BBA - Bioenergetics (v.1767, #3)

Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S0 and S2 states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D2O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S0 and S2 state multiline disappeared after 3 h of D2O incubation in the S0 and S1 states, respectively. The signal with 4.0 MHz hyperfine constants in S0 state multiline disappeared after 3 h of D2O incubation in the S0 state, while the similar signal in S2 state multiline disappeared only after 24 h of D2O incubation in the S1 state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. Based on the point dipole approximation, the distances between the center of electronic spin of the Mn cluster and the exchangeable protons were estimated to be 3.3/3.2 and 2.7 Å, respectively. These short distances suggest the protons belong to the water molecules ligated to the manganese cluster. We propose a model for the binding of water to the manganese cluster based on these results.
Keywords: EPR; ESR; ENDOR; Mn-cluster; Oxygen evolution; Photosystem II;

Redox-linked protonation state changes in cytochrome bc 1 identified by Poisson–Boltzmann electrostatics calculations by Astrid R. Klingen; Hildur Palsdottir; Carola Hunte; G. Matthias Ullmann (204-221).
Cytochrome bc 1 is a major component of biological energy conversion that exploits an energetically favourable redox reaction to generate a transmembrane proton gradient. Since the mechanistic details of the coupling of redox and protonation reactions in the active sites are largely unresolved, we have identified residues that undergo redox-linked protonation state changes. Structure-based Poisson–Boltzmann/Monte Carlo titration calculations have been performed for completely reduced and completely oxidised cytochrome bc 1. Different crystallographically observed conformations of Glu272 and surrounding residues of the cytochrome b subunit in cytochrome bc 1 from Saccharomyces cerevisiae have been considered in the calculations. Coenzyme Q (CoQ) has been modelled into the CoQ oxidation site (Qo-site). Our results indicate that both conformational and protonation state changes of Glu272 of cytochrome b may contribute to the postulated gating of CoQ oxidation. The Rieske iron–sulphur cluster could be shown to undergo redox-linked protonation state changes of its histidine ligands in the structural context of the CoQ-bound Qo-site. The proton acceptor role of the CoQ ligands in the CoQ reduction site (Qi-site) is supported by our results. A modified path for proton uptake towards the Qi-site features a cluster of conserved lysine residues in the cytochrome b (Lys228) and cytochrome c 1 subunits (Lys288, Lys289, Lys296). The cardiolipin molecule bound close to the Qi-site stabilises protons in this cluster of lysine residues.
Keywords: Protonation probability; Titration behaviour; Respiratory chain; Membrane protein; Cardiolipin; Rieske iron–sulphur cluster; Poisson–Boltzmann electrostatics calculation;

Carvedilol inhibits mitochondrial complex I and induces resistance to H2O2-mediated oxidative insult in H9C2 myocardial cells by Paola Sgobbo; Consiglia Pacelli; Ignazio Grattagliano; Gaetano Villani; Tiziana Cocco (222-232).
Carvedilol, a β-adrenoreceptor antagonist with strong antioxidant activity, produces a high degree of cardioprotection in a variety of experimental models of ischemic cardiac injury. Although growing evidences suggest specific effects on mitochondrial metabolism, how carvedilol would exert its overall activity has not been completely disclosed. In the present work we have investigated the impact of carvedilol-treatment on mitochondrial bioenergetic functions and ROS metabolism in H9C2 cells. This analysis has revealed a dose-dependent decrease in respiratory fluxes by NAD-dependent substrates associated with a consistent decline of mitochondrial complex I activity. These changes were associated with an increase in mitochondrial H2O2 production, total glutathione and protein thiols content. To evaluate the antioxidant activity of carvedilol, the effect of the exposure of control and carvedilol-pretreated H9C2 cells to H2O2 were investigated. The H2O2-mediated oxidative insult resulted in a significant decrease of mitochondrial respiration, glutathione and protein thiol content and in an increased level of GSSG. These changes were prevented by carvedilol-pretreatment. A similar protective effect on mitochondrial respiration could be obtained by pre-treatment of the cells with a sub-saturating amount of rotenone, a complex I inhibitor.We therefore suggest that carvedilol exerts its protective antioxidant action both by a direct antioxidant effect and by a preconditioning-like mechanism, via inhibition of mitochondrial complex I.
Keywords: Mitochondria; Respiratory chain; Complex I; Carvedilol; Oxidative stress; Glutathione;

