EPR characterization of ubisemiquinones and iron-sulfur cluster N2, central components of the energy coupling in the NADH-ubiquinone oxidoreductase (complex I) in situ

The proton-translocating NADH-ubiquinone oxidoreductase (complex I) is the largest and least understood respiratory complex. The intrinsic redox components (FMN and iron–sulfur clusters) reside in the promontory part of the complex. Ubiquinone is the most possible key player in proton-pumping reactions in the membrane part. Here we report the presence of three distinct semiquinone species in complex I in situ, showing widely different spin relaxation profiles. As our first approach, the semiquinone forms were trapped during the steady state NADH-ubiquinone-1 (Q₁) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQ_Nf (fast-relaxing component), SQ_Ns (slow-relaxing), and SQ_Nx (very slow relaxing). This indicates the presence of at least three different quinone-binding sites in complex I. In the current study, special attention was placed on the SQ_Nf, because of its high sensitivities to and to specific complex I inhibitors (rotenone and piericidin A) in a unique manner. Rotenone inhibits the forward electron transfer reaction more strongly than the reverse reaction, while piericidine A inhibits both reactions with a similar potency. Rotenone quenched the SQ_Nf signal at a much lower concentration than that required to quench the slower relaxing components (SQ_Ns and SQ_Nx). A close correlation was shown between the line shape alteration of the g = 2.05 signal of the cluster N2 and the quenching of the SQ_Nf signal, using two different experimental approaches: (1) changing the poise by the oligomycin titration which decreases proton leak across the SMP membrane; (2) inhibiting the reverse electron transfer with different concentrations of rotenone. These new experimental results further strengthen our earlier proposal that a direct spin-coupling occurs between SQ_Nf and cluster N2. We discuss the implications of these findings in connection with the energy coupling mechanism in complex I.     

EPR detection of two protein-associated ubiquinone components (SQNf and SQNs) in the membrane in situ and in proteoliposomes of isolated bovine heart complex I

The success of Sazanov's group in determining the X-ray structure of the whole bacterial complex I is a great contribution to the progress of complex I research. In this mini-review of 35 years' history of my laboratory and collaborators, we characterized the function of protein-associated semiquinone molecules in the proton-pumping mechanism in complex I (NADH-quinone oxidoreductase). We have constructed most of the frame work of our hypothesis, utilizing EPR techniques before the X-ray structures of complex I were reported by Sazanov's and Brandt's groups. One of the semiquinones (SQ_Nf) is extremely sensitive to a proton motive force imposed on the energy-transducing membrane, while the other (SQ_Ns) is insensitive. Their sensitivity to rotenone inhibition also differs. These differences were exploited using tightly coupled bovine heart submitochondrial particles with a high respiratory control ratio (> 8). We determined the distance between SQ_Nf and iron–sulfur cluster N2 on the basis of their direct spin–spin interaction. We are extending this line of work using reconstituted bovine heart complex I proteoliposomes which shows a respiratory control ratio > 5. Two frontier research groups support our view point based on their mutagenesis studies. High frequency (33.9 GHz; Q-band) EPR experiments appear to favor our two-semiquinone model. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Roles of semiquinone species in proton pumping mechanism by complex I

