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# Simplified diagram of relative energy levels at different stages of reaction catalyzed by cyt b 6 f (A) and cyt bc 1 (B). Black dots represent electrons on the respective cofactor or quinone molecule. Yellow squares, Q p empty or occupied by substrate; purple squares, Q p with bound DBMIB semiquinone. Green and red arrows, downhill and uphill transitions between the states, respectively. Red frame indicates the states that are inaccessible in antimycin-inhibited cyt bc 1 . State 1 represents the most populated initial state for QH 2 oxidation under steady-state turnover (in antimycin-inhibited cyt bc 1 heme b n is already reduced after the first turnover that takes place within experimental dead time). This reaction results in reduction of FeS and heme b p (transition from 1 to 3 involving unstable SQ in 2). From 3 downhill reactions to 4 or 5 are possible and 5 undergoes further downhill transition to 6 (oxidation of heme b n ) allowing next turnover. State 4 is metastable state containing SQ spin-coupled to reduced FeS. Population of 4 increases when transition to 5 is blocked (antimycin) or transition from 5 to 6 is slow with respect to the transition from 1 to 3. The stationary level of 4 depends on energetic gap between 4 and 5 (gray double arrow), which differs within bc family. State 4 lays below energetic level of 8 in which O 2 − is generated. State 7 involves stable DBMIB semiquinone spincoupled to FeS. The gray square with a gradient depicts uncertainty in E m values for hemes b in cyt b 6 f.

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Significance Photosynthesis and respiration are crucial energy-conserving processes of living organisms. These processes rely on redox reactions that often involve unstable radical intermediates. In an oxygenic atmosphere, such intermediates present a danger of becoming a source of electrons for generation of reactive oxygen species. Here, we disco...

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Context 1
... the results presented here, a mechanism can be proposed for inclusion of the metastable SQ-FeS into the ther- modynamic diagram of electronic bifurcation (Fig. 6). This dia- gram follows the generally accepted scheme of enzymatic cycle but adds a new state, state 4, which is a result of an energetically downhill electron transfer from heme b p to Q at Q p (transition from state 3). This state protects the enzyme against ROS pro- duction by: (i) the fact that electron transfer from SQ-FeS to ...
Context 2
... free energy (ΔG) diagram, shown in Fig. 6, which depends not only on E m but also on other processes, e.g., reconfiguration of H bonds within Q p , provides a possible explanation for sig- nificant occupation of the energetic state representing PSQ-FeS in noninhibited cyt b 6 f. Although the difference in E m between hemes b n and b p in cyt b 6 f is not well defined (shown as ...
Context 3
... E m but also on other processes, e.g., reconfiguration of H bonds within Q p , provides a possible explanation for sig- nificant occupation of the energetic state representing PSQ-FeS in noninhibited cyt b 6 f. Although the difference in E m between hemes b n and b p in cyt b 6 f is not well defined (shown as the width in the level of state 5 in Fig. 6A), the average is somewhat more negative than in hemes b in cyt bc 1 (30,31). This difference, together with the fact that PQ possesses a more positive E m than UQ (13) makes the energetic gap between state 4 (with SQ-FeS) and state 5 (with reduced heme b n ) smaller in cyt b 6 f compared with cyt bc 1 . In other words, in cyt bc 1 , ...
Context 4
... the short circuits (two electrons from QH 2 going to the same cofactor chain) and the leaks of electrons to mo- lecular oxygen (1,7,8). At the molecular level, the stabilization of SQ by its spin-coupling to reduced FeS is likely to occur through the creation of an H bond between SQ and histidine ligating reduced FeS (transition from 2 to 4 in Fig. 6) ...
Context 5
... stabilization of SQ-FeS can occasionally be broken (depic- ted in the Fig. 6 as the transition from state 4 to 2), resulting in the formation of a highly unstable semiquinone (SQ not spin-coupled to FeS), which is able to reduce molecular oxygen. This semi- quinone remains the most likely state responsible for limited su- peroxide release. In the reversible transition between SQ-FeS and SQ that is not ...

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... In this process, the Q o site quinol releases two protons to the P-side of the membrane and the complete reduction and protonation of a quinone molecule in the Q i site needs oxidation of a second quinol at the Q o site and proton uptake from the N-side of the membrane. Consequently, bifurcated electron transfer must be achieved upon quinol oxidation to enable the Q cycle, i.e. the highly reactive SQ • at the Q o site must be controlled to avoid short circuits [2,[29][30][31][32][33][34] which lead to futile bypass reactions which would lower the efficiency of cellular respiration and can generate reactive oxygen species [29] that can cause oxidative damage to the cell [35]. ...
