- Email: [email protected]
Redox-gated NADH oxidation by complex I
Biochimica et Biophysica Acta 1857 (2016?) e1–e7
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a b i o
Plenary Lectures (in order of presentation)
Peter Mitchell Medal Lecture Chemiosmotic coupling in the respiratory chain: cytochrome oxidase past, present and future Peter R. Rich Glynn Laboratory of Bioenergetics, Institute of Structural and Molecular Biology, UCL, Gower Street, London, UK E-mail address: [email protected] (P.R. Rich) The roots of elucidation of the metabolic pathways of energy provision can be traced back to the work of Warburg in Germany and of Hopkins in the UK. Keilin and others provided the link to how reductants produced by the Krebs cycle and other metabolic pathways are oxidised with oxygen by the respiratory electron transfer chain. In 1961 Peter Mitchell proposed the chemiosmotic mechanism for coupling of respiratory chain electron transfer to the synthesis of cellular ATP. However, it took a great deal of time, experimentation and acrimonious exchanges before the central ideas became generally accepted. Since then, major advances have been made in the elucidation of atomic structures of the molecular machines involved. Together with information from a wide range of functional studies with biochemical and biophysical methods, these structures provide insights into the intricate mechanisms, some foreseen and others not by Mitchell, that have evolved to convert redox energy from products of metabolism into the electrochemical gradient of protons across the inner mitochondrial membrane that powers ATP synthesis. I will review the development of ideas on one of these chemiosmotic molecular machines, mitochondrial cytochrome c oxidase, and will highlight structure/ function aspects that remain to be resolved and features of the supernumerary subunits still to be understood that are relevant to health and disease.
Chloroplasts arose from cyanobacteria, mitochondria arose from proteobacteria. Both organelles have conserved their prokaryotic biochemistry, but their genomes are reduced, and most organelle proteins are encoded in the nucleus. Endosymbiotic theory posits that bacterial genes in eukaryotic genomes entered the eukaryotic lineage via organelle ancestors. It predicts episodic influx of prokaryotic genes into the eukaryotic lineage, with acquisition corresponding to endosymbiotic events. Eukaryotic genome sequences, however, increasingly implicate lateral gene transfer, both from prokaryotes to eukaryotes and among eukaryotes, as a source of gene content variation in eukaryotic genomes, which predicts continuous, lineage-specific acquisition of prokaryotic genes in divergent eukaryotic groups. One can discriminate between these two alternatives by clustering and phylogenetic analysis of eukaryotic gene families having prokaryotic homologues. The results indicate (1) that gene transfer from bacteria to eukaryotes is episodic, as revealed by gene distributions, and coincides with major evolutionary transitions at the origin of chloroplasts and mitochondria; (2) that gene inheritance in eukaryotes is vertical, as revealed by extensive topological comparison, sparse gene distributions stemming from differential loss; and (3) that continuous, lineage-specific lateral gene transfer, although it sometimes occurs, does not contribute to longterm gene content evolution in eukaryotic genomes. doi:10.1016/j.bbabio.2016.04.011
Session I - Electron transfer
Redox-gated NADH oxidation by complex I Thorsten Friedrich Institute of Biochemistry, Albert-Ludwigs-Universität, Freiburg, Germany E-mail address: [email protected] (T. Friedrich)
doi:10.1016/j.bbabio.2016.04.010
Opening Lecture
Endosymbiotic gene transfer: What bioenergetic organelles did for eukaryotic chromosomes William Martin Institute for Molecular Evolution, University of Düsseldorf, 40225 Düsseldorf, Germany E-mail address: [email protected] (W. Martin) 0005-2728/$ – see front matter
Respiratory complex I couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. Electron transfer takes place in the peripheral arm of the complex and is spatially separated from proton translocation in the membrane arm [1]. NADH is oxidized at the top of the peripheral arm by FMN. Electrons are transferred via an 80 Å long chain made up of seven iron-sulfur (Fe/S) clusters to the quinone reduction site. Another conserved Fe/S cluster N1a that is off that pathway is located in electron tunneling distance to the FMN. The chain of Fe/S clusters is not a simple electron wire, but controls the electron tunneling rates [2]. Reduction of the last cluster of the chain, N2, leads to a six-fold slower rate, most likely to synchronize fast electron transfer with slow proton translocation. In addition, a
e2
Abstracts
larger fraction of N1a is reduced by NADH when N2 is in the reduced state [2] suggesting a role of N1a in regulating NADH oxidation. We determined the structure of the NADH oxidation module at up to 1.8 Å resolution in the reduced and oxidized states. The two redox states differ by the flip of a peptide bond close to the NADH binding site. Fixation of the peptide bond by side-directed mutagenesis led to an inactivation of electron transfer most likely due to slow release of NAD+ from the NADH oxidation site. Thus, the redox state of N1a seems to be a molecular switch that controls binding of reduced pyrimidine nucleotide. The redox-state of N1a is in turn determined by the redox-state of N2, the last cluster of the chain.