Complex I (NDH-1) translocates protons across the membrane using electron transfer energy. Two different coupling mechanisms are currently being discussed for complex I: direct (redox-driven) and indirect (conformation-driven). Semiquinone (SQ) intermediates are suggested to be key for the coupling mechanism. Recently, using progressive power saturation and simulation techniques, three distinct SQ species were resolved by EPR analysis of E. coli complex I reconstituted into proteoliposomes. The fast-relaxing SQ (SQ_Nf) signals completely disappeared in the presence of the uncoupler gramicidin D or the potent E. coli complex I inhibitor squamotacin. The slow-relaxing SQ (SQ_Ns) signals were insensitive to gramicidin D, but they were sensitive to squamotacin. The very slow-relaxing SQ (SQ_Nvs) signals were insensitive to both gramicidin D and squamotacin. Interestingly, no SQ_Ns signal was observed in the ΔNuoL mutant, which lacks transporter module subunits NuoL and NuoM. Furthermore, we sought out the effect of using menaquinone (which has a lower redox potential compared to that of ubiquinone) as an electron acceptor on the proton pumping stoichiometry by in vitro reconstitution experiments with ubiquinone-rich or menaquinone-rich double knock-out membrane vesicles, which contain neither complex I nor NDH-2 (non-proton translocating NADH dehydrogenase). No difference in the proton pumping stoichiometry between menaquinone and ubiquinone was observed in the ΔNuoL and D178N mutants, which are considered to lack the indirect proton pumping mechanism. However, the proton pumping stoichiometry with menaquinone decreased by half in the wild-type. The roles and relationships of SQ intermediates in the coupling mechanism of complex I are discussed.     

Electron sweep across four b-hemes of cytochrome bc1 revealed by unusual paramagnetic properties of the Qi semiquinone intermediate

Dimeric cytochromes bc are central components of photosynthetic and respiratory electron transport chains. In their catalytic core, four hemes b connect four quinone (Q) binding sites. Two of these sites, Q_i sites, reduce quinone to quinol (QH₂) in a step-wise reaction, involving a stable semiquinone intermediate (SQ_i). However, the interaction of the SQ_i with the adjacent hemes remains largely unexplored. Here, by revealing the existence of two populations of SQ_i differing in paramagnetic relaxation, we present a new mechanistic insight into this interaction. Benefiting from a clear separation of these SQ_i species in mutants with a changed redox midpoint potential of hemes b, we identified that the fast-relaxing SQ_i (SQ_iF) corresponds to the form magnetically coupled with the oxidized heme b_H (the heme b adjacent to the Q_i site), while the slow-relaxing SQ_i (SQ_iS) reflects the form present alongside the reduced (and diamagnetic) heme b_H. This so far unreported SQ_iF calls for a reinvestigation of the thermodynamic properties of SQ_i and the Q_i site. The existence of SQ_iF in the native enzyme reveals a possibility of an extended electron equilibration within the dimer, involving all four hemes b and both Q_i sites. This substantiates the predicted earlier electron transfer acting to sweep the b-chain of reduced hemes b to diminish generation of reactive oxygen species by cytochrome bc₁. In analogy to the Q_i site, we anticipate that the quinone binding sites in other enzymes may contain yet undetected semiquinones which interact magnetically with oxidized hemes upon progress of catalytic reactions.     

Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc1: Implications for the mechanism of superoxide production

In addition to its bioenergetic function of building up proton motive force, cytochrome bc₁ can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQ_o) formed at the quinone oxidation/reduction Q_o site (Q_o) as a result of single-electron oxidation of quinol by the iron-sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme b_L (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc₁ that severely impeded the oxidation of FeS by cytochrome c₁, changed density of FeS around Q_o by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Q_o and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQ_o that present a risk of generation of superoxide by cytochrome bc₁. Isolation of this reaction sequence from multiplicity of possible reactions at Q_o helps to better understand conditions under which complex III might contribute to ROS generation in vivo.     

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa₃) is coupled to translocation of H⁺ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa₃ is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational “strain” in the cytochrome aa₃ complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.     

