Research report:
Mikrobiologie AG Thauer
Rolf Thauer (born 05.10.39)
Physicum (Medicine); University of Frankfurt, 1961
Diplom (Biochemistry), University of Tübingen, 1966
Dr. rer. nat. (Biochemistry), University of Freiburg, 1968
Habilitation (Biochemistry), University of Freiburg, 1971
Postdoc (Biochemistry), Case Western Reserve, Cleveland Ohio, 1972
Professor of Biochemistry, Ruhr University Bochum, 1972-1975
Professor of Microbiology at the Faculty of Biology of the Philipps University Marburg, from 1976 to 2005
Director and Head of the Department of Biochemistry at the MPI Marburg, since 1991 Dr. h.c. of the ETH Zürich (2001)
projects:
- Publications 2004-2005
- Reviews
- Diploma theses
- Doctoral theses
- Guest scientists
- Structure of the group (11/2005)
- Invited lectures abroad
- Grants
- Address
Biochemistry of methanogenic archaea
My research group is presently focused on the unravelling of the structure and catalytic mechanism of methyl-coenzyme M reductase in methanogenic and methanotrophic archaea and of the iron-sulfur-cluster free hydrogenase in these microorganisms. From the results only those obtained for methyl-coenzyme M reductase are described in the following. For the results on the iron-sulfur-cluster free hydrogenase the reader is referred to the report by Seigo Shima.
UIn our work on the structure and function of methyl-coenzyme M reductase my group is collaborating with Seigo Shima from our Institute, with Ulrich Ermler from the Max Planck Institute for Biophysics in Frankfurt, with Friedrich Widdel from the Max Planck Institute for Marine Microbiolgy in Bremen and with Bernhard Jaun and Arthur Schweiger from the ETH Zürich.
Methyl-coenzyme M reductase from methanogenic archaea
Methyl-coenzyme reductase (MCR) catalyzes the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB (ΔGo´= - 30 kJ/mol) (Figure 1A). This is the methane forming reaction in all methanogenic archaea by which more than one billion tons of methane are formed annually. MCR contains nickel bound within cofactor F430 (Fig. 1 B).
Fig. 1. Methyl-coenzyme M reductase (MCR) from methanogenic archaea. (A) The reaction catalyzed by the enzyme. The specific activity for methane oxidation (1 mU/mg; 1U = 1µmol/min) was calculated via the Haldane equation from the maximal specific activity of methane formation (100 U/mg) and the free energy change (ΔGo´ = - 30 kJ/mol) associated with the reaction (Shima and Thauer 2005). (B) The structure of the prosthetic group (coenzyme F430) of the enzyme. The prosthetic group found in MCR from methanotrophic archaea is 172-methylthio-F430.
Structure of MCR: MCR from methanogenic archaea is composed of three different subunits in an α2ß2γ2 arrangement and contains two mol of a nickel porphinoid, designated coenzyme F430, as prosthetic group, which has to be in the Ni(I) oxidation state for the enzyme to be active. The crystal structure of MCR with F430 in the Ni(II) oxidation state was resolved to 1.16 Å. It revealed that the subunits are intertwined such that they form two structurally interlinked active sites made up of the subunits α, α´ß and γ and α´, α, ß´and γ´, respectively. Each active site harbors one F430 molecule buried deeply within the protein and accessible from the outside only via a 50 Å long channel made up of mainly hydrophobic amino acid residues. Near to the active site are five modified amino acids: a thioglycine, a N-methyl-histidine, a S-methyl cysteine, a 5-(S)-methyl arginine and a 2-(S)-methyl glutamine. Labeling studies have shown that the methyl groups are biosynthetically derived from the methyl group of methionine and not from the methyl group of methyl-coenzyme M.The redox state of F430 in the active enzyme: Within the last years there was a major controversy with respect to the number of electrons required to reduce MCR from the inactive EPR silent Ni(II) state to the active Ni(I) state. Evidence was published by others that three electrons are required indicating that besides the nickel the porphinoid ligand was also reduced. It was subsequently unambiguously shown by us that the stoichiometry is 1. Likewise there was controversy over the oxidation state of nickel in the EPR active but enzymatically inactive MCRox forms. Evidence was published by others that the EPR signal exhibited by MCRox could only be derived from Ni(I). More detailed analyses revealed, however, that the nickel in the MCRox form can best be described as a Ni(III) thiolate in equilibrium with a Ni(II) thiyl radical complex (Duin et al., 2004, Goenrich et al., 2004, Harmer et al., 2005).