References [1] R. Baradaran, J.M. Berrisford, G.S. Minhas, L.A. Sazanov, Crystal structure of the entire respiratory complex I, Nature 494 (2013) 441–445. [2] S. De Vries, K. Dörner, M.F.J. Strampraad, T. Friedrich, Electron tunneling Rates in Complex I Are Tuned for Efficient Energy Conversion, Angew. Chem. Int. Ed. 54 (2015) 2844–2848.
doi:10.1016/j.bbabio.2016.04.012
The role of the K-channel and the active-site tyrosine in the catalytic mechanism of cytochrome c oxidase Vivek Sharmaa,b, Mårten Wikströmc, a Department of Physics, Tampere University of Technology, FI-33101, Tampere, Finland b Department of Physics, University of Helsinki, Helsinki, Finland c Institute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland E-mail addresses: vivek.sharma@tut.fi (V. Sharma), marten.wikstrom@ helsinki.fi (M. Wikström) The active site of cytochrome c oxidase (CcO) comprises an oxygen-binding heme, a nearby copper ion (CuB), and a tyrosine residue that is covalently linked to one of the histidine ligands of CuB. Two proton-conducting pathways are observed in CcO, namely the D- and the K-channels, which are used to transfer protons either to the active site of oxygen reduction (substrate protons) or for pumping. Proton transfer through the D-channel is very fast, and its role in efficient transfer of both substrate and pumped protons is well established. However, it has not been fully clear why a separate K-channel is required, apparently for the supply of substrate protons only. In this work, we have analysed the available experimental and computational data, based on which we provide new perspectives on the role of the K-channel. Our analysis suggests that proton transfer in the K-channel may be gated by the protonation state of the active site tyrosine (Tyr244), and that the neutral radical form of this residue has a more general role in the CcO mechanism than thought previously.
Spectroscopic insights into operation and regulation of cytochrome bc1 Artur Osyczka, Marcin Sarewicz, Arkadiusz Borek, Sebastian Pintscher, Patryk Kuleta, Łukasz Bujnowicz, Rafał Pietras, Robert Ekiert Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland E-mail address: [email protected] (A. Osyczka)
doi:10.1016/j.bbabio.2016.04.014
Cytochrome bc1 (mitochondrial complex III), one of the key enzymes of biological energy conversion, is a multi-cofactor and multi-subunit enzyme. Its function is based on a joint action of quinone binding sites at two opposite sides of the membrane. The sites catalyze opposite reactions, differing in the nature of intermediate steps, with relatively stable and easily-detectable semiquinone in the quinone reduction site (Qi) and difficult to trap semiquinone in the quinone oxidation site (Qo). Here we reflect on recent spectroscopic findings that advance our understanding of molecular mechanism of operation and regulation of this complex enzyme. We begin with description of electron paramagnetic resonance (EPR)-detectable signals of uncoupled semiquinone (SQ) and SQ spin-coupled to Rieske cluster (SQ-FeS) at the Qo site. Properties of these signals are discussed in the context of catalysis of quinol oxidation, in particular possible scenarios for proton transfers, and the states associated with reactive oxygen species (ROS) generation by this site. This discussion is extended by spectroscopic analysis of mitochondrial disease-related mutations in cytochrome b affecting the operation of the Qo site. Next, we discuss how to understand the natural engineering of cross-membrane electron transfer connecting the Qo site with the Qi site in light of the properties of hemes b ligand mutants that affect the redox midpoint potentials of hemes. We then describe dynamics of proton transfers for quinone reduction at the Qi site exposed by single and double mutants of protonable side chains that systematically affected the equilibrium levels of electronic reactions. Addressing all these aspects brings us closer to understand how the Qo and Qi sites and hemes b at their interface contribute to secure efficient delivery of electrons from substrate to product coupled with generation of proton motive force.