Charge polarization imposed by the binding site facilitates enzymatic redox reactions of quinone

Quinone reduction site (Q_i) of cytochrome bc₁ represents one of the canonical sites used to explore the enzymatic redox reactions involving semiquinone (SQ) states. However, the mechanism by which Q_i allows the completion of quinone reduction during the sequential transfers of two electrons from the adjacent heme b_H and two protons to C1- and C4-carbonyl remains unclear. Here we established that the SQ coupled to an oxidized heme b_H is a dominant intermediate of catalytic forward reaction and, contrary to the long-standing assumption, represents a significant population of SQ detected across pH 5–9. The pH dependence of its redox midpoint potential implicated proton exchange with histidine. Complementary quantum mechanical calculations revealed that the SQ anion formed after the first electron transfer undergoes charge and spin polarization imposed by the electrostatic field generated by histidine and the aspartate/lysine pair interacting with the C4- and C1-carbonyl, respectively. This favors a barrierless proton exchange between histidine and the C4-carbonyl, which continues until the second electron reaches the SQ_i. Inversion of charge polarization facilitates the uptake of the second proton by the C1-carbonyl. Based on these findings we developed a comprehensive scheme for electron and proton transfers at Q_i featuring the equilibration between the anionic and neutral states of SQ_i as means for a leak-proof stabilization of the radical intermediate. The key catalytic role of the initial charge/spin polarization of the SQ anion at the active site, inherent to the proposed mechanism, may also be applicable to the other quinone oxidoreductases.     

Dissecting the pattern of proton release from partial process involved in ubihydroquinone oxidation in the Q-cycle

A key feature of the modified Q-cycle of the cytochrome bc₁ and related complexes is a bifurcation of QH₂ oxidation involving electron transfer to two different acceptor chains, each coupled to proton release. We have studied the kinetics of proton release in chromatophore vesicles from Rhodobacter sphaeroides, using the pH-sensitive dye neutral red to follow pH changes inside on activation of the photosynthetic chain, focusing on the bifurcated reaction, in which 4H⁺are released on complete turnover of the Q-cycle (2H⁺/ubiquinol (QH₂) oxidized). We identified different partial processes of the Q_o-site reaction, isolated through use of specific inhibitors, and correlated proton release with electron transfer processes by spectrophotometric measurement of cytochromes or electrochromic response. In the presence of myxothiazol or azoxystrobin, the proton release observed reflected oxidation of the Rieske iron?sulfur protein. In the absence of Q_o-site inhibitors, the pH change measured represented the convolution of this proton release with release of protons on turnover of the Q_o-site, involving formation of the ES-complex and oxidation of the semiquinone intermediate. Turnover also regenerated the reduced iron-sulfur protein, available for further oxidation on a second turnover. Proton release was well-matched with the rate limiting step on oxidation of QH₂ on both turnovers. However, a minor lag in proton release found at pH?7 but not at pH?8 might suggest that a process linked to rapid proton release on oxidation of the intermediate semiquinone involves a group with a pK in that range.     

The plastoquinone pool as possible hydrogen pump in photosynthesis

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H⁺ + e^?) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H⁺ uptake and the internal H⁺ release led to the following conclusions:(1) A stoichiometric coupling of 1 : 1 : 1 between the external H⁺ uptake, the electron translocation through the plastoquinone pool and the internal H⁺ release (corrected for H⁺ release due to H₂O oxidation) is valid (pH_out = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation.These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H⁺ + e^?) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.     

Electron acceptors of bacterial photosynthetic reaction centers II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex

The photoreduction of ubiquinone in the electron acceptor complex (Q₁Q₁₁) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H⁺ in the reduction was revealed by the pH dependence of the electron transfer events and by net H⁺ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash Q₁₁ receives an electron via Q₁ to form a stable ubisemiquinone anion (Q?^?₁₁); the second flash generates Q?^?₁. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H⁺, to produce Q₁₁H₂. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H⁺ uptake. Above pH 6 there is a progressive increase in H⁺ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H⁺ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to Q?^?₁₁. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H⁺ binding electron transfer following the second flash.     

