Komlodi 2021 BEC Q

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Komlódi T, Cardoso LHD, Doerrier C, Moore AL, Rich PR, Gnaiger E (2021) Coupling and pathway control of coenzyme Q redox state and respiration in isolated mitochondria. Bioenerg Commun 2021.3. https://doi.org/10.26124/bec:2021-0003

» Bioenerg Commun 2021.3. Open Access pdf published online 2021-11-11

Komlodi Timea, Cardoso Luiza HD, Doerrier Carolina, Moore Anthony L, Rich Peter R, Gnaiger Erich (2021) Bioenerg Commun

Abstract: BEC.png doi:10.26124/bec:2021-0003

Graphical abstract

Redox states of the mitochondrial coenzyme Q pool, which reacts with the electron transfer system, reflect the balance between (1) reducing capacities of electron flow from fuel substrates converging at the Q-junction, (2) oxidative capacities downstream of Q to O2, and (3) the load on the OXPHOS system utilizing or dissipating the protonmotive force.

A three-electrode sensor (Rich 1988; Moore et al 1988) was implemented into the NextGen-O2k to monitor continuously the redox state of CoQ2 added as a Q-mimetic simultaneously with O2 consumption. The Q-Module was optimized for high signal-to-noise ratio, minimum drift, and minimum oxygen diffusion. CoQ2 equilibrates in the same manner as Q at Complexes CI, CII and CIII. The CoQ2 redox state is monitored amperometrically with the working electrode, which is poised at CoQ2 redox peak potentials determined by cyclic voltammetry. The voltammogram also provides quality control of the Q-sensor and reveals chemical interferences.

The CoQ2 redox state and O2 consumption were measured simultaneously in isolated mouse cardiac and brain mitochondria. CoQ2 ― and by implication mitochondrial Q ― was more oxidized when O2 flux was stimulated by coupling control: when energy demand increased from LEAK to OXPHOS and electron transfer capacities in the succinate pathway. In contrast, CoQ2 was more reduced when O2 flux was stimulated by pathway-control of electron input capacities, increasing from the NADH (N)- to succinate (S)-linked pathway which converge at the Q-junction, with CI-Q-CIII and CII-Q-CIII segments, respectively. N- and S- respiratory pathway capacities were not completely additive, compatible with partitioning of Q intermediary between the solid-state and liquid-state models of supercomplex organization. The direct proportionality of CoQ2 reduction and electron input capacities through the CI-Q-CIII and CII-Q-CIII segments suggests that CoQ2 is accurately mimicking mitochondrial Q-redox changes.

Keywords: coenzyme Q CoQ, Q-junction, Q-redox state, electron transfer system ETS, ETS-reactive Q-pool Q, mitochondrial coenzyme Q mtCoQ, supercomplexed Q, free Q-pool according to the fluid-state model Qfree, cyclic voltammetry CV, high-resolution respirometry HRR, isolated mitochondria imt, mouse heart mitochondria, mouse brain mitochondria, oxygen consumption, SUIT protocols, coupling control, pathway control, NS-pathway, additivity Bioblast editor: Gnaiger E, Cardoso LHD O2k-Network Lab: AT Innsbruck Oroboros

ORCID: ORCID.png Komlodi Timea, ORCID.png Cardoso Luiza HD, ORCID.png Doerrier Carolina, Moore Anthony L, ORCID.png Rich Peter R, ORCID.png Gnaiger Erich

Video - NextGen-O2k: the Q-Module

Data availability

Original files are available Open Access at Zenodo repository: 10.5281/zenodo.4478400


Template NextGen-O2k.jpg
Supported by project NextGen-O2k which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770.



