- Email: [email protected]
Structure and mechanism of mitochondrial complex I
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Abstracts
increase in oxygen consumption was observed only under conditions favoring OXPHOS (in the absence of OXPHOS inhibitors, carboxyatractiloside and oligomycin). Additionally, to observe GDP stimulatory effect, the incubation medium should be supplemented with phosphate, magnesium ions and ATP. Thereby, we propose that mitochondrial nucleoside diphosphate kinase (mNDPK)-catalyzed transphosphorylation process of GDP and ATP, generating GTP and ADP, is responsible for an initiation of OXPHOS thus attenuation of GDP inhibitory effect of UCP. Finally, GTP instead of GDP should be commonly used as diagnostic inhibitor of UCPs. This work was supported by grants of the Polish Ministry of Science and Higher Education: Iuventus Plus program 2013–2015 (IP2012 059172) and KNOW RNA Research Centre in Poznan (No. 01/KNOW2/2014). References 1. A.M. Woyda-Ploszczyca, W. Jarmuszkiewicz, Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak, PLoS One 9 (2014) e98969. doi:10.1016/j.bbabio.2016.04.321
03.35 Probing the oxygen reduction cycle of the Alternative Oxidase Luke Young, Benjamin May, Mary S. Albury, Anthony L. Moore. Biochemistry and Biomedicine, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK E-mail address: [email protected] (L. Young) The alternative oxidase (AOX) is a non-protonmotive quinol oxidase which catalyses the reduction of oxygen to water and is located in mitochondria of higher plants, pathogenic fungi and some parasites including Trypanosoma brucei and Cryptosporidium parvum. The former is dependent upon AOX for glycolytic turnover while in its bloodstream form, and the proteins absence from mammalian cells makes it a potent chemotherapeutic target. The crystal structure of the trypanosomal AOX at 2.8A [1] reveals that the oxidase is a homodimer with the non-haem diiron carboxylate active-site buried within a four-helix bundle. In light of these findings we have proposed a mechanism for the AOX O2/2H2O redox cycle [2]. Alignment of the AOX crystal structure to ribonucleotide reductase (RNR), another protein of the diiron carboxylate family, has revealed a number of structural discrepancies around the central core. A double mutation of Y220F and F269Y, and subsequent mutation of E123D has converted AOX to an “RNR like” core. Both mutations retained ~10% catalytic activity, with a triple mutation rescuing activity to ~20% that of the AOX wild type. Preliminary studies have revealed the build-up of a coloured intermediate in all mutants, with further investigations necessary to determine the source. Work will be presented with respect to the effects of these mutations and their affect upon the catalytic cycle of AOX. References 1. Shiba, T., et al., Structure of the trypanosome cyanide-insensitive alternative oxidase. Proceedings of the National Academy of Sciences of the United States of America, 2013. 110(12): p. 4580-5
2. Young, L., et al., The alternative oxidases: simple oxidoreductase proteins with complex functions. Biochem Soc Trans, 2013. 41(5): p. 1305-11. doi:10.1016/j.bbabio.2016.04.322
03.36 Structure and mechanism of mitochondrial complex I Volker Zickermanna,d, Christophe Wirthb, Katarzyna Kmitaa, Carola Hunteb, Ulrich Brandtc,d a Structural Bioenergetics Group, Institute of Biochemistry II, Medical School, Goethe-University, Frankfurt am Main, Germany b Institute for Biochemistry and Molecular Biology, ZBMZ, BIOSS Centre for Biological Signalling Studies, University of Freiburg, Germany c Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands d Cluster of Excellence Frankfurt "Macromolecular Complexes", GoetheUniversity, Germany Corresponding author at: Structural Bioenergetics Group, Institute of Biochemistry II, Medical School, Goethe-University, Frankfurt am Main, Germany. Mitochondrial complex I is the largest and most complex enzyme of the respiratory chain and plays a key role in aerobic energy metabolism. Complex I dysfunction has been linked with hereditary and degenerative disorders and generation of reactive oxygen species by complex I contributes to tissue damage in myocardial infarction. Complex I from many eukaryotic species can undergo a reversible active/deactive (A/D) transition and slowing return to the A form was shown to attenuate reperfusion injury. We have crystallized complex I from the aerobic yeast Yarrowia lipolytica and solved the structure at a resolution of 3.6 to 3.9 Å [1]. The central subunits can be assigned to three functional modules, the N module for NADH oxidation, the Q module for ubiquinone reduction, and the P module for proton pumping. A connection between the ubiquinone reduction site and four putative proton pump elements is formed by a central axis of basic and acidic residues. The X-ray structure offers clues on the structural basis of the A/ D transition and suggests that the redox chemistry of ubiquinone is linked with conformational changes of the ubiquinone binding and exchange domains that ultimately trigger and drive proton translocation. In addition to fourteen central subunits that execute redox-linked proton translocation, complex I comprises some 30 accessory subunits of largely unknown function. We have characterized accessory subunit NUMM and show that it contains a Zn binding site and is required for the assembly of iron-sulfur cluster N4 [2]. References 1. V Zickermann, C Wirth, H Nasiri, K Siegmund, H Schwalbe, C Hunte, U Brandt, Mechanistic insight from the crystal structure of mitochondrial complex I, Science 347 (2015) 44 2. K Kmita, C Wirth, J Warnau, S Guerrero-Castillo, C Hunte, G Hummer, VR Kaila, K Zwicker, U Brandt, V Zickermann, Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I, PNAS 112 (2015) 5685. doi:10.1016/j.bbabio.2016.04.323