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... Its operation is prone to bypass reactions which generate reactive oxygen species (ROS) via a semiquinone radical, resulting in energy waste and deleterious radicals 13 . Unproductive reactions can be triggered through an unbalanced redox-state of the quinone pool, hypoxia, or inherited diseases [14][15][16][17] . In cyt c oxidases, the key to energy conversion is a coupling of dioxygen reduction at the canonical binuclear centre (BNC) with proton pumping 2 . ...
... Furthermore, there is experimental evidence that bifurcation in bc complexes can take place without Rieske movement, as the kinetic characterisation of the cyt bc 1 complex with immobilised Rieske domain from a Rhodobacter capsulatus mutant (plus-two-alanine mutant) still supported bifurcation, whereas the electron transfer to haem c 1 was restricted 53 . The fixed QcrA keeps the Q o site occluded which may minimise the risk of ROS generation, as the reaction intermediate semiquinone can directly reduce dioxygen 17 . Yet, proton release from the closed Q o site is required to fulfil the Q cycle role for PMF generation. ...
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... A general agreement has been reached that a superoxide generation by Cytbc 1 results from a reaction of USQ o with molecular oxygen (Boveris and Cadenas, 1975;Ksenzenko et al., 1983;Muller et al., 2002;Borek et al., 2008;Dröse and Brandt, 2008;Sarewicz et al., 2010;Pagacz et al., 2021). However, trapping the intermediate USQ o radical during the UQH 2 oxidation and its detection by electron paramagnetic resonance (EPR) has been proven difficult and obtained results have often been disputable or contradictory (de Vries et al., 1981;Jünemann et al., 1998;Cape et al., 2006;Cape et al., 2007;Zhang et al., 2007;Zhu et al., 2007;Sarewicz et al., 2013;Vennam et al., 2013;Victoria et al., 2013;Pietras et al., 2016;Crofts et al., 2017;Sarewicz et al., 2017;Sarewicz et al., 2018;Bujnowicz et al., 2019). Additionally, the inability to generate USQ o in the equilibrium redox titrations (Takamiya and Dutton, 1979;Sarewicz et al., 2018) implicated a concept of a high instability of the semiquinone and its reactivity was proposed to be a reason for superoxide production by Cytbc 1 . ...
... Our previous work reported an unusual EPR signal with an average g value less than 2, which originated from the Q o site of the antimycin-inhibited Cytbc 1 , isolated from Rhodobacter capsulatus (Sarewicz et al., 2013;Sarewicz et al., 2017;Bujnowicz et al., 2019). This signal was detected only when the Q o site was able to catalyze the UQH 2 oxidation and the cytochrome c reduction before the system reached equilibrium. ...
... At X-band, the most prominent "derivative-shaped" transition of SQ o -2Fe2S in Cytbc 1 was detected at g ∼1.94 and was found to shift to ∼1.96 at Q-band. Remarkably, the SQ o -2Fe2S signal was also detected in isolated but non-inhibited spinach cytochrome b 6 f during the oxidation of synthetic decylplastoquinol and the reduction of plastocyanin (Sarewicz et al., 2017). ...
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... More recent studies, however, suggest that the oxygen reductant is the semiquinone formed in the so-called semi-reverse reaction in which cytochrome b L reduces the fully oxidized quinone [245,246]. Osyczka [247,248] proposes that a metastable radical state, nonreactive with oxygen, safely holds electrons at a local energetic minimum during the oxidation of ubiquinol. This intermediate state is formed by interaction of a radical with a metal cofactor of a catalytic site, presumably the FeS cluster, under physiological conditions. ...
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... A relatively recent study by Sarewicz/Bujnowicz et al. revealed that the EPR spectrum of 2Fe2S in DBMIB-supplemented spinach Cytb 6 f is frequency-dependent, which is a strong indication that DBMIB semiquinone at the Q p site is spin−spin coupled to the reduced 2Fe2S. 482 Besides being extensively used to characterize electron flow in the photosynthetic chain, DBMIB has also been largely employed to study cell biological responses in photosynthetic organisms. Combining DBMIB with the PSII specific inhibitor DCMU allows the PQ(H 2 ) pool to be either reduced (DBMIB) or oxidized (DCMU) in the light. ...
... Further research showed that a g = 1.94 signal of similar amplitude was observed when the samples were prepared under both aerobic and anaerobic conditions. 482,673 This dismissed concerns raised by others 674 that it might have resulted from oxidative damage to the 2Fe2S. This also pointed toward an interesting possibility that the SQ-2Fe2S state is not reactive toward molecular oxygen (see further discussion). ...