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Aberg F, Appelkvist EL, Dallner G, Ernster L (1992) Distribution and redox state of ubiquinones in rat and human tissues. Arch Biochem Biophys 295:230-4.1992PMID:1586151
Alcázar-Fabra M, Navas P, Brea-Calvo G (2016) Coenzyme Q biosynthesis and its role in the respiratory chain structure. Biochim Biophys Acta 1857:1073-1078.2016PMID:26970214 Open Access
Ausili A, Torrecillas A, Aranda F, de Godos A, Sánchez-Bautista S, Corbalán-García S, Gómez-Fernández JC (2008) Redox state of coenzyme Q10 determines its membrane localization. J Phys Chem B 112:12696-702.2008PMID:18795772
Awad AM, Bradley MC, Fernandez-Del-Rio L, Nag A, Tsui HS, Clarke CF (2018) Coenzyme Q10 deficiencies: pathways in yeast and humans. Essays Biochem 62:361-76.2018PMID:29980630 Open Access
Balaban RS, Nemoto S, Finkel T (2005) Mitochondria, oxidants, and aging. Cell 120:483-95. https://doi.org/10.1016/j.cell.2005.02.0012005PMID:15734681 Open Access
Bentinger M, Brismar K, Dallner G (2007) The antioxidant role of coenzyme Q. Mitochondrion Suppl:S41-50.2007PMID:17482888
Bentinger M, Tekle M, Dallner G (2010) Coenzyme Q--biosynthesis and functions. Biochem Biophys Res Commun 396:74-9.2010PMID:20494114
Bianchi C, Fato R, Genova ML, Parenti Castelli F, Lenaz G (2003) Structural and functional organization of Complex I in the mitochondrial respiratory chain. Biofactors 18:3-9.2003PMID:14695915
Bianchi C, Genova ML, Parenti Castelli G, Lenaz G (2004) The mitochondrial respiratory chain is partially organized in a supercomplex assembly: kinetic evidence using flux control analysis. J Biol Chem 279:36562-9.2004PMID: 15205457 Open Access
Cohen ER, Cvitas T, Frey JG, Holmström B, Kuchitsu K, Marquardt R, Mills I, Pavese F, Quack M, Stohner J, Strauss HL, Takami M, Thor HL (2008) Quantities, Units and Symbols in Physical Chemistry. IUPAC Green Book 3rd Edition, 2nd Printing, IUPAC & RSC Publishing, Cambridge.2008Open Access
Cottingham IR, Moore AL (1983) Ubiquinone pool behaviour in plant mitochondria. Biochim Biophys Acta 724:191-200.1983[1]
Crane FL, Hatefi Y, Lester RL, Widmer C (1957) Isolation of a quinone from beef heart mitochondria. Biochim Biophys Acta 1000:362-3.1957PMID:2673386
Crane FL, Sun IL, Clark MG, Grebing C, Low H (1985) Transplasma-membrane redox systems in growth and development. Biochim Biophys Acta 811:233-64.1985PMID:3893544
Crane FL, Widmer C, Lester RL, Hatefi Y, Fechner W (1959) Studies on the electron transport system: XV. Coenzyme Q (Q275) and the succinoxidase activity of the electron transport particle. Biochim Biophys Acta 31:476-89.1959[2]
Crofts AR (2004) The cytochrome bc1 complex: function in the context of structure. Annu Rev Physiol 66:689-733.2004PMID:14977419
Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. https://doi.org/10.1007/978-1-4939-7831-1_32018PMID: 29850993 »O2k-brief
Dry IB, Moore AL, Day DA, Wiskich JT (1989) Regulation of alternative pathway activity in plant mitochondria: nonlinear relationship between electron flux and the redox poise of the quinone pool. Arch Biochem Biophys 273:148-57.1989PMID: 2757390
Echtay KS, Winkler E, Klingenberg M (2000) Coenzyme Q is an obligatory cofactor for uncoupling protein function. Nature 408:609-13.2000PMID:11117751
Enriquez JA, Lenaz G (2014) Coenzyme Q and the respiratory chain: coenzyme Q pool and mitochondrial supercomplexes. Mol Syndromol 5:119-40.2014PMID: 25126045 Open Access
Ernster L, Lee IY, Norling B, Persson B (1969) Studies with ubiquinone-depleted submitochondrial particles. Essentiality of ubiquinone for the interaction of succinate dehydrogenase, NADH dehydrogenase, and cytochrome b. Eur J Biochem 9:299-310.1969PMID: 4307591 Open Access
Estornell E, Fato R, Castelluccio C, Cavazzoni M, Parenti Castelli G, Lenaz G (1992) Saturation kinetics of coenzyme Q in NADH and succinate oxidation in beef heart mitochondria. FEBS Letters 311:107-9.1992
Fazakerley 1DJ, Chaudhuri R, Yang P, Maghzal GJ, Thomas KC, Krycer JR, Humphrey SJ,Parker BL, Fisher-Wellman KH, Meoli CC, Hoffman NJ, Diskin C, Burchfield JG, Cowley MJ, Kaplan W, Modrusan Z, Kolumam G, Yang JY, Chen DL, Samocha-Bonet D, Greenfield JR, Hoehn KL, Stocker R, James DE (2018) Mitochondrial CoQ deficiency is a common driver of mitochondrial oxidants and insulin resistance. Elife 7:32111.2018PMID:29402381 Open Access
Fontaine E, Ichas F, Bernardi P (1998) A ubiquinone-binding site regulates the mitochondrial permeability transition pore. J Biol Chem 273:25734-40.1998PMID:9748242
Gille L, Nohl H (2000) The existence of a lysosomal redox chain and the role of ubiquinone. Arch Biochem Biophys 375:347-54.2000PMID:10700391
Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. https://doi.org/10.1016/S0034-5687(01)00307-32001Bioblast pdf
Respir Physiol 128:277-97. PMID: 11718759
Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial dysfunction in drug-induced toxicity (Dykens JA, Will Y, eds) John Wiley & Sons, Inc, Hoboken, NJ:327-52.2008Bioblast pdf
O2k-Protocols contents
Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-00022020Open Access pdf published online 2020-12-30

