Publications by authors named "Thorsten Friedrich"

90 Publications

Characterisation of the redox centers of ethylbenzene dehydrogenase.

J Biol Inorg Chem 2021 Nov 29. Epub 2021 Nov 29.

Labor für Mikrobielle Biochemie and Synmikro Zentrum für Synthetische Mikrobiologie, Philipps Universität Marburg, 35043, Marburg, Germany.

Ethylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from the beta-proteobacterium Aromatoleum aromaticum, is a soluble periplasmic molybdenum enzyme consisting of three subunits. It contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor and an 4Fe-4S cluster (FS0) in the α-subunit, three 4Fe-4S clusters (FS1 to FS3) and a 3Fe-4S cluster (FS4) in the β-subunit and a heme b cofactor in the γ-subunit. Ethylbenzene is hydroxylated by a water molecule in an oxygen-independent manner at the Mo-bis-MGD cofactor, which is reduced from the Mo to the Mo state in two subsequent one-electron steps. The electrons are then transferred via the Fe-S clusters to the heme b cofactor. In this report, we determine the midpoint redox potentials of the Mo-bis-MGD cofactor and FS1-FS4 by EPR spectroscopy, and that of the heme b cofactor by electrochemically induced redox difference spectroscopy. We obtained relatively high values of > 250 mV both for the Mo-Mo redox couple and the heme b cofactor, whereas FS2 is only reduced at a very low redox potential, causing magnetic coupling with the neighboring FS1 and FS3. We compare the results with the data on related enzymes and interpret their significance for the function of EbDH.
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http://dx.doi.org/10.1007/s00775-021-01917-0DOI Listing
November 2021

Structure of Escherichia coli cytochrome bd-II type oxidase with bound aurachin D.

Nat Commun 2021 11 11;12(1):6498. Epub 2021 Nov 11.

Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.

Cytochrome bd quinol:O oxidoreductases are respiratory terminal oxidases so far only identified in prokaryotes, including several pathogenic bacteria. Escherichia coli contains two bd oxidases of which only the bd-I type is structurally characterized. Here, we report the structure of the Escherichia coli cytochrome bd-II type oxidase with the bound inhibitor aurachin D as obtained by electron cryo-microscopy at 3 Å resolution. The oxidase consists of subunits AppB, C and X that show an architecture similar to that of bd-I. The three heme cofactors are found in AppC, while AppB is stabilized by a structural ubiquinone-8 at the homologous positions. A fourth subunit present in bd-I is lacking in bd-II. Accordingly, heme b is exposed to the membrane but heme d embedded within the protein and showing an unexpectedly high redox potential is the catalytically active centre. The structure of the Q-loop is fully resolved, revealing the specific aurachin binding.
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http://dx.doi.org/10.1038/s41467-021-26835-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8585947PMC
November 2021

Structural Basis for Inhibition of ROS-Producing Respiratory Complex I by NADH-OH.

Angew Chem Int Ed Engl 2021 Oct 6. Epub 2021 Oct 6.

Institute of Biochemistry, University of Freiburg, 79104, Freiburg, Germany.

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH-OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X-ray crystallographic analysis, we report the structure of NADH-OH bound in the active site of a soluble fragment of complex I at 2.0 Å resolution. We have identified key amino acid residues that are specific and essential for binding NADH-OH. Furthermore, the structure sheds light on the specificity of NADH-OH towards the unique Rossmann-fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH-OH acts as a lead-structure for the synthesis of a novel class of ROS suppressors.
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http://dx.doi.org/10.1002/anie.202112165DOI Listing
October 2021

Structure of the peripheral arm of a minimalistic respiratory complex I.

Structure 2021 Sep 20. Epub 2021 Sep 20.

Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, 79104 Freiburg, Germany. Electronic address:

Respiratory complex I drives proton translocation across energy-transducing membranes by NADH oxidation coupled with (ubi)quinone reduction. In humans, its dysfunction is associated with neurodegenerative diseases. The Escherichia coli complex represents the structural minimal form of an energy-converting NADH:ubiquinone oxidoreductase. Here, we report the structure of the peripheral arm of the E. coli complex I consisting of six subunits, the FMN cofactor, and nine iron-sulfur clusters at 2.7 Å resolution obtained by cryo electron microscopy. While the cofactors are in equivalent positions as in the complex from other species, individual subunits are adapted to the absence of supernumerary proteins to guarantee structural stability. The catalytically important subunits NuoC and D are fused resulting in a specific architecture of functional importance. Striking features of the E. coli complex are scrutinized by mutagenesis and biochemical characterization of the variants. Moreover, the arrangement of the subunits sheds light on the unknown assembly of the complex.
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http://dx.doi.org/10.1016/j.str.2021.09.005DOI Listing
September 2021

Biochemical consequences of two clinically relevant ND-gene mutations in Escherichia coli respiratory complex I.

