The Experts below are selected from a list of 279 Experts worldwide ranked by ideXlab platform
David Kramer - One of the best experts on this subject based on the ideXlab platform.
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a caged destabilized free radical intermediate in the Q Cycle
ChemBioChem, 2013Co-Authors: Preethi Vennam, David Kramer, Nicholas Fisher, Matthew D Krzyaniak, Michael K BowmanAbstract:The Rieske/cytochrome b complexes, also known as cytochrome bc complexes, catalyze a uniQue oxidant-induced reduction reaction at their Quinol oxidase (Qo ) sites, in which substrate hydroQuinone reduces two distinct electron transfer chains, one through a series of high-potential electron carriers, the second through low-potential cytochrome b. This reaction is a critical step in energy storage by the Q-Cycle. The semiQuinone intermediate in this reaction can reduce O2 to produce deleterious superoxide. It is yet unknown how the enzyme controls this reaction, though numerous models have been proposed. In previous work, we trapped a Q-Cycle semiQuinone anion intermediate, termed SQo , in bacterial cytochrome bc1 by rapid freeze-Quenching. In this work, we apply pulsed-EPR techniQues to determine the location and properties of SQo in the mitochondrial complex. In contrast to semiQuinone intermediates in other enzymes, SQo is not thermodynamically stabilized, and can even be destabilized with respect to solution. It is trapped in Qo at a site that is distinct from previously described inhibitor-binding sites, yet sufficiently close to cytochrome bL to allow rapid electron transfer. The binding site and EPR analyses show that SQo is not stabilized by hydrogen bonds to proteins. The formation of SQo involves "stripping" of both substrate -OH protons during the initial oxidation step, as well as conformational changes of the semiQuinone and Qo proteins. The resulting charged radical is kinetically trapped, rather than thermodynamically stabilized (as in most enzymatic semiQuinone species), conserving redox energy to drive electron transfer to cytochrome bL while minimizing certain Q-Cycle bypass reactions, including oxidation of prereduced cytochrome b and reduction of O2 .
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a semiQuinone intermediate generated at the Qo site of the cytochrome bc1 complex importance for the Q Cycle and superoxide production
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Jonathan L Cape, Michael K Bowman, David KramerAbstract:The cytochrome bc1 and related complexes are essential energy-conserving components of mitochondrial and bacterial electron transport chains. They orchestrate a complex seQuence of electron and proton transfer reactions resulting in the oxidation of Quinol, the reduction of a mobile electron carrier, and the translocation of protons across the membrane to store energy in an electrochemical proton gradient. The enzyme can also catalyze substantial rates of superoxide production, with deleterious physiological conseQuences. Progress on understanding these processes has been hindered by the lack of observable enzymatic intermediates. We report the first direct detection of a semiQuinone radical generated by the Qo site using continuous wave and pulsed EPR spectroscopy. The radical is a ubisemiQuinone anion and is sensitive to both specific inhibitors and mutations within the Qo site as well as O2, suggesting that it is the elusive intermediate responsible for superoxide production. Paramagnetic interactions show that the new semiQuinone species is buried in the protein, probably in or near the Qo site but not strongly interacting with the 2Fe2S cluster. The semiQuinone is substoichiometric, even with conditions optimized for its accumulation, consistent with recently proposed models where the semiQuinone is destabilized to limit superoxide production. The discovery of this intermediate provides a critical tool to directly probe the elusive chemistry that takes place within the Qo site.
