BBA - Bioenergetics (v.1827, #7)

Proteomic analysis of F1F0-ATP synthase super-assembly in mitochondria of cardiomyoblasts undergoing differentiation to the cardiac lineage by Elena Bisetto; Marina Comelli; Anna Maria Salzano; Paola Picotti; Andrea Scaloni; Giovanna Lippe; Irene Mavelli (807-816).
Mitochondria are essential organelles with multiple functions, especially in energy metabolism. An increasing number of data highlighted their role for cellular differentiation processes. We investigated differences in ATP synthase supra-molecular organization occurring in H9c2 cardiomyoblasts in the course of cardiac-like differentiation, along with ATP synthase biogenesis and maturation of mitochondrial cristae morphology. Using BN-PAGE analysis combined with one-step mild detergent extraction from mitochondria, a significant increase in dimer/monomer ratio was observed, indicating a distinct rise in the stability of the enzyme super-assembly. Remarkably, sub-stoichiometric mean values for ATP synthase subunit e were determined in both parental and cardiac-like H9c2 by an MS-based quantitative proteomics approach. This indicates a similar high proportion of complex molecules lacking subunit e in both cell types, and suggests a minor contribution of this component in the observed changes. 2D BN-PAGE/immunoblotting analysis and MS/MS analysis on single BN-PAGE band showed that the amount of inhibitor protein IF1 bound within the ATP synthase complexes increased in cardiac-like H9c2 and appeared greater in the dimer. In concomitance, a consistent improvement of enzyme activity, measured as both ATP synthesis and ATP hydrolysis rate, was observed, despite the increase of bound IF1 evocative of a greater inhibitory effect on the enzyme ATPase activity. The results suggest i) a role for IF1 in promoting dimer stabilization and super-assembly in H9c2 with physiological IF1 expression levels, likely unveiled by the fact that the contacts through accessory subunit e appear to be partially destabilized, ii) a link between dimer stabilization and enzyme activation.
Keywords: F1F0-ATP synthase; Supramolecular organization; IF1; BN-PAGE; Cardiomyocyte-like differentiation; H9c2;

Mitochondria play an integral role in cell death signaling, yet how mitochondrial defects disrupt this important function is not well understood. We have used a mouse L-cell fibroblast model harboring Rattus norvegicus mtDNA (Rn xenocybrids) to examine the effects of multiple oxidative phosphorylation (OXPHOS) defects on reactive oxygen species (ROS) generation and cell death signaling. Blue native-PAGE analyses of Rn xenocybrids revealed defects in OXPHOS complex biogenesis with reduced steady-state levels of complexes I, III and IV. Isolated Rn xenocybrid mitochondria exhibited deficiencies in complex II + III and III activities, with CIII-stimulated ROS generation 66% higher than in control mitochondria. Rn xenocybrid cells were resistant to staurosporine-induced cell death, but exhibited a four-fold increase in sensitivity to ceramide-induced cell death that was caspase-3 independent and did not induce chromosomal DNA degradation. Furthermore, ceramide directly inhibited Rn xenocybrid complex II + III activity by 97%, although this inhibition could be completely abolished by exogenous decylubiquinone. Ceramide also induced a further increase in ROS output from Rn xenocybrid complex III by 42%. These results suggest that the interaction of ceramide with OXPHOS complex III is significantly enhanced by the presence of the xenotypic Rattus cytochrome b in complex III, likely due to the increased affinity for ceramide at the ubiquinone binding site. We propose a novel mechanism of altered mitochondrial cell death signaling due to mtDNA mutations whereby ceramide directly induces OXPHOS complex ROS generation to initiate cell death pathways.
Keywords: Oxidative phosphorylation; Reactive oxygen species; Mitochondrial DNA; Ceramide; Mouse fibroblast; Mitochondrial disease;

The membrane-bound enzyme cNOR (cytochrome c dependent nitric oxide reductase) catalyzes the reduction of NO in a non-electrogenic process. This is in contrast to the reduction of O2 in cytochrome c oxidase (CcO), the other member of the heme-copper oxidase family, which stores energy by the generation of a membrane gradient. This difference between the two enzymes has not been understood, but it has been speculated to be of kinetic origin, since per electron the NO reduction is more exergonic than the O2 reduction, and the energy should thus be enough for an electrogenic process. However, it has not been clear how and why electrogenicity, which mainly affects the thermodynamics, would slow down the very exergonic NO reduction. Quantum chemical calculations are used to construct a free energy profile for the catalytic reduction of NO in the active site of cNOR. The energy profile shows that the reduction of the NO molecules by the enzyme and the formation of N2O are very exergonic steps, making the rereduction of the enzyme endergonic and rate-limiting for the entire catalytic cycle. Therefore the NO reduction cannot be electrogenic, i.e. cannot take electrons and protons from the opposite sides of the membrane, since it would increase the endergonicity of the rereduction when the gradient is present, thereby increasing the rate-limiting barrier, and the reaction would become too slow. It also means that proton pumping coupled to electron transfer is not possible in cNOR. In CcO the corresponding rereduction of the enzyme is very exergonic.
Keywords: Nitric oxide reductase; Cytochrome c oxidase; Density functional theory; Electrogenicity; Proton pumping;