Sci Rep 2021 06 16;11(1):12641. Epub 2021 Jun 16.

Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.

NADH:ubiquinone oxidoreductase (respiratory complex I) plays a major role in energy metabolism by coupling electron transfer from NADH to quinone with proton translocation across the membrane. Complex I deficiencies were found to be the most common source of human mitochondrial dysfunction that manifest in a wide variety of neurodegenerative diseases. Seven subunits of human complex I are encoded by mitochondrial DNA (mtDNA) that carry an unexpectedly large number of mutations discovered in mitochondria from patients' tissues. However, whether or how these genetic aberrations affect complex I at a molecular level is unknown. Here, we used Escherichia coli as a model system to biochemically characterize two mutations that were found in mtDNA of patients. The V253A mutation completely disturbed the assembly of complex I, while the mutation D199G led to the assembly of a stable complex capable to catalyze redox-driven proton translocation. However, the latter mutation perturbs quinone reduction leading to a diminished activity. D199 is part of a cluster of charged amino acid residues that are suggested to be important for efficient coupling of quinone reduction and proton translocation. A mechanism considering the role of D199 for energy conservation in complex I is discussed.
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http://dx.doi.org/10.1038/s41598-021-91631-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209014PMC
June 2021

A Quinol Anion as Catalytic Intermediate Coupling Proton Translocation With Electron Transfer in Respiratory Complex I.

Front Chem 2021 7;9:672969. Epub 2021 May 7.

Institut für Biochemie, Albert-Ludwigs-Universität, Freiburg, Germany.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism. It couples NADH oxidation and quinone reduction with the translocation of protons across the membrane, thus contributing to the protonmotive force. Complex I has an overall L-shaped structure with a peripheral arm catalyzing electron transfer and a membrane arm engaged in proton translocation. Although both reactions are arranged spatially separated, they are tightly coupled by a mechanism that is not fully understood. Using redox-difference UV-vis spectroscopy, an unknown redox component was identified in complex I as reported earlier. A comparison of its spectrum with those obtained for different quinone species indicates features of a quinol anion. The re-oxidation kinetics of the quinol anion intermediate is significantly slower in the D213G variant that was previously shown to operate with disturbed quinone chemistry. Addition of the quinone-site inhibitor piericidin A led to strongly decreased absorption peaks in the difference spectrum. A hypothesis for a mechanism of proton-coupled electron transfer with the quinol anion as catalytically important intermediate in complex I is discussed.
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http://dx.doi.org/10.3389/fchem.2021.672969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138167PMC
May 2021

Electrocatalytic evidence of the diversity of the oxygen reaction in the bacterial bd oxidase from different organisms.

Biochim Biophys Acta Bioenerg 2021 08 30;1862(8):148436. Epub 2021 Apr 30.

Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, Chimie de la Matière Complexe, Université de Strasbourg - CNRS 4, rue Blaise Pascal, 67081 Strasborg, France; USIAS, University of Strasbourg Institute for Advanced Studies, Strasbourg, France. Electronic address:

Cytochrome bd oxidase is a bacterial terminal oxygen reductase that was suggested to enable adaptation to different environments and to confer resistance to stress conditions. An electrocatalytic study of the cyt bd oxidases from Escherichia coli, Corynebacterium glutamicum and Geobacillus thermodenitrificans gives evidence for a different reactivity towards oxygen. An inversion of the redox potential values of the three hemes is found when comparing the enzymes from different bacteria. This inversion can be correlated with different protonated glutamic acids as evidenced by reaction induced FTIR spectroscopy. The influence of the microenvironment of the hemes on the reactivity towards oxygen is discussed.
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http://dx.doi.org/10.1016/j.bbabio.2021.148436DOI Listing
August 2021

Mass Photometry of Membrane Proteins.

Chem 2021 Jan 14;7(1):224-236. Epub 2021 Jan 14.