... 258,575,597 Interestingly the SQ-2Fe2S state is not restricted to Cytbc 1 but has as it was also observed in isolated spinach Cytb 6 f exposed to its substrates, PQH 2 and PC. 482 In this case, however, unlike in Cytbc 1 , inhibition of the Q n site was not required. This revealed that the probability of SQ-2Fe2S state formation when the enzyme operates without inhibition is higher in the Q p site of Cytb 6 f compared to the Q o site Cytbc 1 . ...
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This review focuses on key components of respiratory and photosynthetic energy-transduction systems: the cytochrome bc1 and b6f (Cytbc1/b6f) 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. Cytsbc1/b6f 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 Cytbc1/b6f, 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 Cytsbc1/b6f. In addressing these topics, we point out existing uncertainties and controversies, which, as suggested, will drive further research in this field.
... Recent work on the complex III SQ allows us to further qualify the type of complex III SQ we are observing. First, we consider the recently discovered Q o site metastable SQ [45] to be an unlikely contributor to the complex III g = 2.00 signal because Q o site SQs have not been reported under steady-state aerobic conditions in SMPs as employed here [43]. Two populations of SQ are in principle possible in the Q i site, one with a dipolar coupling interaction to the neighbouring oxidised (paramagnetic) heme b H and another without magnetic coupling to the reduced (diamagnetic) state of the heme [46]. ...
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Background: For decades, semiquinone intermediates have been suggested to play an essential role in catalysis by one of the most enigmatic proton-pumping enzymes, respiratory complex I, and different mechanisms have been proposed on their basis. However, the difficulty in investigating complex I semiquinones, due to the many different enzymes embedded in the inner mitochondrial membrane, has resulted in an ambiguous picture and no consensus. Results: In this paper, we re-examine the highly debated origin of semiquinone species in mitochondrial membranes using a novel approach. Our combination of a semi-artificial chimeric respiratory chain with pulse EPR spectroscopy (HYSCORE) has enabled us to conclude, unambiguously and for the first time, that the majority of the semiquinones observed in mitochondrial membranes originate from complex III. We also identify a minor contribution from complex II. Conclusions: We are unable to attribute any semiquinone signals unambiguously to complex I and, reconciling our observations with much of the previous literature, conclude that they are likely to have been misattributed to it. We note that, for this earlier work, the tools we have relied on here to deconvolute overlapping EPR signals were not available. Proposals for the mechanism of complex I based on the EPR signals of semiquinone species observed in mitochondrial membranes should thus be treated with caution until future work has succeeded in isolating any complex I semiquinone EPR spectroscopic signatures present.
... The bound chlorophyll adjacent to PQ1 may fulfil a gating function at the Q p pocket, either controlling access of PQH 2 and/or increasing the retention time of the reactive semiplastoquinone (SPQ) intermediate species formed following electron transfer to the 2Fe-2S cluster. Indeed, spin-coupling between the SPQ and the 2Fe-2S cluster has been detected during enzymatic turnover of cytb 6 f but is absent in cytbc 1 complexes that lack the chlorophyll molecule 24 . SPQ in the 2Fe-2S-bound state does not react with oxygen, providing a potential mechanism to control the release of superoxide from the Q p site 24 and regulate the activity of the LHCII kinase STN7 25 , which is proposed to bind to cytb 6 f between transmembrane helices F and H of subunit IV 26 . ...
... Indeed, spin-coupling between the SPQ and the 2Fe-2S cluster has been detected during enzymatic turnover of cytb 6 f but is absent in cytbc 1 complexes that lack the chlorophyll molecule 24 . SPQ in the 2Fe-2S-bound state does not react with oxygen, providing a potential mechanism to control the release of superoxide from the Q p site 24 and regulate the activity of the LHCII kinase STN7 25 , which is proposed to bind to cytb 6 f between transmembrane helices F and H of subunit IV 26 . ...
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The cytochrome b6 f (cytb6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1,2,3. Electron transfer within cytb6 f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation². In higher plants, cytb6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses³. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 f monomer near the PQ oxidation site (Qp) adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis.
... He and his co-authors-Jonathan Townsend and George C. Pake-wrote in 1954: "In the light of the suggestion of Michaelis and others that free radicals occur as intermediates in biological electron-transfer processes, it is of considerable interest to investigate the association between free radical content and metabolic activity" ( [45], p. 690). When reading this paper nowadays, it appears striking up to date and sharpens the view of the engagement of unpaired electrons in the metabolic processes, in particular in two principle processes of life intensively studied at present with EPR: photosynthesis [54,55] and oxygen respiration [56][57][58][59][60]. This is exactly what Barry Commoner wrote about when analysing his EPR spectra. ...