Photosynthesis in Chondrus crispus: The contribution of energy spill-over in the regulation of excitonic flux by Nathalie Kowalczyk; Fabrice Rappaport; Catherine Boyen; Francis-André Wollman; Jonas Collén; Pierre Joliot (834-842).
Chondrus crispus is a species of red algae that grows on rocks from the middle intertidal into the subtidal zones of the North Atlantic coasts. As such, it has to cope with strongly variable abiotic conditions. Here we studied the response of the photosynthetic apparatus of this red alga to illumination. We found that, as previously described in the case of the unicellular alga Rhodella violacea (E. Delphin et al., Plant Physiol. 118 (1998) 103–113), a single multi-turnover saturating pulse of light is sufficient to induce a strong quenching of fluorescence. To elucidate the mechanisms underlying this fluorescence quenching, we combined room temperature and 77 K fluorescence measurements with absorption spectroscopy to monitor the redox state of the different electron carriers in the chain. In addition, we studied the dependence of these various observables upon the excitation wavelength. This led us to identify energy spill-over from Photosystem II to Photosystem I rather than a qE-type non-photochemical quenching as the major source of fluorescence quenching that develops upon a series of 200 ms pulses of saturating light results, in line with the conclusion of Ley and Butler (Biochim. Biophys. Acta 592 (1980) 349–363) from their studies of the unicellular red alga Porphyridium cruentum. In addition, we show that the onset of this spill-over is triggered by the reduction of the plastoquinone pool.
Keywords: Photosynthesis; Red algae; Chondrus crispus; Chlorophyll fluorescence; Spill-over; Plastoquinone pool;

Cytochrome oxidase is one of the functionally most intriguing redox-driven proton pumps. During the last decade our increased understanding of the system has greatly benefited from theoretical calculations and modeling in the framework of three-dimensional structures of cytochrome c oxidases from different species. Because these studies are based on results from experiments, it is important that any ambiguities in the conclusions extracted from these experiments are discussed and elucidated. In a recent study Szundi et al. (Szundi et al. Biochemistry 2012, 51, 9302) investigated the reaction of the reduced Rhodobacter sphaeroides cytochrome c oxidase with O2 and arrived at conclusions different from those derived from earlier investigations. In this short communication we compare these very recent data to those obtained from earlier studies and discuss the origin of the differences.
Keywords: Electron transfer; Proton transfer; Membrane protein; Respiration; Redox reaction; Cytochrome aa 3;

Dissecting the molecular mechanism by which NH2htau and Aβ1-42 peptides impair mitochondrial ANT-1 in Alzheimer disease by A. Bobba; G. Amadoro; V.A. Petragallo; P. Calissano; A. Atlante (848-860).
To find out whether and how the adenine nucleotide translocator-1 (ANT-1) inhibition due to NH2htau and Aβ1-42 is due to an interplay between these two Alzheimer's peptides, ROS and ANT-1 thiols, use was made of mersalyl, a reversible alkylating agent of thiol groups that are oriented toward the external hydrophilic phase, to selectively block and protect, in a reversible manner, the –SH groups of ANT-1. The rate of ATP appearance outside mitochondria was measured as the increase in NADPH absorbance which occurs, following external addition of ADP, when ATP is produced by oxidative phosphorylation and exported from mitochondria in the presence of glucose, hexokinase and glucose-6-phosphate dehydrogenase. We found that the mitochondrial superoxide anions, whose production is induced at the level of Complex I by externally added Aβ1-42 and whose release from mitochondria is significantly reduced by the addition of the VDAC inhibitor DIDS, modify the thiol group/s present at the active site of mitochondrial ANT-1, impair ANT-1 in a mersalyl-prevented manner and abrogate the toxic effect of NH2htau on ANT-1 when Aβ1-42 is already present. A molecular mechanism is proposed in which the pathological Aβ-NH2htau interplay on ANT-1 in Alzheimer's neurons involves the thiol redox state of ANT-1 and the Aβ1-42-induced ROS increase. This result represents an important innovation because it suggests the possibility of using various strategies to protect cells at the mitochondrial level, by stabilizing or restoring mitochondrial function or by interfering with the energy metabolism providing a promising tool for treating or preventing AD.
Keywords: Adenine nucleotide translocator; Mitochondria; β-amyloid; Tau fragment; Thiol group;

Forty-three years ago, Kok and coworkers introduced a phenomenological model describing period-four oscillations in O2 flash yields during photosynthetic water oxidation (WOC), which had been first reported by Joliot and coworkers. The original two-parameter Kok model was subsequently extended in its level of complexity to better simulate diverse data sets, including intact cells and isolated PSII-WOCs, but at the expense of introducing physically unrealistic assumptions necessary to enable numerical solutions. To date, analytical solutions have been found only for symmetric Kok models (inefficiencies are equally probable for all intermediates, called “S-states”). However, it is widely accepted that S-state reaction steps are not identical and some are not reversible (by thermodynamic restraints) thereby causing asymmetric cycles. We have developed a mathematically more rigorous foundation that eliminates unphysical assumptions known to be in conflict with experiments and adopts a new experimental constraint on solutions. This new algorithm termed STEAMM for S-state Transition Eigenvalues of Asymmetric Markov Models enables solutions to models having fewer adjustable parameters and uses automated fitting to experimental data sets, yielding higher accuracy and precision than the classic Kok or extended Kok models. This new tool provides a general mathematical framework for analyzing damped oscillations arising from any cycle period using any appropriate Markov model, regardless of symmetry. We illustrate applications of STEAMM that better describe the intrinsic inefficiencies for photon-to-charge conversion within PSII-WOCs that are responsible for damped period-four and period-two oscillations of flash O2 yields across diverse species, while using simpler Markov models free from unrealistic assumptions.
Keywords: Photosystem II; Photosynthetic efficiency; Oxygen evolution; Markov chain; Kok model;