On the mechanism of photosynthetic electron transfer in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b₅₀ and cytochrome b_?90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q^?_I) and the secondary acceptor ubiquinone (Q_II) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b₅₀ reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b₅₀ reduction increased about 10-fold as the E_h(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with E_m(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100–500 μs before cytochrome b₅₀ reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q^?_I and Q_II. It was an effective inhibitor of cytochrome b₅₀ photoreduction at E_h(7.0) 390 mV, but not at E_h(7.0) 100 mV. At the latter E_h, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q^?_I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At E_h(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b₅₀ reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b₅₀ oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high E_h values. However, at lower redox potentials, cytochrome b₅₀ oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b_?90 in the rapid reactions of the cyclic electron transfer chain     

The Q-cycle reviewed: How well does a monomeric mechanism of the bc1 complex account for the function of a dimeric complex?

Recent progress in understanding the Q-cycle mechanism of the bc₁ complex is reviewed. The data strongly support a mechanism in which the Q_o-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron–sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe–2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q_o-site, and the reduced iron–sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c₁ and liberate the H⁺. When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O₂ is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b_L to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b_L to enhance the rate constant. The acceptor reactions at the Q_i-site are still controversial, but likely involve a “two-electron gate” in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b₁₅₀ phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed.The mechanism discussed is applicable to a monomeric bc₁ complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b_L hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.     

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ−B and free energy and kinetic relations between Q−AQB and QAQ−B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: We found that the charge recombination pathway of D⁺Q_AQ^?_B proceeds via the intermediate state D⁺Q^?_AQ_B, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product k_AD-times the fraction of reaction centers in the Q^?_AQ_B state) with the observed recombination rate, k^obs_{D⁺ →D}. The kinetic measurements were used to obtain the pH dependence (6.1 ? pH ? 11.7) of the free energy difference between the states Q^?_AQ_B and Q_AQ^?_B. At low pH (less than 9) Q_AQ^?_B is stabilized relative to Q^?_AQ_B by 67 meV, whereas at high pH Q^?_AQ_B is energetically favored. Both Q^?_A and Q^?_B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q^?_B provides the driving force for the forward electron transfer.     

Hydrated electrons and prebiotic evolution

It is proposed that reactions of the hydrated electron e_aq^? in the “primordial soup” were an evolutionary pressure which selected biopolyelectrolytes, coacervates, “organelles” and lipid-membrane-bound structures of a pattern found in the biosphere today.The two physical restrictions of e_aq^? reactivity which determined its evolutionary specificity are (a) its exclusion from polyanion domains in Donman fashion, and (b) its inability to solvate in alkanes. Thus, (1) polyanions (as opposed to polyanions) and (2) lipid-membrane-bound (as opposed to free) structures are protected from e_aq^? reactivity and degradation. The polyanionic nature of the current biosphere, and the prevalence of anionic polyanion-polycation complexes (such as ribosomes and chromatin), are attributed to (1). (2) co-operated in protecting the products of (1) as salinity in the “primordial soup” increased and (a) was no longer important. The evolution of DNA, and the existence of electron transport mechanisms are seen as logical extensions of the properties of e_aq^? Experimental evidence is already available to support all the postulated processes.     

How rapid are the internal reactions of the ubiquinol:cytochrome c 2 oxidoreductase?