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similar transition states mediate the Q Cycle and superoxide production by the cytochrome bc1 complex
Journal of Biological Chemistry, 2006Co-Authors: Isaac P Forquer, Michael K Bowman, Bernard L Trumpower, Raul Covian, David KramerAbstract:Abstract The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria play critical roles in their respective electron transport chains. The Quinol oxidase (Qo) site in this complex oxidizes a hydroQuinone (Quinol), reducing two one-electron carriers, a low potential cytochrome b heme and the “Rieske” iron-sulfur cluster. The overall electron transfer reactions are coupled to transmembrane translocation of protons via a “Q-Cycle” mechanism, which generates proton motive force for ATP synthesis. Since semiQuinone intermediates of Quinol oxidation are generally highly reactive, one of the key Questions in this field is: how does the Qo site oxidize Quinol without the production of deleterious side reactions including superoxide production? We attempt to test three possible general models to account for this behavior: 1) The Qo site semiQuinone (or Quinol-imidazolate complex) is unstable and thus occurs at a very low steady-state concentration, limiting O2 reduction; 2) the Qo site semiQuinone is highly stabilized making it unreactive toward oxygen; and 3) the Qo site catalyzes a Quantum mechanically coupled two-electron/two-proton transfer without a semiQuinone intermediate. Enthalpies of activation were found to be almost identical between the uninhibited Q-Cycle and superoxide production in the presence of antimycin A in wild type. This behavior was also preserved in a series of mutants with altered driving forces for Quinol oxidation. Overall, the data support models where the rate-limiting step for both Q-Cycle and superoxide production is essentially identical, consistent with model 1 but reQuiring modifications to models 2 and 3.
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understanding the cytochrome bc complexes by what they don t do the Q Cycle at 30
Trends in Plant Science, 2006Co-Authors: Jonathan L Cape, Michael K Bowman, David KramerAbstract:The cytochrome (cyt) bc 1 , b 6 f and related complexes are central components of the respiratory and photosynthetic electron transport chains. These complexes carry out an extraordinary seQuence of electron and proton transfer reactions that conserve redox energy in the form of a trans-membrane proton motive force for use in synthesizing ATP and other processes. Thirty years ago, Peter Mitchell proposed a general turnover mechanism for these complexes, which he called the Q-Cycle. Since that time, many opposing schemes have challenged the Q-Cycle but, with the accumulation of large amounts of biochemical, kinetic, thermodynamic and high-resolution structural data, the Q-Cycle has triumphed as the accepted model, although some of the intermediate steps are poorly understood and still controversial. One of the major research Questions concerning the cyt bc 1 and b 6 f complexes is how these enzymes suppress deleterious and dissipative side reactions. In particular, most Q-Cycle models involve reactive semiQuinone radical intermediates that can reduce O 2 to superoxide and lead to cellular oxidative stress. Current models to explain the avoidance of side reactions involve unprecedented or unusual enzyme mechanisms, the testing of which will involve new theoretical and experimental approaches.
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the respiratory substrate rhodoQuinol induces Q Cycle bypass reactions in the yeast cytochrome bc1 complex mechanistic and physiological implications
Journal of Biological Chemistry, 2005Co-Authors: Jonathan L Cape, Michael K Bowman, Jeff Strahan, Michael J Lenaeus, Brook A Yuknis, Jennifer N Shepherd, David KramerAbstract:Abstract The mitochondrial cytochrome bc1 complex catalyzes the transfer of electrons from ubiQuinol to cyt c while generating a proton motive force for ATP synthesis via the “Q-Cycle” mechanism. Under certain conditions electron flow through the Q-Cycle is blocked at the level of a reactive intermediate in the Quinol oxidase site of the enzyme, resulting in “bypass reactions,” some of which lead to superoxide production. Using analogs of the respiratory substrates ubiQuinol-3 and rhodoQuinol-3, we show that the relative rates of Q-Cycle bypass reactions in the Saccharomyces cerevisiae cyt bc1 complex are highly dependent by a factor of up to 100-fold on the properties of the substrate Quinol. Our results suggest that the rate of Q-Cycle bypass reactions is dependent on the steady state concentration of reactive intermediates produced at the Quinol oxidase site of the enzyme. We conclude that normal operation of the Q-Cycle reQuires a fairly narrow window of redox potentials with respect to the Quinol substrate to allow normal turnover of the complex while preventing potentially damaging bypass reactions.