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers-in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.
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http://dx.doi.org/10.1016/j.chempr.2020.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815066PMC
January 2021

ErpA is important but not essential for the Fe/S cluster biogenesis of Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

Biochim Biophys Acta Bioenerg 2020 12 8;1861(12):148286. Epub 2020 Aug 8.

Albert-Ludwigs-Universität, Institut für Biochemie, Albertstr. 21, D-79104 Freiburg, Germany. Electronic address:

Energy converting NADH:ubiquinone oxidoreductase, complex I, is the first enzyme of respiratory chains in most eukaryotes and many bacteria. The complex comprises a peripheral arm catalyzing electron transfer and a membrane arm involved in proton-translocation. In Escherichia coli, the peripheral arm features a non-covalently bound flavin mononucleotide and nine iron-sulfur (Fe/S)-clusters. Very little is known about the incorporation of the Fe/S-clusters into the E. coli complex I. ErpA, an A-type carrier protein is discussed to act as a Fe/S-cluster carrier protein. To contribute to the understanding of ErpA for the assembly of E. coli complex I, we analyzed an erpA knock-out strain. Deletion of erpA decreased the complex I content in cytoplasmic membranes to approximately one third and the NADH oxidase activity to one fifth. EPR spectroscopy showed the presence of all Fe/S-clusters of the complex in the membrane but only in minor quantities. Sucrose gradient centrifugation and native PAGE revealed the presence of a marginal amount of a stable and fully assembled complex extractable from the membrane. Thus, ErpA is not essential for the assembly of complex I but its absence leads to a strong decrease of a functional complex in the cytoplasmic membrane due to a major lack of all EPR-detectable Fe/S-clusters.
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http://dx.doi.org/10.1016/j.bbabio.2020.148286DOI Listing
December 2020

Stabilization of the Highly Hydrophobic Membrane Protein, Cytochrome Oxidase, on Metallic Surfaces for Direct Electrochemical Studies.

Molecules 2020 Jul 16;25(14). Epub 2020 Jul 16.

Laboratoire de Bioelectrochimie et Spectroscopie, UMR 7140, Chimie de la Matière Complexe, Université de Strasbourg, CNRS, 67081 Strasbourg, France.

The cytochrome oxidase catalyzes the reduction of oxygen to water in bacteria and it is thus an interesting target for electrocatalytic studies and biosensor applications. The oxidase is completely embedded in the phospholipid membrane. In this study, the variation of the surface charge of thiol-modified gold nanoparticles, the length of the thiols and the other crucial parameters including optimal phospholipid content and type, have been performed, giving insight into the role of these factors for the optimal interaction and direct electron transfer of an integral membrane protein. Importantly, all three tested factors, the lipid type, the electrode surface charge and the thiol length mutually influenced the stability of films of the cytochrome oxidase. The best electrocatalytic responses were obtained on the neutral gold surface when the negatively charged phosphatidylglycerol (PG) was used and on the charged gold surface when the zwitterionic phosphatidylethanolamine (PE) was used. The advantages of the covalent binding of the membrane protein to the electrode surface over the non-covalent binding are also discussed.
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http://dx.doi.org/10.3390/molecules25143240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397230PMC
July 2020

The Rnf complex from the acetogenic bacterium Acetobacterium woodii: Purification and characterization of RnfC and RnfB.

Biochim Biophys Acta Bioenerg 2020 11 11;1861(11):148263. Epub 2020 Jul 11.

Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany. Electronic address:

rnf genes are widespread in anaerobic bacteria and hypothesized to encode a respiratory enzyme that couples exergonic reduction of NAD with reduced ferredoxin as a reductant to vectorial ion (Na, H) translocation across the cytoplasmic membrane. However, despite its importance for the physiology of these bacteria, little is known about the subunit composition and the function of subunits. Here, we have purified the entire Rnf complex from the acetogen Acetobacterium woodii or after its production in Escherichia coli. These studies revealed covalently bound flavin in RnfB and RnfD. Unfortunately, the complex did not catalyze electron transfer from reduced ferredoxin to NAD. We, therefore, concentrated on the two cytosolic subunits RnfC and RnfB. RnfC was produced in E. coli, purified and shown to have 8.3 mol iron and 8.6 mol sulfur per mol of the subunit, consistent with the presence of two [4Fe-4S] centers, which were verified by EPR analysis. Flavins could not be detected, but RnfC catalyzed NADH-dependent FMN reduction. These data confirm RnfC as NADH-binding subunit and FMN as an intermediate in the electron transport chain. RnfB could only be produced as a fusion to the maltose-binding protein. It contained 25 mol iron and 26 mol sulfur, consistent with the predicted six [4Fe4S] centers. The FeS centers in RnfB were reduced with reduced ferredoxin as reductant. These data are consistent with RnfB as the ferredoxin-binding subunit of the complex.
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http://dx.doi.org/10.1016/j.bbabio.2020.148263DOI Listing
November 2020

Water-Gated Proton Transfer Dynamics in Respiratory Complex I.