The dynamic energy budget of photosynthesis David Kramer Departments of Plant Biology, Biochemistry and Molecular Biology and DOE-Plant Research Laboratory, Michigan State University, East Lansing MI 48824, USA E-mail address: [email protected] (D. Kramer)
doi:10.1016/j.bbabio.2016.04.013
Session II - Photosynthesis
Abstract not received doi:10.1016/j.bbabio.2016.04.015
Intracellular signaling - how does the chloroplast talk to the nucleus? Dario Leister Department of Biology, Ludwig-Maximilians-University Munich, Germany E-mail address: [email protected] (D. Leister) Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis in Arabidopsis thaliana seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a b i o
Plenary Lectures (in order of presentation)
Peter Mitchell Medal Lecture Chemiosmotic coupling in the respiratory chain: cytochrome oxidase past, present and future Peter R. Rich Glynn Laboratory of Bioenergetics, Institute of Structural and Molecular Biology, UCL, Gower Street, London, UK E-mail address: [email protected] (P.R. Rich) The roots of elucidation of the metabolic pathways of energy provision can be traced back to the work of Warburg in Germany and of Hopkins in the UK. Keilin and others provided the link to how reductants produced by the Krebs cycle and other metabolic pathways are oxidised with oxygen by the respiratory electron transfer chain. In 1961 Peter Mitchell proposed the chemiosmotic mechanism for coupling of respiratory chain electron transfer to the synthesis of cellular ATP. However, it took a great deal of time, experimentation and acrimonious exchanges before the central ideas became generally accepted. Since then, major advances have been made in the elucidation of atomic structures of the molecular machines involved. Together with information from a wide range of functional studies with biochemical and biophysical methods, these structures provide insights into the intricate mechanisms, some foreseen and others not by Mitchell, that have evolved to convert redox energy from products of metabolism into the electrochemical gradient of protons across the inner mitochondrial membrane that powers ATP synthesis. I will review the development of ideas on one of these chemiosmotic molecular machines, mitochondrial cytochrome c oxidase, and will highlight structure/ function aspects that remain to be resolved and features of the supernumerary subunits still to be understood that are relevant to health and disease.
Chloroplasts arose from cyanobacteria, mitochondria arose from proteobacteria. Both organelles have conserved their prokaryotic biochemistry, but their genomes are reduced, and most organelle proteins are encoded in the nucleus. Endosymbiotic theory posits that bacterial genes in eukaryotic genomes entered the eukaryotic lineage via organelle ancestors. It predicts episodic influx of prokaryotic genes into the eukaryotic lineage, with acquisition corresponding to endosymbiotic events. Eukaryotic genome sequences, however, increasingly implicate lateral gene transfer, both from prokaryotes to eukaryotes and among eukaryotes, as a source of gene content variation in eukaryotic genomes, which predicts continuous, lineage-specific acquisition of prokaryotic genes in divergent eukaryotic groups. One can discriminate between these two alternatives by clustering and phylogenetic analysis of eukaryotic gene families having prokaryotic homologues. The results indicate (1) that gene transfer from bacteria to eukaryotes is episodic, as revealed by gene distributions, and coincides with major evolutionary transitions at the origin of chloroplasts and mitochondria; (2) that gene inheritance in eukaryotes is vertical, as revealed by extensive topological comparison, sparse gene distributions stemming from differential loss; and (3) that continuous, lineage-specific lateral gene transfer, although it sometimes occurs, does not contribute to longterm gene content evolution in eukaryotic genomes. doi:10.1016/j.bbabio.2016.04.011
Session I - Electron transfer
Redox-gated NADH oxidation by complex I Thorsten Friedrich Institute of Biochemistry, Albert-Ludwigs-Universität, Freiburg, Germany E-mail address: [email protected] (T. Friedrich)
doi:10.1016/j.bbabio.2016.04.010
Opening Lecture
Endosymbiotic gene transfer: What bioenergetic organelles did for eukaryotic chromosomes William Martin Institute for Molecular Evolution, University of Düsseldorf, 40225 Düsseldorf, Germany E-mail address: [email protected] (W. Martin) 0005-2728/$ – see front matter
Respiratory complex I couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. Electron transfer takes place in the peripheral arm of the complex and is spatially separated from proton translocation in the membrane arm [1]. NADH is oxidized at the top of the peripheral arm by FMN. Electrons are transferred via an 80 Å long chain made up of seven iron-sulfur (Fe/S) clusters to the quinone reduction site. Another conserved Fe/S cluster N1a that is off that pathway is located in electron tunneling distance to the FMN. The chain of Fe/S clusters is not a simple electron wire, but controls the electron tunneling rates [2]. Reduction of the last cluster of the chain, N2, leads to a six-fold slower rate, most likely to synchronize fast electron transfer with slow proton translocation. In addition, a
e2
Abstracts
larger fraction of N1a is reduced by NADH when N2 is in the reduced state [2] suggesting a role of N1a in regulating NADH oxidation. We determined the structure of the NADH oxidation module at up to 1.8 Å resolution in the reduced and oxidized states. The two redox states differ by the flip of a peptide bond close to the NADH binding site. Fixation of the peptide bond by side-directed mutagenesis led to an inactivation of electron transfer most likely due to slow release of NAD+ from the NADH oxidation site. Thus, the redox state of N1a seems to be a molecular switch that controls binding of reduced pyrimidine nucleotide. The redox-state of N1a is in turn determined by the redox-state of N2, the last cluster of the chain.