Michael K Bowman - One of the best experts on this subject based on the ideXlab platform.
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a caged destabilized free radical intermediate in the Q Cycle
ChemBioChem, 2013Co-Authors: Preethi Vennam, David Kramer, Nicholas Fisher, Matthew D Krzyaniak, Michael K BowmanAbstract:The Rieske/cytochrome b complexes, also known as cytochrome bc complexes, catalyze a uniQue oxidant-induced reduction reaction at their Quinol oxidase (Qo ) sites, in which substrate hydroQuinone reduces two distinct electron transfer chains, one through a series of high-potential electron carriers, the second through low-potential cytochrome b. This reaction is a critical step in energy storage by the Q-Cycle. The semiQuinone intermediate in this reaction can reduce O2 to produce deleterious superoxide. It is yet unknown how the enzyme controls this reaction, though numerous models have been proposed. In previous work, we trapped a Q-Cycle semiQuinone anion intermediate, termed SQo , in bacterial cytochrome bc1 by rapid freeze-Quenching. In this work, we apply pulsed-EPR techniQues to determine the location and properties of SQo in the mitochondrial complex. In contrast to semiQuinone intermediates in other enzymes, SQo is not thermodynamically stabilized, and can even be destabilized with respect to solution. It is trapped in Qo at a site that is distinct from previously described inhibitor-binding sites, yet sufficiently close to cytochrome bL to allow rapid electron transfer. The binding site and EPR analyses show that SQo is not stabilized by hydrogen bonds to proteins. The formation of SQo involves "stripping" of both substrate -OH protons during the initial oxidation step, as well as conformational changes of the semiQuinone and Qo proteins. The resulting charged radical is kinetically trapped, rather than thermodynamically stabilized (as in most enzymatic semiQuinone species), conserving redox energy to drive electron transfer to cytochrome bL while minimizing certain Q-Cycle bypass reactions, including oxidation of prereduced cytochrome b and reduction of O2 .
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a semiQuinone intermediate generated at the Qo site of the cytochrome bc1 complex importance for the Q Cycle and superoxide production
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Jonathan L Cape, Michael K Bowman, David KramerAbstract:The cytochrome bc1 and related complexes are essential energy-conserving components of mitochondrial and bacterial electron transport chains. They orchestrate a complex seQuence of electron and proton transfer reactions resulting in the oxidation of Quinol, the reduction of a mobile electron carrier, and the translocation of protons across the membrane to store energy in an electrochemical proton gradient. The enzyme can also catalyze substantial rates of superoxide production, with deleterious physiological conseQuences. Progress on understanding these processes has been hindered by the lack of observable enzymatic intermediates. We report the first direct detection of a semiQuinone radical generated by the Qo site using continuous wave and pulsed EPR spectroscopy. The radical is a ubisemiQuinone anion and is sensitive to both specific inhibitors and mutations within the Qo site as well as O2, suggesting that it is the elusive intermediate responsible for superoxide production. Paramagnetic interactions show that the new semiQuinone species is buried in the protein, probably in or near the Qo site but not strongly interacting with the 2Fe2S cluster. The semiQuinone is substoichiometric, even with conditions optimized for its accumulation, consistent with recently proposed models where the semiQuinone is destabilized to limit superoxide production. The discovery of this intermediate provides a critical tool to directly probe the elusive chemistry that takes place within the Qo site.