J Am Chem Soc 2020 08 30;142(32):13718-13728. Epub 2020 Jul 30.

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.

The respiratory complex I transduces redox energy into an electrochemical proton gradient in aerobic respiratory chains, powering energy-requiring processes in the cell. However, despite recently resolved molecular structures, the mechanism of this gigantic enzyme remains poorly understood. By combining large-scale quantum and classical simulations with site-directed mutagenesis and biophysical experiments, we show here how the conformational state of buried ion-pairs and water molecules control the protonation dynamics in the membrane domain of complex I and establish evolutionary conserved long-range coupling elements. We suggest that an electrostatic wave propagates in forward and reverse directions across the 200 Å long membrane domain during enzyme turnover, without significant dissipation of energy. Our findings demonstrate molecular principles that enable efficient long-range proton-electron coupling (PCET) and how perturbation of this PCET machinery may lead to development of mitochondrial disease.
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http://dx.doi.org/10.1021/jacs.0c02789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659035PMC
August 2020

Photolysis of Caged Inositol Pyrophosphate InsP Directly Modulates Intracellular Ca Oscillations and Controls C2AB Domain Localization.

J Am Chem Soc 2020 06 3;142(24):10606-10611. Epub 2020 Jun 3.

Department of Chemistry and Pharmacy, Albert-Ludwigs University Freiburg, Albertstrasse 21, 79104 Freiburg i.B., Germany.

Inositol pyrophosphates constitute a family of hyperphosphorylated signaling molecules involved in the regulation of glucose uptake and insulin sensitivity. While our understanding of the biological roles of inositol heptaphosphates (PP-InsP) has greatly improved, the functions of the inositol octaphosphates ((PP)-InsP) have remained unclear. Here we present the synthesis of two enantiomeric cell-permeant and photocaged (PP)-InsP derivatives and apply them to study the functions in living β-cells. Photorelease of the naturally occurring isomer 1,5-(PP)-InsP led to an immediate and concentration-dependent reduction of intracellular calcium oscillations, while other caged inositol pyrophosphates (3,5-(PP)-InsP, 5-PP-InsP, 1-PP-InsP, 3-PP-InsP) showed no immediate effect. Furthermore, uncaging of 1,5-(PP)-InsP but not 3,5-(PP)-InsP induced translocation of the C2AB domain of granuphilin from the plasma membrane to the cytosol. Granuphilin is involved in membrane docking of secretory vesicles. This suggests that 1,5-(PP)-InsP impacts β-cell activity by regulating granule localization and/or priming and calcium signaling in concert.
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http://dx.doi.org/10.1021/jacs.0c01697DOI Listing
June 2020

Quantifying the heterogeneity of macromolecular machines by mass photometry.

Nat Commun 2020 04 14;11(1):1772. Epub 2020 Apr 14.

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford, OX1 3QZ, UK.

Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization.
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http://dx.doi.org/10.1038/s41467-020-15642-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156492PMC
April 2020

The long Q-loop of Escherichia coli cytochrome bd oxidase is required for assembly and structural integrity.

FEBS Lett 2020 05 13;594(10):1577-1585. Epub 2020 Feb 13.

Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Germany.

Cytochrome bd-I oxidase is a terminal reductase of bacterial respiratory chains produced under low oxygen concentrations, oxidative stress, and during pathogenicity. While the bulk of the protein forms transmembrane helices, a periplasmic domain, the Q-loop, is expected to be involved in binding and oxidation of (ubi)quinol. According to the length of the Q-loop, bd oxidases are classified into the S (short)- and the L (long)-subfamilies. Here, we show that either shortening the Q-loop of the Escherichia coli oxidase from the L-subfamily or replacing it by one from the S-subfamily leads to the production of labile and inactive variants, indicating a role for the extended Q-loop in the stability of the enzyme.
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http://dx.doi.org/10.1002/1873-3468.13749DOI Listing
May 2020

The N-terminal domains of the paralogous HycE and NuoCD govern assembly of the respective formate hydrogenlyase and NADH dehydrogenase complexes.