Gnaiger E (2021) Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts. MitoFit Preprints 2021.08. https://doi.org/10.26124/mitofit:2021-00082021MitoFit Preprints 2021.08.
MitoFit pdf
Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts
Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Berlin, Heidelberg:431-42. https://doi.org/10.1007/978-3-662-04162-8_452000Bioblast pdf - Springer link
O2k-Protocols contents
Goldstein JL, Brown MS (1990) Regulation of the mevalonate pathway. Nature 343:425-30.1990PMID:1967820
González-Mariscal I, García-Testón E, Padilla S, Martín-Montalvo A, Pomares-Viciana T, Vazquez-Fonseca L, Gandolfo-Domínguez P, Santos-Ocaña C (2014) Regulation of coenzyme Q biosynthesis in yeast: a new complex in the block. IUBMB Life 66:63-70.2014PMID:24470391 Open Access
Graham D (2018) Standard operating procedures for cyclic voltammetry. ISBN 978-1-387-51430-4.2018http://sop4cv.com/
Gulaboski R, Markovski V, Jihe Z (2016) Redox chemistry of coenzyme Q—a short overview of the voltammetric features. J Solid State Electrochem 20:3229–3238.2016[3]
Gutman M (1985) Kinetic analysis of electron flux through the mitochondrial system. Coenzyme Q, Chichester, UK: Wiley 10:215-34.1985[4]
Gvozdjáková A, Sumbalová Z, Kucharská J, Szamosová M, Čápová L, Rausová Z, Vančová O, Mojto V, Langsjoen P, Palacka P (2021) Platelet mitochondrial respiration and coenzyme Q10 could be used as new diagnostic strategy for mitochondrial dysfunction in rheumatoid diseases. PLoS ONE 16:e0256135.2021PMID: 34582480 Open Access
Gvozdjáková A, Sumbalová Z, Kucharská J, Komlósi M, Rausová Z, Vančová O, Számošová M, Mojto V (2020) Platelet mitochondrial respiration, endogenous coenzyme Q10 and oxidative stress in patients with chronic kidney disease. Diagnostics (Basel) 10:176.2020PMID: 32210203 Open Access »O2k-brief
Hackenbrock CR, Chazotte B, Gupte SS (1986) The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport. J Bioenerg Biomembr 18:331–68.1986PMID:3021714
Hatefi Y, Lester RL, Crane FL, Widmer C (1959) Studies on the electron transport system. XVI. Enzymic oxidoreduction reactions of coenzyme Q. Biochim Biophys Acta 31:490-501.1959PMID:13628678
Hernández-Camacho JD, Bernier M, López-Lluch G, Navas P (2018) Coenzyme Q10 Supplementation in aging and disease. Front Physiol 9:44.2018PMID:29459830
Hunte C, Palsdottir H, Trumpower BL (2003) Protonmotive pathways and mechanisms in the cytochrome bc1 complex. FEBS Letters 545:39-46.2003PMID:12788490
Jørgensen BM, Rasmussen HN, Rasmussen UF (1985) Ubiquinone reduction pattern in pigeon heart mitochondria. Identification of three distinct ubiquinone pools. Biochem J 229:621-9.1985PMID:4052014 Open Access
Kalén A, Appelkvist EL, Dallner G (1987) Biosynthesis of ubiquinone in rat liver. Acta Chem Scand B 41:70-2.1987PMID:3577554
Kröger A, Klingenberg M (1966) On the role of ubiquinone in mitochondria. II. Redox reactions of ubiquinone under the control of oxidative phosphorylation. Biochem Z 344:317-36.1966PMID:4295856
Kröger A, Klingenberg M (1973) Further evidence for the pool function of ubiquinone as derived from the inhibition of the electron transport by antimycin. Eur J Biochem 39:313-23.1973PMID:4359626 Open Access
Kröger A, Klingenberg M (1973) The kinetics of the redox reactions of ubiquinone related to the electron-transport activity in the respiratory chain. Eur J Biochem 34:358-68.1973PMID:4351161 Open Access
Laboratory Protocol: isolation of rat brain mitochondria.
2016-06-18Bioblast pdf »Versions
Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840. doi:10.1038/s41598-017-02789-82017PMID: 28588260 Sci Rep Open Access