... 1. Free radicals and hydrated electrons [89]-products of metabolism [45,[53][54][55][56][57][58][59][60], products of action of radiation (including UV) [63] on other "-omes", and products of secondary reactions of the primary products with other "-omes" [ [45,115,116] 5. Some defects in crystals of biominerals [66,117] 6. Donor-acceptor complexes with strong charge transfer, e.g. in photosynthetic centres [118,119]. ...
Chapter
EPR spectroscopy and imaging remain still a branch of physical sciences, while its application to biology and medicine is wide and valuable. The EPR-measureable species create in biological systems important and well-defined pool, unique on the background of metabolomes and metallomes. At the same time, they seem to be these constituents of the system, which make it deserving to be called “alive”. I propose to coin the phrase “the paramagnetome” to name this pool and to replace the common, descriptive name of “biologically and medically-oriented EPR/ESR spectroscopy” with “paramagnetomics”, per analogiam to other “-omes” and “-omics”. A short characteristic of these two newly defined terms is proposed, which makes the paramagnetomics closely related to other branches of the systems biology. Relations to the problems of genomics and the central problems of molecular genetics, genetic information, as well as biological evolution are also discussed. The position of EPR spectroscopy and a special role that it plays in defining and understanding the phenomenon of life seem to accomplish the long expected establishing the paramagnetomics and research on paramagnetomes as a branch of biology.
... The genome of Thermus thermophilus, for example, encodes both a bc ⁠ 1 complex [6] and an ACIII. Whereas there have been many studies of the bc ⁠ 1 /b ⁠ 6 f complexes [7][8][9], including X-ray structures [10][11][12][13][14], little is known about the ACIII complexes [15,16]. Although the bc ⁠ 1 /b ⁠ 6 f complexes and ACIII catalyze the same reaction, there is no structural similarity between the sub units, and the two families have independent phylogenetic histories [2]. ...
... Both reactions proceed as two concerted processes: (1) PQH 2 + ISP ox → SPQ + ISP red , and (2) SPQ + (b L 6 ) ox → PQ + (b L 6 ) red , where SPQ denotes the semiquinone form of PQ (semi-plastoquinone). The concerted (almost simultaneous) mechanism of PQH 2 oxidation precludes the accumulation of SPQ, thereby avoiding the formation of harmful superoxide radicals (SPQ + O 2 → PQ + O ⋅− 2 ) (Cape et al. 2006(Cape et al. , 2007Sarewicz et al. 2017). The oxidation of PQH 2 strongly depends on the possibility of SPQ to be oxidized by heme b L 6 . ...
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In plants, the short-term regulation (STR, seconds to minute time scale) of photosynthetic apparatus is associated with the energy-dependent control in the chloroplast electron transport, the distribution of light energy between photosystems (PS) II and I, activation/deactivation of the Calvin–Benson cycle (CBC) enzymes, and relocation of chloroplasts within the plant cell. In this work, using a dual-PAM technique for measuring the time-courses of P700 photooxidation and Chl a fluorescence, we have investigated the STR events in Tradescantia fluminensis leaves. The comparison of Chl a fluorescence and $${\text{P}}_{{700}}^{+}$$ induction allowed us to investigate the contribution of the trans-thylakoid pH difference (ΔpH) to the STR events. Two parameters were used as the indicators of ΔpH generation: pH-dependent component of non-photochemical quenching of Chl a fluorescence, and pHin-dependent rate of electron transfer from plastoquinol (PQH2) to $${\text{P}}_{{700}}^{+}$$ (via the Cyt b6f complex and plastocyanin). In dark-adapted leaves, kinetics of $${\text{P}}_{{700}}^{+}$$ induction revealed three phases. Initial phase is characterized by rapid electron flow to $${\text{P}}_{{700}}^{+}$$ (τ1/2 ~ 5–10 ms), which is likely related to cyclic electron flow around PSI, while the outflow of electrons from PSI is restricted by slow consumption of NADPH in the CBC. The light-induced generation of ΔpH and activation of the CBC promote photooxidation of P700 and concomitant retardation of $${\text{P}}_{{700}}^{+}$$ reduction (τ1/2 ~ 20 ms). Prolonged illumination induces additional slowing down of electron transfer to $${\text{P}}_{{700}}^{+}$$ (τ1/2 ≥ 30–35 ms). The latter effect is not accompanied by changes in the Chl a fluorescence parameters which are sensitive to ΔpH generation. We suggest the tentative explanation of the latter results by the reversal of Q-cycle, which causes the deceleration of PQH2 oxidation due to the back pressure of stromal reductants.