FEBS Open Bio 2020 03 4;10(3):371-385. Epub 2020 Feb 4.

Institute of Biology/Microbiology, Martin-Luther University Halle-Wittenberg, Germany.

Formate hydrogenlyase (FHL) is the main hydrogen-producing enzyme complex in enterobacteria. It converts formate to CO and H via a formate dehydrogenase and a [NiFe]-hydrogenase. FHL and complex I are evolutionarily related and share a common core architecture. However, complex I catalyses the fundamentally different electron transfer from NADH to quinone and pumps protons. The catalytic FHL subunit, HycE, resembles NuoCD of Escherichia coli complex I; a fusion of NuoC and NuoD present in other organisms. The C-terminal domain of HycE harbours the [NiFe]-active site and is similar to other hydrogenases, while this domain in NuoCD is involved in quinone binding. The N-terminal domains of these proteins do not bind cofactors and are not involved in electron transfer. As these N-terminal domains are separate proteins in some organisms, we removed them in E. coli and observed that both FHL and complex I activities were essentially absent. This was due to either a disturbed assembly or to complex instability. Replacing the N-terminal domain of HycE with a 180 amino acid E. coli NuoC protein fusion did not restore activity, indicating that the domains have complex-specific functions. A FHL complex in which the N- and C-terminal domains of HycE were physically separated still retained most of its FHL activity, while the separation of NuoCD abolished complex I activity completely. Only the FHL complex tolerates physical separation of the HycE domains. Together, the findings strongly suggest that the N-terminal domains of these proteins are key determinants in complex assembly.
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http://dx.doi.org/10.1002/2211-5463.12787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050243PMC
March 2020

Homologous bd oxidases share the same architecture but differ in mechanism.

Nat Commun 2019 11 13;10(1):5138. Epub 2019 Nov 13.

Institut für Biochemie, Albert-Ludwigs-Universität, Freiburg, Germany.

Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.
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http://dx.doi.org/10.1038/s41467-019-13122-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853902PMC
November 2019

The -type cytochrome oxidase assembly factor CcoG is a widely distributed cupric reductase.

Proc Natl Acad Sci U S A 2019 10 30;116(42):21166-21175. Epub 2019 Sep 30.

Institute of Biochemistry and Molecular Biology, Zentrum für Biochemie und Molekulare Zellforschung, Faculty of Medicine, Albert Ludwigs University of Freiburg, 79104 Freiburg, Germany;

Copper (Cu)-containing proteins execute essential functions in prokaryotic and eukaryotic cells, but their biogenesis is challenged by high Cu toxicity and the preferential presence of Cu(II) under aerobic conditions, while Cu(I) is the preferred substrate for Cu chaperones and Cu-transport proteins. These proteins form a coordinated network that prevents Cu accumulation, which would lead to toxic effects such as Fenton-like reactions and mismetalation of other metalloproteins. Simultaneously, Cu-transport proteins and Cu chaperones sustain Cu(I) supply for cuproprotein biogenesis and are therefore essential for the biogenesis of Cu-containing proteins. In eukaryotes, Cu(I) is supplied for import and trafficking by cell-surface exposed metalloreductases, but specific cupric reductases have not been identified in bacteria. It was generally assumed that the reducing environment of the bacterial cytoplasm would suffice to provide sufficient Cu(I) for detoxification and cuproprotein synthesis. Here, we identify the proposed -type cytochrome oxidase ( -Cox) assembly factor CcoG as a cupric reductase that binds Cu via conserved cysteine motifs and contains 2 low-potential [4Fe-4S] clusters required for Cu(II) reduction. Deletion of or mutation of the cysteine residues results in defective -Cox assembly and Cu sensitivity. Furthermore, anaerobically purified CcoG catalyzes Cu(II) but not Fe(III) reduction in vitro using an artificial electron donor. Thus, CcoG is a bacterial cupric reductase and a founding member of a widespread class of enzymes that generate Cu(I) in the bacterial cytosol by using [4Fe-4S] clusters.
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http://dx.doi.org/10.1073/pnas.1913803116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800367PMC
October 2019

Visualizing the movement of the amphipathic helix in the respiratory complex I using a nitrile infrared probe and SEIRAS.