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similar transition states mediate the Q Cycle and superoxide production by the cytochrome bc1 complex
Journal of Biological Chemistry, 2006Co-Authors: Isaac P Forquer, Michael K Bowman, Bernard L Trumpower, Raul Covian, David KramerAbstract:Abstract The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria play critical roles in their respective electron transport chains. The Quinol oxidase (Qo) site in this complex oxidizes a hydroQuinone (Quinol), reducing two one-electron carriers, a low potential cytochrome b heme and the “Rieske” iron-sulfur cluster. The overall electron transfer reactions are coupled to transmembrane translocation of protons via a “Q-Cycle” mechanism, which generates proton motive force for ATP synthesis. Since semiQuinone intermediates of Quinol oxidation are generally highly reactive, one of the key Questions in this field is: how does the Qo site oxidize Quinol without the production of deleterious side reactions including superoxide production? We attempt to test three possible general models to account for this behavior: 1) The Qo site semiQuinone (or Quinol-imidazolate complex) is unstable and thus occurs at a very low steady-state concentration, limiting O2 reduction; 2) the Qo site semiQuinone is highly stabilized making it unreactive toward oxygen; and 3) the Qo site catalyzes a Quantum mechanically coupled two-electron/two-proton transfer without a semiQuinone intermediate. Enthalpies of activation were found to be almost identical between the uninhibited Q-Cycle and superoxide production in the presence of antimycin A in wild type. This behavior was also preserved in a series of mutants with altered driving forces for Quinol oxidation. Overall, the data support models where the rate-limiting step for both Q-Cycle and superoxide production is essentially identical, consistent with model 1 but reQuiring modifications to models 2 and 3.
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understanding the cytochrome bc complexes by what they don t do the Q Cycle at 30
Trends in Plant Science, 2006Co-Authors: Jonathan L Cape, Michael K Bowman, David KramerAbstract:The cytochrome (cyt) bc 1 , b 6 f and related complexes are central components of the respiratory and photosynthetic electron transport chains. These complexes carry out an extraordinary seQuence of electron and proton transfer reactions that conserve redox energy in the form of a trans-membrane proton motive force for use in synthesizing ATP and other processes. Thirty years ago, Peter Mitchell proposed a general turnover mechanism for these complexes, which he called the Q-Cycle. Since that time, many opposing schemes have challenged the Q-Cycle but, with the accumulation of large amounts of biochemical, kinetic, thermodynamic and high-resolution structural data, the Q-Cycle has triumphed as the accepted model, although some of the intermediate steps are poorly understood and still controversial. One of the major research Questions concerning the cyt bc 1 and b 6 f complexes is how these enzymes suppress deleterious and dissipative side reactions. In particular, most Q-Cycle models involve reactive semiQuinone radical intermediates that can reduce O 2 to superoxide and lead to cellular oxidative stress. Current models to explain the avoidance of side reactions involve unprecedented or unusual enzyme mechanisms, the testing of which will involve new theoretical and experimental approaches.
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the respiratory substrate rhodoQuinol induces Q Cycle bypass reactions in the yeast cytochrome bc1 complex mechanistic and physiological implications
Journal of Biological Chemistry, 2005Co-Authors: Jonathan L Cape, Michael K Bowman, Jeff Strahan, Michael J Lenaeus, Brook A Yuknis, Jennifer N Shepherd, David KramerAbstract:Abstract The mitochondrial cytochrome bc1 complex catalyzes the transfer of electrons from ubiQuinol to cyt c while generating a proton motive force for ATP synthesis via the “Q-Cycle” mechanism. Under certain conditions electron flow through the Q-Cycle is blocked at the level of a reactive intermediate in the Quinol oxidase site of the enzyme, resulting in “bypass reactions,” some of which lead to superoxide production. Using analogs of the respiratory substrates ubiQuinol-3 and rhodoQuinol-3, we show that the relative rates of Q-Cycle bypass reactions in the Saccharomyces cerevisiae cyt bc1 complex are highly dependent by a factor of up to 100-fold on the properties of the substrate Quinol. Our results suggest that the rate of Q-Cycle bypass reactions is dependent on the steady state concentration of reactive intermediates produced at the Quinol oxidase site of the enzyme. We conclude that normal operation of the Q-Cycle reQuires a fairly narrow window of redox potentials with respect to the Quinol substrate to allow normal turnover of the complex while preventing potentially damaging bypass reactions.