References [1] R. Baradaran, J.M. Berrisford, G.S. Minhas, L.A. Sazanov, Crystal structure of the entire respiratory complex I, Nature 494 (2013) 441–445. [2] S. De Vries, K. Dörner, M.F.J. Strampraad, T. Friedrich, Electron tunneling Rates in Complex I Are Tuned for Efficient Energy Conversion, Angew. Chem. Int. Ed. 54 (2015) 2844–2848.
doi:10.1016/j.bbabio.2016.04.012
The role of the K-channel and the active-site tyrosine in the catalytic mechanism of cytochrome c oxidase Vivek Sharmaa,b, Mårten Wikströmc, a Department of Physics, Tampere University of Technology, FI-33101, Tampere, Finland b Department of Physics, University of Helsinki, Helsinki, Finland c Institute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland E-mail addresses: vivek.sharma@tut.fi (V. Sharma), marten.wikstrom@ helsinki.fi (M. Wikström) The active site of cytochrome c oxidase (CcO) comprises an oxygen-binding heme, a nearby copper ion (CuB), and a tyrosine residue that is covalently linked to one of the histidine ligands of CuB. Two proton-conducting pathways are observed in CcO, namely the D- and the K-channels, which are used to transfer protons either to the active site of oxygen reduction (substrate protons) or for pumping. Proton transfer through the D-channel is very fast, and its role in efficient transfer of both substrate and pumped protons is well established. However, it has not been fully clear why a separate K-channel is required, apparently for the supply of substrate protons only. In this work, we have analysed the available experimental and computational data, based on which we provide new perspectives on the role of the K-channel. Our analysis suggests that proton transfer in the K-channel may be gated by the protonation state of the active site tyrosine (Tyr244), and that the neutral radical form of this residue has a more general role in the CcO mechanism than thought previously.
Spectroscopic insights into operation and regulation of cytochrome bc1 Artur Osyczka, Marcin Sarewicz, Arkadiusz Borek, Sebastian Pintscher, Patryk Kuleta, Łukasz Bujnowicz, Rafał Pietras, Robert Ekiert Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland E-mail address: [email protected] (A. Osyczka)
doi:10.1016/j.bbabio.2016.04.014
Cytochrome bc1 (mitochondrial complex III), one of the key enzymes of biological energy conversion, is a multi-cofactor and multi-subunit enzyme. Its function is based on a joint action of quinone binding sites at two opposite sides of the membrane. The sites catalyze opposite reactions, differing in the nature of intermediate steps, with relatively stable and easily-detectable semiquinone in the quinone reduction site (Qi) and difficult to trap semiquinone in the quinone oxidation site (Qo). Here we reflect on recent spectroscopic findings that advance our understanding of molecular mechanism of operation and regulation of this complex enzyme. We begin with description of electron paramagnetic resonance (EPR)-detectable signals of uncoupled semiquinone (SQ) and SQ spin-coupled to Rieske cluster (SQ-FeS) at the Qo site. Properties of these signals are discussed in the context of catalysis of quinol oxidation, in particular possible scenarios for proton transfers, and the states associated with reactive oxygen species (ROS) generation by this site. This discussion is extended by spectroscopic analysis of mitochondrial disease-related mutations in cytochrome b affecting the operation of the Qo site. Next, we discuss how to understand the natural engineering of cross-membrane electron transfer connecting the Qo site with the Qi site in light of the properties of hemes b ligand mutants that affect the redox midpoint potentials of hemes. We then describe dynamics of proton transfers for quinone reduction at the Qi site exposed by single and double mutants of protonable side chains that systematically affected the equilibrium levels of electronic reactions. Addressing all these aspects brings us closer to understand how the Qo and Qi sites and hemes b at their interface contribute to secure efficient delivery of electrons from substrate to product coupled with generation of proton motive force.
The dynamic energy budget of photosynthesis David Kramer Departments of Plant Biology, Biochemistry and Molecular Biology and DOE-Plant Research Laboratory, Michigan State University, East Lansing MI 48824, USA E-mail address: [email protected] (D. Kramer)
doi:10.1016/j.bbabio.2016.04.013
Session II - Photosynthesis
Abstract not received doi:10.1016/j.bbabio.2016.04.015
Intracellular signaling - how does the chloroplast talk to the nucleus? Dario Leister Department of Biology, Ludwig-Maximilians-University Munich, Germany E-mail address: [email protected] (D. Leister) Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis in Arabidopsis thaliana seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the