FEBS Lett 2020 02 7;594(3):491-496. Epub 2019 Oct 7.

Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, CMC, Université de Strasbourg CNRS, France.

Conformational movements play an important role in enzyme catalysis. Respiratory complex I, an L-shaped enzyme, connects electron transfer from NADH to ubiquinone in its peripheral arm with proton translocation through its membrane arm by a coupling mechanism still under debate. The amphipathic helix across the membrane arm represents a unique structural feature. Here, we demonstrate a new way to study conformational changes by introducing a small and highly flexible nitrile infrared (IR) label to this helix to visualize movement with surface-enhanced IR absorption spectroscopy. We find that labeled residues K551C and Y590C move to a more hydrophobic environment upon NADH reduction of the enzyme, likely as a response to the reorganization of the antiporter-like subunits in the membrane arm.
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http://dx.doi.org/10.1002/1873-3468.13620DOI Listing
February 2020

A mechanism to prevent production of reactive oxygen species by Escherichia coli respiratory complex I.

Nat Commun 2019 06 11;10(1):2551. Epub 2019 Jun 11.

Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstr. 21, 79104, Freiburg, Germany.

Respiratory complex I plays a central role in cellular energy metabolism coupling NADH oxidation to proton translocation. In humans its dysfunction is associated with degenerative diseases. Here we report the structure of the electron input part of Aquifex aeolicus complex I at up to 1.8 Å resolution with bound substrates in the reduced and oxidized states. The redox states differ by the flip of a peptide bond close to the NADH binding site. The orientation of this peptide bond is determined by the reduction state of the nearby [Fe-S] cluster N1a. Fixation of the peptide bond by site-directed mutagenesis led to an inactivation of electron transfer and a decreased reactive oxygen species (ROS) production. We suggest the redox-gated peptide flip to represent a previously unrecognized molecular switch synchronizing NADH oxidation in response to the redox state of the complex as part of an intramolecular feed-back mechanism to prevent ROS production.
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http://dx.doi.org/10.1038/s41467-019-10429-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560083PMC
June 2019

CyaC, a redox-regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme-B membrane anchor domain.

Mol Microbiol 2019 07 10;112(1):16-28. Epub 2019 Apr 10.

Microbiology and Wine Research, Institute for Molecular Physiology, Johannes Gutenberg-University of Mainz, Becherweg 15, 55099, Mainz, Germany.

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane-integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra-histidine signature that is universally present in the membrane anchors of bacterial diheme-B succinate-quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme-B protein and carried a binuclear iron-sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme-B in the membrane and loss of AC activity. Heme-B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q or Q H ). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q and O , or NADH and Q H respectively. We conclude that CyaC-like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.
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http://dx.doi.org/10.1111/mmi.14251DOI Listing
July 2019

Iron-sulfur cluster carrier proteins involved in the assembly of Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

Mol Microbiol 2019 01 23;111(1):31-45. Epub 2018 Oct 23.

Albert-Ludwigs-Universität, Institut für Biochemie, Albertstr. 21, D-79104, Freiburg, Germany.

The NADH:ubiquinone oxidoreductase (respiratory complex I) is the main entry point for electrons into the Escherichia coli aerobic respiratory chain. With its sophisticated setup of 13 different subunits and 10 cofactors, it is anticipated that various chaperones are needed for its proper maturation. However, very little is known about the assembly of E. coli complex I, especially concerning the incorporation of the iron-sulfur clusters. To identify iron-sulfur cluster carrier proteins possibly involved in the process, we generated knockout strains of NfuA, BolA, YajL, Mrp, GrxD and IbaG that have been reported either to be involved in the maturation of mitochondrial complex I or to exert influence on the clusters of bacterial complex. We determined the NADH and succinate oxidase activities of membranes from the mutant strains to monitor the specificity of the individual mutations for complex I. The deletion of NfuA, BolA and Mrp led to a decreased stability and partially disturbed assembly of the complex as determined by sucrose gradient centrifugation and native PAGE. EPR spectroscopy of cytoplasmic membranes revealed that the BolA deletion results in the loss of the binuclear Fe/S cluster N1b.
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http://dx.doi.org/10.1111/mmi.14137DOI Listing
January 2019

Charge transfer through a fragment of the respiratory complex I and its regulation: an atomistic simulation approach.