Bernard L Trumpower - One of the best experts on this subject based on the ideXlab platform.
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similar transition states mediate the Q Cycle and superoxide production by the cytochrome bc1 complex
Journal of Biological Chemistry, 2006Co-Authors: Isaac P Forquer, Michael K Bowman, Bernard L Trumpower, Raul Covian, David KramerAbstract:Abstract The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria play critical roles in their respective electron transport chains. The Quinol oxidase (Qo) site in this complex oxidizes a hydroQuinone (Quinol), reducing two one-electron carriers, a low potential cytochrome b heme and the “Rieske” iron-sulfur cluster. The overall electron transfer reactions are coupled to transmembrane translocation of protons via a “Q-Cycle” mechanism, which generates proton motive force for ATP synthesis. Since semiQuinone intermediates of Quinol oxidation are generally highly reactive, one of the key Questions in this field is: how does the Qo site oxidize Quinol without the production of deleterious side reactions including superoxide production? We attempt to test three possible general models to account for this behavior: 1) The Qo site semiQuinone (or Quinol-imidazolate complex) is unstable and thus occurs at a very low steady-state concentration, limiting O2 reduction; 2) the Qo site semiQuinone is highly stabilized making it unreactive toward oxygen; and 3) the Qo site catalyzes a Quantum mechanically coupled two-electron/two-proton transfer without a semiQuinone intermediate. Enthalpies of activation were found to be almost identical between the uninhibited Q-Cycle and superoxide production in the presence of antimycin A in wild type. This behavior was also preserved in a series of mutants with altered driving forces for Quinol oxidation. Overall, the data support models where the rate-limiting step for both Q-Cycle and superoxide production is essentially identical, consistent with model 1 but reQuiring modifications to models 2 and 3.
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protonmotive pathways and mechanisms in the cytochrome bc1 complex
FEBS Letters, 2003Co-Authors: Carola Hunte, Hildur Palsdottir, Bernard L TrumpowerAbstract:The cytochrome bc(1) complex catalyzes electron transfer from ubiQuinol to cytochrome c by a protonmotive Q Cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q Cycle mechanism proton translocation is the net result of topographically segregated reduction of Quinone and reoxidation of Quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the Quinol. The linkage of proton chemistry to electron transfer during Quinol oxidation and Quinone reduction reQuires pathways for moving protons to and from the aQueous phase and the hydrophobic environment in which the Quinol and Quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc(1) complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiQuinone redox reactions with particular reference to recently determined structures of the yeast bc(1) complex.
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role of the rieske iron sulfur protein midpoint potential in the protonmotive Q Cycle mechanism of the cytochrome bc1 complex
Journal of Bioenergetics and Biomembranes, 1999Co-Authors: Christopher H Snyder, Thomas A. Link, Torsten Merbitzzahradnik, Bernard L TrumpowerAbstract:The midpoint potential of the [2Fe–2S] cluster of the Rieske iron–sulfurprotein (Em7 = +280mV) is the primary determinant of the rate of electron transfer from ubiQuinol to cytochromec catalyzed by the cytochrome bc1 complex. As the midpoint potential of the Rieske clusteris lowered by altering the electronic environment surrounding the cluster, theubiQuinol-cytochrome c reductase activity of the bc1 complex decreases; between 220 and 280 mV therate changes 2.5-fold. The midpoint potential of the Rieske cluster also affects thepresteady-state kinetics of cytochrome b and c1 reduction. When the midpoint potential of the Rieskecluster is more positive than that of the heme of cytochrome c1, reduction of cytochrome bis biphasic. The fast phase of b reduction is linked to the optically invisible reduction of theRieske center, while the rate of the second, slow phase matches that of c1 reduction. The ratesof b and c1 reduction become slower as the potential of the Rieske cluster decreases andchange from biphasic to monophasic as the Rieske potential approaches that of theubiQuinone/ubiQuinol couple. Reduction of b and c1 remain kinetically linked as the midpoint potentialof the Rieske cluster is varied by 180 mV and under conditions where the presteady statereduction is biphasic or monophasic. The persistent linkage of the rates of b and c1 reduction isaccounted for by the bifurcated oxidation of ubiQuinol that is uniQue to the Q-Cycle mechanism.