The temperature dependence of the partial reactions leading to turn-over of the UQH₂:cyt c ₂ oxidoreductase of Rhodobacter sphaeroides have been studied. The redox properties of the cytochrome components show a weak temperature dependence over the range 280–330 K, with coefficients of about 1 m V per degree; our results suggest that the other components show similar dependencies, so that no significant change in the gradient of standard free-energy between components occurs over this temperature range. The rates of the reactions of the high potential chain (the Rieske iron sulfur center, cytochromes c ₁ and c ₂, reaction center primary donor) show a weak temperature dependence, indicating an activation energy < 8 kJ per mole for electron transfer in this chain. The oxidation of ubiquinol at the Q_z-site of the complex showed a strong temperature dependence, with an activation energy of about 32 kJ mole^–1. The electron transfer from cytochrome b-566 to cytochrome b-561 was not rate determining at any temperature, and did not contribute to the energy barrier. The activation energy of 32 kJ mole^–1 for quinol oxidation was the same for all states of the quinone pool (fully oxidized, partially reduced, or fully reduced before the flash). We suggest that the activation barrier is in the reaction by which ubiquinol at the catalytic site is oxidized to semiquinone. The most economical scheme for this reaction would have the semiquinone intermediate at the energy level indicated by the activation barrier. We discuss the plausibility of this simple model, and the values for rate constants, stability constant, the redox potentials of the intermediate couples, and the binding constant for the semiquinone, which are pertinent to the mechanism of the ubiquinol oxidizing site.Abbreviations (BChl)₂ P870, primary donor of the photochemical reaction center - b/c ₁ complex ubiquinol: cytochrome c ₂ oxidoreductase - cyt b _H cytochrome b-561 or higher potential cytochrome b - cyt b _L cytochrome b-566, or low potential cytochrome b - cyt c ₁, cyt c ₂, cyt c _t cytochromes c ₁ and c ₂, and total cytochrome c (cyt c ₁ and cyt c ₂) - Fe.S Rieske-type iron sulfur center, Q - QH₂ ubiquinone, ubiquinol - Q_z, Q_zH₂, Q_z ^– ubiquinone, ubiquinol, and semiquinone anion of ubiquinone, bound at quinol oxidizing site - Q_z-site ubiquinol oxidizing site (also called Q_o-(outside) - Q_o (Oxidizing) - Q_P (Positive proton potential) site) - Q_c-site uubiquinone reductase site (also called the Q_i-(inside) - Q_R (Reducing), or - Q_N (Negative proton potential) site) - UHDBT 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazol     

The pathway of electrons through QH2:cytochrome c oxidoreductase studied by pre-steady-state kinetics

(1) The kinetic behaviour of the prosthetic groups and the semiquinones in QH₂:cytochrome c oxidoreductase has been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) In the absence of antimycin, cytochrome b-562 is reduced in two phases separated by a lag time. The initial very rapid reduction phase, that coincides with the formation of the antimycin-sensitive Q^?_in, is ascribed to high-potential cytochrome b-562 and the slow phase to low-potential cytochrome b-562. The two cytochromes are present in a 1:1 molar ratio. The lag time between the two reduction phases decreases with increasing pH. Both the [2 Fe-2 S] clusters and cytochrome c₁ are reduced monophasically under these conditions, but at a rate lower than that of the initial rapid reduction of cytochrome b-562. (3) In the presence of antimycin and absence of oxidant, cytochrome b-562 is still reduced biphasically, but there is no lag between the two phases. No Q^?_in is formed and both the Fe-S clusters and cytochrome c₁ are reduced biphasically, one-half being reduced at the same rate as in the absence of antimycin and the other half 10-times slower. (4) In the presence of antimycin and oxidant, the recently described antimycin-insensitive species of semiquinone anion, Q^?_out (De Vries, S., Albracht, S.P.J., Berden, J.A. and Slater, E.C. (1982) J. Biol. Chem. 256, 11996–11998) is formed at the same rate as that of the reduction of all species of cytochrome b. In this case cytochrome b is reduced in a single phase. (5) The reversible change of the line shape of the EPR spectrum of the [2Fe-2S] cluster 1 is caused by ubiquinone bound in the vicinity of this cluster. (6) The experimental results are consistent with the basic principles of the Q cycle. Because of the multiplicity, stoicheiometry and heterogeneous kinetics of the prosthetic groups, a Q cycle model describing the pathway of electrons through a dimeric QH₂:cytochrome c oxidoreductase is proposed.     