Phys Chem Chem Phys 2018 Aug;20(30):20023-20032

Institut für Physikalische Chemie, Universität Freiburg, Albertstraße23a, 79104 Freiburg im Breisgau, Germany.

We simulate electron transfer within a fragment of the NADH:ubiquinone oxidoreductase (respiratory complex I) of the hyperthermophilic bacterium Aquifex aeolicus. We apply molecular dynamics simulations, thermodynamic integration, and a thermodynamic network least squares analysis to compute two key parameters of Marcus' theory of charge transfer, the thermodynamic driving force and the reorganization energy. Intramolecular contributions to the Gibbs free energy differences of electron and hydrogen transfer processes, ΔG, are accessed by calibrating against experimental redox titration data. This approach permits the computation of the interactions between the species NAD+, FMNH2, N1a-, and N3-, and the construction of a free energy surface for the flow of electrons within the fragment. We find NAD+ to be a strong candidate for the regulation of charge transfer.
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http://dx.doi.org/10.1039/c8cp02420kDOI Listing
August 2018

Low cost, microcontroller based heating device for multi-wavelength differential scanning fluorimetry.

Sci Rep 2018 01 23;8(1):1457. Epub 2018 Jan 23.

Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstr. 21, 79104, Freiburg i. Br., Germany.

Differential scanning fluorimetry is a popular method to estimate the stability of a protein in distinct buffer conditions by determining its 'melting point'. The method requires a temperature controlled fluorescence spectrometer or a RT-PCR machine. Here, we introduce a low-budget version of a microcontroller based heating device implemented into a 96-well plate reader that is connected to a standard fluorescence spectrometer. We demonstrate its potential to determine the 'melting point' of soluble and membranous proteins at various buffer conditions.
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http://dx.doi.org/10.1038/s41598-018-19702-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780519PMC
January 2018

Reduction of the off-pathway iron-sulphur cluster N1a of Escherichia coli respiratory complex I restrains NAD dissociation.

Sci Rep 2017 08 18;7(1):8754. Epub 2017 Aug 18.

Albert-Ludwigs-Universität, Institut für Biochemie, Albertstr. 21, Chemie-Hochhaus, 79104, Freiburg i. Br., Germany.

Respiratory complex I couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The reaction starts with NADH oxidation by a flavin cofactor followed by transferring the electrons through a chain of seven iron-sulphur clusters to quinone. An eighth cluster called N1a is located proximally to flavin, but on the opposite side of the chain of clusters. N1a is strictly conserved although not involved in the direct electron transfer to quinone. Here, we show that the NADH:ferricyanide oxidoreductase activity of E. coli complex I is strongly diminished when the reaction is initiated by an addition of ferricyanide instead of NADH. This effect is significantly less pronounced in a variant containing N1a with a 100 mV more negative redox potential. Detailed kinetic analysis revealed that the reduced activity is due to a lower dissociation constant of bound NAD. Thus, reduction of N1a induces local structural rearrangements of the protein that stabilise binding of NAD. The variant features a considerably enhanced production of reactive oxygen species indicating that bound NAD represses this process.
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http://dx.doi.org/10.1038/s41598-017-09345-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562879PMC
August 2017

Significance of [2Fe-2S] Cluster N1a for Electron Transfer and Assembly of Escherichia coli Respiratory Complex I.

Biochemistry 2017 06 25;56(22):2770-2778. Epub 2017 May 25.

Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-University , Freiburg, Germany.

NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with proton translocation across the membrane. NADH reduces a noncovalently bound FMN, and the electrons are transported further to the quinone reduction site by a 95 Å long chain of seven iron-sulfur (Fe-S) clusters. Binuclear Fe-S cluster N1a is not part of this long chain but is located within electron transfer distance on the opposite site of FMN. The relevance of N1a to the mechanism of complex I is not known. To elucidate its role, we individually substituted the cysteine residues coordinating N1a of Escherichia coli complex I by alanine and serine residues. The mutations led to a significant loss of the NADH oxidase activity of the mutant membranes, while the amount of the complex was only slightly diminished. N1a could not be detected by electron paramagnetic resonance spectroscopy, and unexpectedly, the content of binuclear cluster N1b located on a neighboring subunit was significantly decreased. Because of the lack of N1a and the partial loss of N1b, the variants did not survive detergent extraction from the mutant membranes. Only the C97A variant retained N1a and was purified by chromatographic steps. The preparation showed a slightly diminished NADH/ferricyanide oxidoreductase activity, while the NADH:decyl-ubiquinone oxidoreductase activity was not affected. N1a of this preparation showed unusual spectroscopic properties indicating a different ligation. We discuss whether N1a is involved in the physiological electron transfer reaction.
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http://dx.doi.org/10.1021/acs.biochem.6b01058DOI Listing
June 2017

Wide Distribution of Foxicin Biosynthetic Gene Clusters in Strains - An Unusual Secondary Metabolite with Various Properties.