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energy transduction in mitochondrial respiration by the proton motive Q Cycle mechanism of the cytochrome bc1 complex
1999Co-Authors: Bernard L TrumpowerAbstract:The cytochrome bc 1 complex is an oligomeric electron transfer enzyme located in the inner membrane of mitochondria, where it participates in respiration, and the plasma membrane of bacteria, where it participates in respiration, denitrification, and nitrogen fixation (Trumpower and Gennis, 1994). The cytochrome bc 1 complex transfers electrons from ubiQuinol to cytochrome c and links this electron transfer to translocation of protons across the membrane in which it resides, thus converting the available free energy of the oxidation-reduction reaction into an electrochemical proton gradient. The relationship between the cytochrome bc 1 complex, the cytochrome c oxidase complex, and some of the dehydrogenases that form the ubiQuinol substrate for the cytochrome bc 1 complex is shown in Fig. 1.
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mechanism of ubiQuinol oxidation by the cytochrome bc1 complex pre steady state kinetics of cytochrome bc1 complexes containing site directed mutants of the rieske iron sulfur protein
Biochimica et Biophysica Acta, 1998Co-Authors: Chris Snyder, Bernard L TrumpowerAbstract:To facilitate characterization of mutated cytochrome bc1 complexes in S. cerevisiae we have developed a new approach using a rapid scanning monochromator to examine pre-steady-state reduction of the enzyme with menaQuinol. The RSM records optical spectra of cytochromes b and c1 at 1-ms intervals after a dead time of 2 ms, and menaQuinol fully reduces both cytochromes bH and c1 and a portion of cytochrome bL. The rapid-mixing, rapid-scanning monochromator methodology obviates limitations inherent in previous rapid kinetics methods and permits measurements of rates exceeding 200 s−1. To document the validity of this methodology we have examined the reduction kinetics of the cytochrome bc1 complexes from wild-type yeast and yeast that lack ubiQuinone. The results establish that menaQuinol reacts via the Q Cycle pathway both in the presence and absence of ubiQuinone. From analyzing bc1 complexes containing Rieske proteins in which the midpoint potential of the iron-sulfur cluster has been altered from +280 to +105 mV, we propose a mechanism in which the protonated Quinol displaces a proton from the imidazole nitrogen of one of the histidines that is a ligand to the iron-sulfur cluster and forms a Quinol-imidazolate complex that is the electron donor to the redox active iron.
Antony R Crofts - One of the best experts on this subject based on the ideXlab platform.
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the modified Q Cycle a look back at its development and forward to a functional model
Biochimica et Biophysica Acta, 2021Co-Authors: Antony R CroftsAbstract:Abstract On looking back at a lifetime of research, it is interesting to see, in the light of current progress, how things came to be, and to speculate on how things might be. I am delighted in the context of the Mitchell prize to have that excuse to present this necessarily personal view of developments in areas of my interests. I have focused on the Q-Cycle and a few examples showing wider ramifications, since that had been the main interest of the lab in the 20 years since structures became available, - a watershed event in determining our molecular perspective. I have reviewed the evidence for our model for the mechanism of the first electron transfer of the bifurcated reaction at the Qo-site, which I think is compelling. In reviewing progress in understanding the second electron transfer, I have revisited some controversies to justify important conclusions which appear, from the literature, not to have been taken seriously. I hope this does not come over as nitpicking. The conclusions are important to the final section in which I develop an internally consistent mechanism for turnovers of the complex leading to a state similar to that observed in recent rapid-mix/freeze-Quench experiments, reported three years ago. The final model is necessarily speculative but is open to test.