Inhibition of cytochrome b563-oxidation by triorganotins in spinach chloroplasts

The triorganotin compounds triphenyltin chloride and tributyltin chloride have been known as inhibitors of the transmembrane proton channel forming F₀-domain of ATPases at micromolar concentrations. We show that these compounds at higher concentrations (10–100 µM) also inhibit uncoupled electron transport in chloroplasts within the low potential chain of the cytochrome bf complex. They cause high levels of transiently reduced cytochrome b563 as they decelerate the reoxidation process in flash illuminated chloroplasts. At the same time they slow down the flash induced slow electrogenic step generated at the cytochrome bf complex. The inhibitory effect of triphenyltin chloride on cytochrome b563 turnover in chloroplasts is comparable to that of the Q_n-inhibitor MOA-stilbene, with even less side effects on the high potential chain. Studies on the isolated bf complex suggest different binding sites for triorganotins and the quinone analogue type Q_n-inhibitors. The results are interpreted within the framework of the modified Q-cycle model by a putative organotin sensitive proton translocating site which enables proton transfer from the outer aqueous face of the membrane to the hydrophobic quinone reduction site within the complex. Hence, cytochrome b563 oxidation and plastoquinone reduction may be inhibited as a consequence of proton transfer being suppressed by triorganotins. In analogy, the previously described inhibitory effect of Val/K⁺ at the n-side of the cytochrome bf complex [Klughammer and Schreiber (1993) FEBS 336: 491–495] may be rationalised by binding of the cyclic depsipeptide at the entrance of the proton path to the Q_n-site.     

The proton pumping mechanism of the bc1 complex

The bc₁ complex is a proton pump of the mitochondrial electron transport chain which transfers electrons from ubiquinol to cytochrome c. It operates via the modified Q cycle in which the two electrons from oxidation of ubiquinol at the Q_o center are bifurcated such that the first electron is passed to Cytc via an iron sulfur center and c₁ whereas the second electron is passed across the membrane by b_L and b_H to reduce ubiquinone at the Q_i center. Proton pumping occurs because oxidation of ubiquinol at the Q_o center releases protons to the P-side and reduction of ubiquinone at the Q_i center takes up protons from the N-side. However, the mechanisms which prevent the thermodynamically more favorable short circuit reactions and so ensure precise bifurcation and proton pumping are not known. Here we use statistical thermodynamics to show that reaction steps that originate from high energy states cannot support high flux even when they have large rate constants. We show how the chemistry of ubiquinol oxidation and the structure of the Q_o site can result in free energy profiles that naturally suppress flux through the short circuit pathways while allowing high rates of bifurcation. These predictions are confirmed through in-silico simulations using a Markov state model.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

Kinetics of photosystem II electron transport: a mathematical analysis based on chlorophyll fluorescence induction

The OJDIP rise in chlorophyll fluorescence during induction at different light intensities was mathematically modeled using 24 master equations describing electron transport through photosystem II (PSII) plus ordinary differential equations for electron budgets in plastoquinone, cytochrome f, plastocyanin, photosystem I, and ferredoxin. A novel feature of the model is consideration of electron in- and outflow budgets resulting in changes in redox states of Tyrosine Z, P680, and Q_A as sole bases for changes in fluorescence yield during the transient. Ad hoc contributions by transmembrane electric fields, protein conformational changes, or other putative quenching species were unnecessary to account for primary features of the phenomenon, except a peculiar slowdown of intra-PSII electron transport during induction at low light intensities. The lower than F _m post-flash fluorescence yield F _f was related to oxidized tyrosine Z. The transient J peak was associated with equal rates of electron arrival to and departure from Q_A and requires that electron transfer from Q_A ^? to Q_B be slower than that from Q_A ^? to Q_B ^?. Strong quenching by oxidized P680 caused the dip D. Reduced plastoquinone, a competitive product inhibitor of PSII, blocked electron transport proportionally with its concentration. Electron transport rate indicated by fluorescence quenching was faster than the rate indicated by O₂ evolution, because oxidized donor side carriers quench fluorescence but do not transport electrons. The thermal phase of the fluorescence rise beyond the J phase was caused by a progressive increase in the fraction of PSII with reduced Q_A and reduced donor side.