Front Microbiol 2017 21;8:221. Epub 2017 Feb 21.

Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University of Freiburg Freiburg im Breisgau, Germany.

Tü6028 is known to produce the polyketide antibiotic polyketomycin. The deletion of the oxygenase gene led to a non-polyketomycin-producing mutant. Instead, novel compounds were produced by the mutant, which have not been detected before in the wild type strain. Four different compounds were identified and named foxicins A-D. Foxicin A was isolated and its structure was elucidated as an unusual nitrogen-containing quinone derivative using various spectroscopic methods. Through genome mining, the foxicin biosynthetic gene cluster was identified in the draft genome sequence of . The cluster spans 57 kb and encodes three PKS type I modules, one NRPS module and 41 additional enzymes. A gene-inactivated mutant of Tü6028 Δ is unable to produce foxicins. Homologous biosynthetic gene clusters were found in more than 20 additional strains, overall in about 2.6% of all sequenced genomes. However, the production of foxicin-like compounds in these strains has never been described indicating that the clusters are expressed at a very low level or are silent under fermentation conditions. Foxicin A acts as a siderophore through interacting with ferric ions. Furthermore, it is a weak inhibitor of the aerobic respiratory chain and shows moderate antibiotic activity. The wide distribution of the cluster and the various properties of the compound indicate a major role of foxicins in strains.
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http://dx.doi.org/10.3389/fmicb.2017.00221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5318452PMC
February 2017

Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism.

Sci Rep 2016 11 25;6:37743. Epub 2016 Nov 25.

Aix-Marseille Univ, CNRS, IMM, LCB UMR7283, Marseille, France.

A major gap of knowledge in metalloproteins is the identity of the prefolded state of the protein before cofactor insertion. This holds for molybdoenzymes serving multiple purposes for life, especially in energy harvesting. This large group of prokaryotic enzymes allows for coordination of molybdenum or tungsten cofactors (Mo/W-bisPGD) and Fe/S clusters. Here we report the structural data on a cofactor-less enzyme, the nitrate reductase respiratory complex and characterize the conformational changes accompanying Mo/W-bisPGD and Fe/S cofactors insertion. Identified conformational changes are shown to be essential for recognition of the dedicated chaperone involved in cofactors insertion. A solvent-exposed salt bridge is shown to play a key role in enzyme folding after cofactors insertion. Furthermore, this salt bridge is shown to be strictly conserved within this prokaryotic molybdoenzyme family as deduced from a phylogenetic analysis issued from 3D structure-guided multiple sequence alignment. A biochemical analysis with a distantly-related member of the family, respiratory complex I, confirmed the critical importance of the salt bridge for folding. Overall, our results point to a conserved cofactors insertion mechanism within the Mo/W-bisPGD family.
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http://dx.doi.org/10.1038/srep37743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123574PMC
November 2016

Creation of a gold nanoparticle based electrochemical assay for the detection of inhibitors of bacterial cytochrome bd oxidases.

Bioelectrochemistry 2016 Oct 7;111:109-14. Epub 2016 Jun 7.

Laboratoire de Bioelectrochimie et Spectroscopie, UMR 7140, Chimie de la Matière Complexe, Université de Strasbourg, CNRS, Strasbourg, France. Electronic address:

Cytochrome bd oxidases are membrane proteins expressed by bacteria including a number of pathogens, which make them an attractive target for the discovery of new antibiotics. An electrochemical assay is developed to study the activity of these proteins and inhibition by quinone binding site tool compounds. The setup relies on their immobilization at electrodes specifically modified with gold nanoparticles, which allows achieving a direct electron transfer to/from the heme cofactors of this large enzyme. After optimization of the protein coverages, the assay shows at pH7 a good reproducibility and readout stability over time, and it is thus suitable for further screening of small molecule collections.
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http://dx.doi.org/10.1016/j.bioelechem.2016.06.001DOI Listing
October 2016
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