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dissecting the pattern of proton release from partial process involved in ubihydroQuinone oxidation in the Q Cycle
Biochimica et Biophysica Acta, 2018Co-Authors: Charles A Wilson, Antony R CroftsAbstract:Abstract 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 Qo-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 Qo-site inhibitors, the pH change measured represented the convolution of this proton release with release of protons on turnover of the Qo-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.
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the Q Cycle mechanism of the bc1 complex a biologist s perspective on atomistic studies
Journal of Physical Chemistry B, 2017Co-Authors: Antony R Crofts, Stuart Rose, Rodney L Burton, A V Desai, Paul J A Kenis, Sergei A DikanovAbstract:The Q-Cycle mechanism of the bc1 complex is now well enough understood to allow application of advanced computational approaches to the study of atomistic processes. In addition to the main features of the mechanism, these include control and gating of the bifurcated reaction at the Qo-site, through which generation of damaging reactive oxygen species is minimized. We report a new molecular dynamics model of the Rhodobacter sphaeroides bc1 complex implemented in a native membrane, and constructed so as to eliminate blemishes apparent in earlier Rhodobacter models. Unconstrained MD simulations after eQuilibration with ubiQuinol and ubiQuinone respectively at Qo- and Qi-sites show that substrate binding configurations at both sites are different in important details from earlier models. We also demonstrate a new Qo-site intermediate, formed in the sub-ms time range, in which semiQuinone remains complexed with the reduced iron sulfur protein. We discuss this, and a spring-loaded mechanism for modulating inte...
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the Q Cycle reviewed how well does a monomeric mechanism of the bc1 complex account for the function of a dimeric complex
Biochimica et Biophysica Acta, 2008Co-Authors: Antony R Crofts, Sergei A Dikanov, Derrick R J Kolling, Todd J Holland, Doreen Victoria, Ryan Gilbreth, Sangmoon Lhee, Richard Kuras, Mariana Guergova KurasAbstract:Recent progress in understanding the Q-Cycle mechanism of the bc(1) 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 c(1) and liberate the H(+). When oxidation of the semiQuinone is prevented, it participates in bypass reactions, including superoxide generation if O(2) 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 b(150) 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 bc(1) 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.
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the cytochrome bc1 complex function in the context of structure
Annual Review of Physiology, 2004Co-Authors: Antony R CroftsAbstract:▪ Abstract The bc 1 complexes are intrinsic membrane proteins that catalyze the oxidation of ubihydroQuinone and the reduction of cytochrome c in mitochondrial respiratory chains and bacterial photosynthetic and respiratory chains. The bc 1 complex operates through a Q-Cycle mechanism that couples electron transfer to generation of the proton gradient that drives ATP synthesis. Genetic defects leading to mutations in proteins of the respiratory chain, including the subunits of the bc 1 complex, result in mitochondrial myopathies, many of which are a direct result of dysfunction at catalytic sites. Some myopathies, especially those in the cytochrome b subunit, exacerbate free-radical damage by enhancing superoxide production at the ubihydroQuinone oxidation site. This bypass reaction appears to be an unavoidable feature of the reaction mechanism. Cellular aging is largely attributable to damage to DNA and proteins from the reactive oxygen species arising from superoxide and is a major contributing factor i...