Lenaz G, Genova ML (2009) Structural and functional organization of the mitochondrial respiratory chain: A dynamic super-assembly. Int J Biochem Cell Biol 41:1750-72.2009PMID:19711505 Open Access
Lopez-Lluch G, Rodriguez-Aguilera JC, Santos-Ocana C, Navas P (2010) Is coenzyme Q a key factor in aging? Mech Ageing Dev 131:225-35.2010PMID:20193705
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-75.1951PMID:14907713 Open Access
Miles MV (2007) The uptake and distribution of coenzyme Q(10). Mitochondrion 7 Suppl:S72-7.2007PMID:17446143
Mitchell P (1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191:144-8.1961PMID: 13771349
Mitchell P (1975) The protonmotive Q cycle: A general formulation. FEBS Lett 59:137-9.1975Open Access
Moore AL, Dry IB, Wiskich JT (1991) Regulation of electron transport in plant mitochondria under state 4 conditions. Plant Physiol 95:34-40.1991PMID: 16667977 Open Access
Moore AL, Dry IB, Wiskich TJ (1988) Measurement of the redox state of the ubiquinone pool in plant mitochondria. FEBS Lett 235:76-80.1988Open Access
Morré DJ, Morré DM (1989) Preparation of mammalian plasma membranes by aqueous two-phase partition. Biotechniques 7:946-58.1989PMID:2483665
Morré DJ, Morré DM (2011) Non-mitochondrial coenzyme Q. Biofactors 37:355-60.2011PMID:21674641
Noh YH, Kim K-Y, Shim MS, Choi S-H, Choi S, Ellisman MH, Weinreb RN, Perkins GA, Ju W-K (2013) Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes. Cell Death Dis 4:e820.2013PMID:24091663 Open Access
Nyquist SE, Barr R, Morré DJ (1970) Ubiquinone from rat liver Golgi apparatus fractions. Biochim Biophys Acta 208:532-4.1970PMID:5506581
Osakai T, Yamamoto T, Ueki M (2019) Directional electron transfer from ubiquinone-10 to cytochrome c at a biomimetic self-assembled monolayer modified electrode. Electrochemistry 87:59-64.2019Open Access
Petrova EV, Korotkova EI, Kratochvil B, Voronova OA, Dorozhko EV, Bulycheva EV (2014) Investigation of coenzyme Q10 by voltammetry. Proc Chem 10:173-8. https://doi.org/10.1016/j.proche.2014.10.030.2014Open Access
Rauchová H, Fato R, Drahota Z, Lenaz G (1997) Steady-state kinetics of reduction of coenzyme Q analogs by glycerol-3-phosphate dehydrogenase in brown adipose tissue mitochondria. Arch Biochem Biophys 344:235-41.1997PMID:9244403 Open Access
Reed JS, Ragan CI (1987) The effect of rate limitation by cytochrome c on the redox state of the ubiquinone pool in reconstituted NADH: cytochrome c reductase. Biochem J 247:657-62.1987PMC:1148462
Rich PR (1982) Electron and proton transfers in chemical and biological quinone systems. Faraday Discuss Chem Soc 74:349-64.1982PMID:7183455
Rich PR (1984) Electron and proton transfers through quinones and cytochrome bc complexes. Biochim Biophys Acta 768:53-79.1984PMID: 6322844
Rich PR (1988) Patent of Q-electrode. Glynn Res. Ph. European Patent no.85900699.1.1988
Rich PR (2004) The quinone chemistry of bc complexes. Biochim Biophys Acta 1658:165-71.2004PMID:15282188 Open Access
Rodríguez-Hernández A, Cordero MD, Salviati L, Artuch R, Pineda M, Briones P, Izquierdo LG, Cotán D, Navas P, Sánchez-Alcázar JA (2009) Coenzyme Q deficiency triggers mitochondria degradation by mitophagy. Autophagy 5:19-32.2009PMID: 19115482 Open Access
Sastry PS, Jayaraman J, Ramasarma T (1961) Distribution of coenzyme Q in rat liver cell fractions. Nature 189:577.1961Open Access
Song Y, Buettner GR (2011) Thermodynamic and kinetic considerations for the reaction of semiquinone radicals to form superoxide and hydrogen peroxide. Free Radical Biol Med 919-62.2011PMID: 20493944 Open Access
Stefely JA, Pagliarini DJ (2018) Biochemistry of Mitochondrial Coenzyme Q Biosynthesis. Trends Biochem Sci 42: 824–843.2018PMID:28927698 Open Access
