This review focuses on key components of respiratory and photosynthetic energy-transduction systems: the cytochrome bc 1 and b 6 f (Cytbc 1 /b 6 f) membranous multisubunit homodimeric complexes. These remarkable molecular machines catalyze electron transfer from membranous quinones to water-soluble electron carriers (such as cytochromes c or plastocyanin), coupling electron flow to proton translocation across the energy-transducing membrane and contributing to the generation of a transmembrane electrochemical potential gradient, which powers cellular metabolism in the majority of living organisms. Cytsbc 1 /b 6 f share many similarities but also have significant differences. While decades of research have provided extensive knowledge on these enzymes, several important aspects of their molecular mechanisms remain to be elucidated. We summarize a broad range of structural, mechanistic, and physiological aspects required for function of Cytbc 1 /b 6 f, combining textbook fundamentals with new intriguing concepts that have emerged from more recent studies. The discussion covers but is not limited to (i) mechanisms of energy-conserving bifurcation of electron pathway and energy-wasting superoxide generation at the quinol oxidation site, (ii) the mechanism by which semiquinone is stabilized at the quinone reduction site, (iii) interactions with substrates and specific inhibitors, (iv) intermonomer electron transfer and the role of a dimeric complex, and (v) higher levels of organization and regulation that involve Cytsbc 1 /b 6 f. In addressing these topics, we point out existing uncertainties and controversies, which, as suggested, will drive further research in this field.
Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.
Oxygenic respiration and photosynthesis based on quinone redox reactions face a danger of wasteful energy dissipation by diversion of the productive electron transfer pathway through the generation of reactive oxygen species (ROS). Nevertheless, the widespread quinone oxido-reductases from the cytochrome bc family limit the amounts of released ROS to a low, perhaps just signaling, level through an as-yet-unknown mechanism. Here, we propose that a metastable radical state, nonreactive with oxygen, safely holds electrons at a local energetic minimum during the oxidation of plastohydroquinone catalyzed by the chloroplast cytochrome b 6 f. This intermediate state is formed by interaction of a radical with a metal cofactor of a catalytic site. Modulation of its energy level on the energy landscape in photosynthetic vs. respiratory enzymes provides a possible mechanism to adjust electron transfer rates for efficient catalysis under different oxygen tensions.cytochrome b 6 f | reactive oxygen species | semiquinone | electron paramagnetic resonance | electron transport P hotosynthetic and respiratory cytochromes bc 1 /b 6 f (Fig. 1A) generate a proton-motive force (pmf) that powers cellular metabolism by using the Gibbs free energy difference (ΔG) between hydroquinone (QH 2 ) derivatives (Fig. 1B) and oxidized soluble electron transfer proteins (e.g., cytochrome c or plastocyanin) (1, 2). To increase the efficiency of this process, which is critical for the yield of the generated pmf, one part of the enzyme recirculates electrons to the quinone pool in the membrane (Q pool), whereas the second part steers the electrons to the cytochrome c pool, powering the electron recirculation (Fig. 1C). This mechanism, which is best established for the cytochrome bc 1 (cyt bc 1 ) (3, 4), with supporting data for the cytochrome b 6 f (cyt b 6 f) (5), discussed in ref. 2, is based on bifurcation of the route for two electrons released upon oxidation of QH 2 at one of the catalytic sites-the Q p site, (Q p ), (Fig. 1D) (3-5). A model for the energetics of this reaction assumes that one electron derived from the two-electron QH 2 donor is transferred, through the high-potential cofactor chain ("steering part" in Fig. 1C) to plastocyanin or cytochrome c, whereas the second electron is transferred across the membrane through low-potential cofactors ("recirculation" part in Fig. 1C).The electronic bifurcation process requires formation of a short-lived and reducing redox intermediate-ubisemiquinone (USQ) or plastosemiquinone (PSQ) (4, 6, 7). However, such an intermediate in an oxygenic environment would readily reduce oxygen to form superoxide radical, (O 2 − ), compromising the efficiency of energy conservation (8). Even in cyt b 6 f where the level of superoxide production through this pathway is at least an order of magnitude greater than that from yeast cyt bc 1 , the branching ratio for electron transfer to O 2 forming O 2 − is only 1-2% of the total flux (6). The low absolute level of O 2 − production in native proteins implies the e...
One of the less understood parts of the catalytic cycle of cytochrome bc/bf complexes is the mechanism of electronic bifurcation occurring within the hydroquinone oxidation site (Q site). Several models describing this mechanism invoke a phenomenon of formation of an unstable semiquinone. Recent studies with isolated cytochrome bc or bf revealed that a relatively stable semiquinone spin-coupled to the reduced Rieske cluster (SQ-FeS) is generated at the Q site during the oxidation of ubi- or plastohydroquinone analogs under conditions of continuous turnover. Here, we identified the EPR transition of SQ-FeS formed upon oxidation of ubihydroquinone in native photosynthetic membranes from purple bacterium Rhodobacter capsulatus. We observed a significant amount of SQ-FeS generated when the antimycin-inhibited enzyme experiences conditions of non-equilibrium caused by the continuous light activation of the reaction center. We also noted that SQ-FeS cannot be detected under equilibrium redox titrations in dark. The non-equilibrium redox titrations of SQ-FeS indicate that this center has a higher apparent redox midpoint potential when compared to the redox midpoint potential of the quinone pool. This suggests that SQ-FeS is stabilized, which corroborates a recently proposed mechanism in which the SQ-FeS state is metastable and functions to safely hold electrons at the local energy minimum during the oxidation of ubihydroquinone and limits superoxide formation. Our results open new possibilities to study the formation and properties of this state in cytochromes bc under close to physiological conditions in which non-equilibrium is attained by the light activation of bacterial reaction centers or photosystems.
Catalytic reactions of quinol oxidoreductases may lead to the generation of superoxide due to electron leaks from unstable semiquinone intermediates (SQ). For cytochrome bc1, the mechanism of suppression of superoxide generation remains unknown. We analyzed conditions of formation of a spin‐spin–coupled state between SQ and the Rieske cluster (SQ‐FeS) associated with catalysis of the quinol oxidation site of cytochrome bc1. We reveal that mutants that preclude direct interaction between SQ and the Rieske cluster do not form SQ‐FeS and release enhanced superoxide. In the enzymes generating SQ‐FeS, little or no superoxide is detected. We propose that SQ‐FeS suppresses superoxide generation, becoming an element modulating superoxide release under physiologically relevant conditions slowing electron flow through the enzyme.
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