skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Similar Transition States Mediate the Q-cycle and Superoxide Production by the Cytochrome bc1 Complex

Abstract

The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria catalyze a critical reaction 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 a ''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 towards 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 alsomore » preserved in a series of mutants with altered driving forces for quinol oxidation. Overall, the data supports models where the rate-limiting step for both Q-cycle and superoxide production are essentially identical, consistent with model 1 but requiring modifications to models 2 and 3.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
897679
Report Number(s):
PNWD-SA-7426
19832; TRN: US0701500
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry, 281(50):38459-38465
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; 72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS; ANTIBIOTICS; BACTERIA; CHLOROPLASTS; CYTOCHROMES; ELECTRON TRANSFER; ELECTRONS; HEME; MITOCHONDRIA; MODIFICATIONS; MUTANTS; OXIDASES; OXIDATION; OXYGEN; PROTONS; SYNTHESIS; TRANSLOCATION; Environmental Molecular Sciences Laboratory

Citation Formats

Forquer, Isaac P., Covian, Raul, Bowman, Michael K., Trumpower, Bernard, and Kramer, David M. Similar Transition States Mediate the Q-cycle and Superoxide Production by the Cytochrome bc1 Complex. United States: N. p., 2006. Web. doi:10.1074/jbc.M605119200.
Forquer, Isaac P., Covian, Raul, Bowman, Michael K., Trumpower, Bernard, & Kramer, David M. Similar Transition States Mediate the Q-cycle and Superoxide Production by the Cytochrome bc1 Complex. United States. doi:10.1074/jbc.M605119200.
Forquer, Isaac P., Covian, Raul, Bowman, Michael K., Trumpower, Bernard, and Kramer, David M. Fri . "Similar Transition States Mediate the Q-cycle and Superoxide Production by the Cytochrome bc1 Complex". United States. doi:10.1074/jbc.M605119200.
@article{osti_897679,
title = {Similar Transition States Mediate the Q-cycle and Superoxide Production by the Cytochrome bc1 Complex},
author = {Forquer, Isaac P. and Covian, Raul and Bowman, Michael K. and Trumpower, Bernard and Kramer, David M.},
abstractNote = {The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria catalyze a critical reaction 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 a ''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 towards 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 supports models where the rate-limiting step for both Q-cycle and superoxide production are essentially identical, consistent with model 1 but requiring modifications to models 2 and 3.},
doi = {10.1074/jbc.M605119200},
journal = {Journal of Biological Chemistry, 281(50):38459-38465},
number = ,
volume = ,
place = {United States},
year = {Fri Dec 15 00:00:00 EST 2006},
month = {Fri Dec 15 00:00:00 EST 2006}
}
  • Although several X-ray structures have been solved for the mitochondrial cytochrome (cyt) bc1 complex, none yet shows the position of substrate, ubiquinol, in the quinol oxidase (Qo) site. In the present study, the interaction of molecular oxygen with the reactive intermediate Qo semiquinone is used to probe Qo site. It has been known for some time that partial turnover of the cyt bc1 complex in the presence of antimycin A, a Qi site inhibitor, results in accumulation of a semiquinone at the Qo site, which can reduce O2 to superoxide (O2?-). It was more recently shown that myxothiazol, which bindsmore » close to the cyt bL heme in the proximal Qo niche, also induces O2?- production. In this work we show that, in addition to myxothiazol, a number of other proximal Qo inhibitors (including E-b-methoxyacrylate-stilbene, mucidin and famoxadone) also induce O2?- production in isolated yeast cyt bc1 complex, at about 50% the Vmax observed in the presence of antimycin A. We propose that proximal Qo site inhibitors induce O2?- production because they allow formation, but not oxidation of the semiquinone at the distal niche of the Qo site pocket. The apparent Km for ubiquinol at the Qo site in the presence of Qo proximal inhibitors suggests that the distal niche of the Qo pocket can act as a fully independent quinol binding and oxidation site. Together with the X-ray structures these results suggest substrate ubiquinol binds in essentially the same position as stigmatellin with H-bonds between H161 of the Rieske iron-sulfur protein and E272 of the cyt b protein. When modeled in this way, mucidin, and ubiquinol can bind simultaneously to the Qo site with virtually no steric hindrance, whereas progressively bulkier inhibitors show increasing overlap. The fact that partial turnover of the Qo site is possible even with bound proximal Qo site inhibitors is consistent with the participation of two separate functional Qo binding niches, occupied simultaneously or sequentially.« less