The mechanism of charge recombination was studied in Photosystem II by using flash induced chlorophyll fluorescence and thermoluminescence measurements. The experiments were performed in intact cells of the cyanobacterium Synechocystis 6803 in which the redox properties of the primary pheophytin electron acceptor, Phe, the primary electron donor, P680, and the first quinone electron acceptor, QA, were modified. In the D1Gln130Glu or D1His198Ala mutants, which shift the free energy of the primary radical pair to more positive values, charge recombination from the S2QA and S2QB states was accelerated relative to the wild type as shown by the faster decay of chlorophyll fluorescence yield, and the downshifted peak temperature of the thermoluminescence Q and B bands. The opposite effect, i.e. strong stabilization of charge recombination from both the S2QA and S2QB states was observed in the D1Gln130Leu or D1His198Lys mutants, which shift the free energy level of the primary radical pair to more negative values, as shown by the retarded decay of flash induced chlorophyll fluorescence and upshifted thermoluminescence peak temperatures. Importantly, these mutations caused a drastic change in the intensity of thermoluminescence, manifested by 8- and 22-fold increase in the D1Gln130Leu and D1His198Lys mutants, respectively, as well as by a 4- and 2.5-fold decrease in the D1Gln130Glu and D1His198Ala mutants, relative to the wild type, respectively. In the presence of the electron transport inhibitor bromoxynil, which decreases the redox potential of QA/QA relative to that observed in the presence of DCMU, charge recombination from the S2QA state was accelerated in the wild type and all mutant strains. Our data confirm that in PSII the dominant pathway of charge recombination goes through the P680 +Phe radical pair. This indirect recombination is branched into radiative and non-radiative pathways, which proceed via repopulation of P680 * from 1[P680 +Ph] and direct recombination of the 3[P680 +Ph] and 1[P680 +Ph] radical states, respectively. An additional non-radiative pathway involves direct recombination of P680 +QA . The yield of these charge recombination pathways is affected by the free energy gaps between the Photosystem II electron transfer components in a complex way: Increase of ΔG(P680 *  ↔ P680 +Phe) decreases the yield of the indirect radiative pathway (in the 22–0.2% range). On the other hand, increase of ΔG(P680 +Phe  ↔ P680 +QA ) increases the yield of the direct pathway (in the 2–50% range) and decreases the yield of the indirect non-radiative pathway (in the 97–37% range).
Keywords: Photosystem II; Charge recombination; Thermoluminescence; Flash-induced chlorophyll fluorescence;

Gaining a detailed understanding of the energetics of the proton pumping process in cytochrome c oxidase (CcO) is one of the challenges of modern biophysics. Although there are several current mechanistic proposals, most of these ideas have not been subjected to consistent structure–function considerations. In particular most works have not related the activation barriers for different mechanistic proposals to the protein structure. The present work describes a general approach for exploring the energetics of different feasible models of the action of CcO, using the observed protein structure, established simulation methods and a modified Marcus' formulation. We start by reviewing our methods for evaluation of the energy diagrams for different proton translocation paths and then present a systematic analysis of various constraints that should be imposed on any energy diagram for the pumping process. After the general analysis we turn to the actual computational study, where we construct energy diagrams for forward and backward paths, using the estimated calculated reduction potentials and pK a values of all the relevant sites (including internal water molecules). We then explore the relationship between the calculated energy diagrams and key experimental constraints. This comparison allows us to identify some barriers that are not fully consistent with the overall requirement for an efficient pumping. In particular we identify back leakage channels, which are hard to block without stopping the forward channels. This helps to identify open problems that will require further experimental and theoretical studies. We also consider reasonable adjustments of the calculated barriers that may lead to a working pump. Although the present analysis does not establish a unique and workable model for the mechanism of CcO, it presents what is probably the most consistent current analysis of the barriers for different feasible pathways. Perhaps more importantly, the framework developed here should provide a general way for examining any proposal for the action of CcO as well as for the analysis of further experimental findings about the action of this fascinating system.
Keywords: Cytochrome c oxidase; Coupled electron transfer-proton transfer; Protonpumps; Dielectric of proteins; Computer simulations; Electrostatic effects;