Stoner CD (1984) Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria. Determination of the coupling relationships between the respiratory reactions and miscellaneous observations concerning rate-limiting steps. J Bioenerg Biomembr 16:115-41.1984PMID:6100296
Takada M, Ikenoya S, Yuzuriha T, Katayama K (1984) Simultaneous determination of reduced and oxidized ubiquinones. Methods Enzymol 105:147-55.1984
Tang PH, Miles MV (2012) Measurement of oxidized and reduced coenzyme Q in biological fluids, cells, and tissues: an HPLC-EC method. Methods Mol Biol 837:149-68.2012PMID:22215546
Tran UC, Clarke CF (2007) Endogenous synthesis of coenzyme Q in eukaryotes. Mitochondrion 7 Suppl:S62-71.2007PMID:17482885 Open Access
Trumpower BL (1990) The protonmotive Q cycle. Energy transduction by coupling of proton translocation to electron transfer by the cytochrome bc1 complex. J Biol Chem 265:11409-12.1990PMID:2164001
Trumpower BL, Gennis RB (1994) Energy transduction by cytochrome complexes in mitochondrial and bacterial respiration: the enzymology of coupling electron transfer reactions to transmembrane proton translocation. . Annu Rev Biochem 63:675-716.1994PMID:7979252
Turunen M, Olsson J, Dallner G (2004) Metabolism and function of coenzyme Q. Biochim Biophys Acta 1660:171-99.2004PMID:14757233 Open Access
Urban PF, Klingenberg M (1969) On the redox potentials of ubiquinone and cytochrome b in the respiratory chain. Eur J Biochem 9:519-25.1969Open Access
Van den Bergen CW, Wagner AM, Krab K, Moore AL (1994) The relationship between electron flux and the redox poise of the quinone pool in plant mitochondria. Interplay between quinol-oxidizing and quinone-reducing pathways. Eur J Biochem 226:1071-8.1994PMID: 7813462 Open Access
Watts JL, Ristow M (2017) Lipid and Carbohydrate Metabolism in Caenorhabditis elegans. Genetics 207:413-46.2017PMID:28978773 Open Access
Wolf DE, Hoffman CH, Trenner NR, Arison BH, Shunk CH, Linn BO, McPherson JF, Folkers K (1958) Coenzyme Q. I. Structure studies on the coenzyme Q group. J Am Chem Soc 80:4752.1958Open Access
Zannoni D, Moore AL (1990) Measurement of the redox state of the ubiquinone pool in Rhodobacter capsulatus membrane fragments. FEBS Lett 271:123-7.1990PMID: 2171997 Open Access
Mitochondrial Respiration Medium - MiR05-Kit.
2022-03-03Bioblast pdf » Versions
NextGen-O2k: Q-Module manual
2021-10-29PDF.jpg » Versions
Laboratory protocol: isolation of mouse heart mitochondria.
2021-08-09Bioblast pdf »Versions


Komlodi 2021 MitoFit Q

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Gnaiger 2021 Bioenerg Commun

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  • Komlodi et al (2022) Hydrogen peroxide production, mitochondrial membrane potential and the coenzyme Q redox state measured at tissue normoxia and experimental hyperoxia in heart mitochondria. MitoFit Preprints 2021 (in prep)
  • Komlódi T, Schmitt S, Zdrazilova L, Donnelly C, Zischka H, Gnaiger E. Oxygen dependence of hydrogen peroxide production in isolated mitochondria and permeabilized cells. MitoFit Preprints (in prep).

Labels: MiParea: Respiration, Instruments;methods 

Organism: Mouse  Tissue;cell: Heart, Nervous system  Preparation: Isolated mitochondria 

Regulation: Redox state, Q-junction effect  Coupling state: LEAK, OXPHOS, ET  Pathway: N, S, NS  HRR: Oxygraph-2k, NextGen-O2k, O2k-Protocol 

Additivity, SUIT-006 Q mt D071, SUIT-006 Q ce-pce D073, SUIT-031 Q mt D072, SUIT-031 Q ce-pce D074, BEC2021.5, MitoFit 2022 NADH, MitoFit 2021 AmR, Redox, O2k-Demo 

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