent reports, we found in trypsin-treated Na+/K+-ATPase additionally to α-helix also β-sheet structures in the transmembrane segments. However, binding of fluorescein 5′-isothiocyanate (FITC), the pseudo-ATP analog, to the ATP-binding site did not affect the secondary structure of undigested Na+/K+-ATPase. Consequently, fluorescence intensity changes of FITC-labeled Na+/K+-ATPase commonly used to observe conformational transitions of the enzyme reflect physiological changes of the native structure. The metal complex analogues of ATP, Cr(H2O)4ATP and Co(NH3)4ATP, on the other hand, affected the secondary structure of Na+/K+-ATPase. We propose that these changes in the secondary structure are responsible for inhibition of backdoor phosphorylation.
Keywords: Na+/K+-ATPase; ATP analog; Backdoor phosphorylation; Ouabain-binding; FT-IR;