Abstracts
increase in oxygen consumption was observed only under conditions favoring OXPHOS (in the absence of OXPHOS inhibitors, carboxyatractiloside and oligomycin). Additionally, to observe GDP stimulatory effect, the incubation medium should be supplemented with phosphate, magnesium ions and ATP. Thereby, we propose that mitochondrial nucleoside diphosphate kinase (mNDPK)-catalyzed transphosphorylation process of GDP and ATP, generating GTP and ADP, is responsible for an initiation of OXPHOS thus attenuation of GDP inhibitory effect of UCP. Finally, GTP instead of GDP should be commonly used as diagnostic inhibitor of UCPs. This work was supported by grants of the Polish Ministry of Science and Higher Education: Iuventus Plus program 2013–2015 (IP2012 059172) and KNOW RNA Research Centre in Poznan (No. 01/KNOW2/2014). References 1. A.M. Woyda-Ploszczyca, W. Jarmuszkiewicz, Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak, PLoS One 9 (2014) e98969. doi:10.1016/j.bbabio.2016.04.321
03.35 Probing the oxygen reduction cycle of the Alternative Oxidase Luke Young, Benjamin May, Mary S. Albury, Anthony L. Moore. Biochemistry and Biomedicine, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK E-mail address: [email protected] (L. Young) The alternative oxidase (AOX) is a non-protonmotive quinol oxidase which catalyses the reduction of oxygen to water and is located in mitochondria of higher plants, pathogenic fungi and some parasites including Trypanosoma brucei and Cryptosporidium parvum. The former is dependent upon AOX for glycolytic turnover while in its bloodstream form, and the proteins absence from mammalian cells makes it a potent chemotherapeutic target. The crystal structure of the trypanosomal AOX at 2.8A [1] reveals that the oxidase is a homodimer with the non-haem diiron carboxylate active-site buried within a four-helix bundle. In light of these findings we have proposed a mechanism for the AOX O2/2H2O redox cycle [2]. Alignment of the AOX crystal structure to ribonucleotide reductase (RNR), another protein of the diiron carboxylate family, has revealed a number of structural discrepancies around the central core. A double mutation of Y220F and F269Y, and subsequent mutation of E123D has converted AOX to an “RNR like” core. Both mutations retained ~10% catalytic activity, with a triple mutation rescuing activity to ~20% that of the AOX wild type. Preliminary studies have revealed the build-up of a coloured intermediate in all mutants, with further investigations necessary to determine the source. Work will be presented with respect to the effects of these mutations and their affect upon the catalytic cycle of AOX. References 1. Shiba, T., et al., Structure of the trypanosome cyanide-insensitive alternative oxidase. Proceedings of the National Academy of Sciences of the United States of America, 2013. 110(12): p. 4580-5
2. Young, L., et al., The alternative oxidases: simple oxidoreductase proteins with complex functions. Biochem Soc Trans, 2013. 41(5): p. 1305-11. doi:10.1016/j.bbabio.2016.04.322
03.36 Structure and mechanism of mitochondrial complex I Volker Zickermanna,d, Christophe Wirthb, Katarzyna Kmitaa, Carola Hunteb, Ulrich Brandtc,d a Structural Bioenergetics Group, Institute of Biochemistry II, Medical School, Goethe-University, Frankfurt am Main, Germany b Institute for Biochemistry and Molecular Biology, ZBMZ, BIOSS Centre for Biological Signalling Studies, University of Freiburg, Germany c Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands d Cluster of Excellence Frankfurt "Macromolecular Complexes", GoetheUniversity, Germany Corresponding author at: Structural Bioenergetics Group, Institute of Biochemistry II, Medical School, Goethe-University, Frankfurt am Main, Germany. Mitochondrial complex I is the largest and most complex enzyme of the respiratory chain and plays a key role in aerobic energy metabolism. Complex I dysfunction has been linked with hereditary and degenerative disorders and generation of reactive oxygen species by complex I contributes to tissue damage in myocardial infarction. Complex I from many eukaryotic species can undergo a reversible active/deactive (A/D) transition and slowing return to the A form was shown to attenuate reperfusion injury. We have crystallized complex I from the aerobic yeast Yarrowia lipolytica and solved the structure at a resolution of 3.6 to 3.9 Å [1]. The central subunits can be assigned to three functional modules, the N module for NADH oxidation, the Q module for ubiquinone reduction, and the P module for proton pumping. A connection between the ubiquinone reduction site and four putative proton pump elements is formed by a central axis of basic and acidic residues. The X-ray structure offers clues on the structural basis of the A/ D transition and suggests that the redox chemistry of ubiquinone is linked with conformational changes of the ubiquinone binding and exchange domains that ultimately trigger and drive proton translocation. In addition to fourteen central subunits that execute redox-linked proton translocation, complex I comprises some 30 accessory subunits of largely unknown function. We have characterized accessory subunit NUMM and show that it contains a Zn binding site and is required for the assembly of iron-sulfur cluster N4 [2]. References 1. V Zickermann, C Wirth, H Nasiri, K Siegmund, H Schwalbe, C Hunte, U Brandt, Mechanistic insight from the crystal structure of mitochondrial complex I, Science 347 (2015) 44 2. K Kmita, C Wirth, J Warnau, S Guerrero-Castillo, C Hunte, G Hummer, VR Kaila, K Zwicker, U Brandt, V Zickermann, Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I, PNAS 112 (2015) 5685. doi:10.1016/j.bbabio.2016.04.323