  • 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 ''Qcycle'' 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 onemore » hundred-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.« less
  • We have previously reported that mutant strains of Rhodobacter capsulatus that have alanine insertions (+nAla mutants) in the hinge region of the iron sulfur (Fe-S) containing subunit of the bc1 complex have increased redox midpoint potentials (Em) for their[2Fe2S] clusters. The alteration of the Em in these strains, that contain mutations far from the metal binding site, implied that the local environment of the metal center is indirectly altered by a change in the interaction of this subunit with the hydroquinone oxidizing (Qo) site (Darrouzet, E., Valkova-Valchanova, M., and Daldal, F. (2002) Journal of Biological Chemistry 277, 3464- 3470). Subsequently,more » the Em changes have been proposed to be predominantly due to a stronger or more stabilized hydrogen bonding between the reduced[2Fe2S] cluster and the Qo site inhabitant ubiquinone (Q) (Shinkarev, V. P., Kolling, D. R. J., Miller, T. J., and Crofts, A. R. (2002) Biochemistry 41, 14372-14382). To further investigate this issue, Fe-S protein-Q interactions were monitored by EPR spectroscopy and the findings indicated that the wild type and mutant proteins interactions with Q are similar. Moreover, when the Qpool was chemically depleted, the Em of the[2Fe2S] cluster in mutant bc1 complexes remained more positive than a similarly treated native enzyme (e.g., the[2Fe2S] Em of the+2Ala mutant was 55 mV more positive than the wild type). These data suggest that the increased Em of the[2Fe2S] cluster in the+nAla mutants is in part due to the clusters interaction with Q, and in part to additional factors that are independent of hydrogen bonding to Q. One such factor, the possibility of a different position of the Fe-S at the Qo site of the mutant proteins versus the native enzyme, was addressed by determining the orientation of the[2Fe2S] cluster in the membrane using EPR spectroscopy. In the case of the+2Ala mutant, the[2Fe2S] cluster orientation in the absence of inhibitor is different than that seen in the native enzyme. However, the+2Ala mutant cluster shared a similar orientation with the native enzyme when both samples were exposed to either stigmatellin or myxothiazol. In addition, Qpool extracted membranes of+2Ala mutant exhibited fewer overall orientations, with the predominant one being more similar to that observed in the non Q-depleted membranes of+2Ala mutant than the Q-depleted membranes of a wild type strain. Therefore, additional component(s) that are independent of Qo site inhabitants and that originate from the newly observed orientations of the[2Fe2S] clusters in the+nAla mutants also contribute to the increased midpoint potentials of their[2Fe2S] clusters. While the molecular basis of these components remains to be determined, salient implications of these findings in terms of Qo site catalysis are discussed.« less
  • The chicken mitochondrial ubiquinol cytochrome c oxidoreductase (bc1 complex) is inhibited by Zn{sup 2+} ions, but with higher Ki ({approximately}3 {micro}M) than the corresponding bovine enzyme. When equilibrated with mother liquor containing 200 mM ZnCl{sub 2} for 7 days, the crystalline chicken bc1 complex specifically binds Zn{sup 2+} at 4 sites representing two sites on each monomer in the dimer. These two sites are close to the stigmatellin-binding site, taken to be center Qo of the Q-cycle mechanism, and are candidates for the inhibitory site. One binding site is actually in the hydrophobic channel between the Qo site and themore » bulk lipid phase, and may interfere with quinone binding. The other is in a hydrophilic area between cytochromes b and c1, and might interfere with the egress of protons from the Qo site to the intermembrane aqueous medium. No zinc was bound near the putative proteolytic active site of subunits 1 and 2 (homologous to mitochondrial processing peptidase) under these conditions.« less