Di Xia - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of bacterial cytochrome bc1 in complex with azoxystrobin reveals a conformation switch of the rieske iron sulfur protein subunit
Journal of Biological Chemistry, 2019Co-Authors: Lothar Esser, Fei Zhou, Di XiaAbstract:Cytochrome bc 1 complexes (cyt bc 1), also known as complex III in mitochondria, are components of the cellular respiratory chain and of the photosynthetic apparatus of non-oxygenic photosynthetic bacteria. They catalyze electron transfer (ET) from ubiQuinol to cytochrome c and concomitantly translocate protons across the membrane, contributing to the cross-membrane potential essential for a myriad of cellular activities. This ET-coupled proton translocation reaction reQuires a gating mechanism that ensures bifurcated electron flow. Here, we report the observation of the Rieske iron-sulfur protein (ISP) in a mobile state, as revealed by the crystal structure of cyt bc 1 from the photosynthetic bacterium Rhodobacter sphaeroides in complex with the fungicide azoxystrobin. Unlike cyt bc 1 inhibitors stigmatellin and famoxadone that immobilize the ISP, azoxystrobin causes the ISP-ED to separate from the cyt b subunit and to remain in a mobile state. Analysis of anomalous scattering signals from the iron-sulfur cluster of the ISP suggests the existence of a trajectory for electron delivery. This work supports and solidifies the hypothesis that the bimodal conformation switch of the ISP provides a gating mechanism for bifurcated ET, which is essential to the Q-Cycle mechanism of cyt bc 1 function.
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crystal structure of bacterial cytochrome bc1 in complex with azoxystrobin reveals a conformational switch of the rieske iron sulfur protein subunit
Journal of Biological Chemistry, 2019Co-Authors: Lothar Esser, Fei Zhou, Di XiaAbstract:Cytochrome bc1 complexes (cyt bc1), also known as complex III in mitochondria, are components of the cellular respiratory chain and of the photosynthetic apparatus of non-oxygenic photosynthetic bacteria. They catalyze electron transfer (ET) from ubiQuinol to cytochrome c and concomitantly translocate protons across the membrane, contributing to the cross-membrane potential essential for a myriad of cellular activities. This ET-coupled proton translocation reaction reQuires a gating mechanism that ensures bifurcated electron flow. Here, we report the observation of the Rieske iron–sulfur protein (ISP) in a mobile state, as revealed by the crystal structure of cyt bc1 from the photosynthetic bacterium Rhodobacter sphaeroides in complex with the fungicide azoxystrobin. Unlike cyt bc1 inhibitors stigmatellin and famoxadone that immobilize the ISP, azoxystrobin causes the ISP-ED to separate from the cyt b subunit and to remain in a mobile state. Analysis of anomalous scattering signals from the iron–sulfur cluster of the ISP suggests the existence of a trajectory for electron delivery. This work supports and solidifies the hypothesis that the bimodal conformation switch of the ISP provides a gating mechanism for bifurcated ET, which is essential to the Q-Cycle mechanism of cyt bc1 function.
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structural basis for the Quinone reduction in the bc1 complex a comparative analysis of crystal structures of mitochondrial cytochrome bc1 with bound substrate and inhibitors at the Qi site
Biochemistry, 2003Co-Authors: Xiugong Gao, Lothar Esser, Xiaoling Wen, Byron Quinn, Di XiaAbstract:Cytochrome bc1 is an integral membrane protein complex essential to cellular respiration and photosynthesis. The Q Cycle reaction mechanism of bc1 postulates a separated Quinone reduction (Qi) and Quinol oxidation (Qo) site. In a complete catalytic Cycle, a Quinone molecule at the Qi site receives two electrons from the bH heme and two protons from the negative side of the membrane; this process is specifically inhibited by antimycin A and NQNO. The structures of bovine mitochondrial bc1 in the presence or absence of bound substrate ubiQuinone and with either the bound antimycin A1 or NQNO were determined and refined. A ubiQuinone with its first two isoprenoid repeats and an antimycin A1 were identified in the Qi pocket of the substrate and inhibitor bound structures, respectively; the NQNO, on the other hand, was identified in both Qi and Qo pockets in the inhibitor complex. The two inhibitors occupied different portions of the Qi pocket and competed with substrate for binding. In the Qo pocket, the NQNO...