The catalytic mechanism: The active site structure indicates that methyl-coenzyme M reduction to methane takes place in a hydrophobic pocket from which water is completely excluded. When entering the active site via the hydrophobic channel methyl-coenzyme is stripped of water and after the reaction the heterodisulfide is expelled into the water phase. The reaction most probably starts with a conformational change within the active site which is induced upon binding of coenzyme B. This is indicated by the finding that upon addition of coenzyme B to active MCR in the presence of coenzyme M the enzyme is converted from the MCRred1c state exhibiting an axial EPR signal into the MCRred2 state exhibiting a highly rhombic EPR signal (Goenrich et al. 2005).
There is evidence that the two active sites are structurally and functionally interlinked. The two α subunits in the enzyme are intertwined such that a conformational change in the one active site (made up of the subunits α, α´,ß and γ) can be transmitted to the other active site (made up of the subunits α´, α, ß´and γ´) and vice versa. An indication for the coupling of the two active sites is the finding that at most 50% of the enzyme are converted from the MCRred1c state into the MCRred2 state upon addition of coenzyme B (Goenrich et al., 2005). MCR thus shows “half-of-the-sites” reactivity. Based on these findings it has been proposed that the enzyme operates according to a dual stroke engine mechanism. This would allow the coupling of endergonic steps of the catalytic cycle in one active site to the exergonic steps in the other site. The coupling is predicted to lower the activation energy for both the forward and the back reaction (Goenrich et al., 2005) (Fig. 2).
Fig. 2. Dual stroke engine mechanism proposed for methyl-coenzyme M reductase. The mechanism allows the coupling of endergonic steps of the catalytic cycle in the one active site to exergonic steps in the other active site. The coupling is predicted to lower the activation energy both in the forward and the back reaction.
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Two different mechanisms for methyl-coenzyme M reduction have been proposed. In mechanism 1 the methyl group of methyl-coenzyme M reacts with the Ni(I) in a nucleophilic substitution reaction yielding methyl-Ni(III) and coenzyme M, which in turn react to methyl-Ni(II) and the thiyl radical of coenzyme M. Methyl-Ni(II) then reacts with a proton in an electrophilic substitution reaction to methane and Ni(II) and the coenzyme M thiyl radical reacts with coenzyme B yielding a disulfide anion radical, which is a strong reductant and which reduces Ni(II) back to Ni(I) thus closing the catalytic cycle. This mechanism is mainly supported by the findings that MCR-catalyzed methyl-coenzyme M reduction proceeds with inversion of stereoconfiguration, that enzyme bound Ni(I)F430 reacts with 3-bromopropane sulfonate to alkyl Ni(III) (Goenrich et al., 2004) and that free Ni(I)F430 in aprotic solvents reacts with methylbromide to methyl-Ni(II)F430, which subsequently can be protonolyzed to methane and Ni(II)F430. Also, the finding that MCR catalyzes the reduction of ethyl-coenzyme M with less than 1% of the catalytic efficiency of methyl-coenzyme M reduction is consistent with a nucleophilic substitution as first step in the catalytic cycle (Goenrich et al., 2004).
However, density function calculations have revealed that mechanism 1 is energetically not favorable, although the calculations have not taken into account that the two active sites could be energetically coupled. Based on their calculations Ghosh and Siegbahn have proposed mechanism 2, in which as first step in the catalytic cycle the methyl thioether bond in methyl-coenzyme M is reductively cleaved yielding a Ni(II) thiolate and a methyl radical which in turn reacts with HS-CoB yielding methane and a CoB-S. thiyl radical. The latter reacts with coenzyme M thiolate to the disulfide anion radical which like in mechanism 1 is used to re-reduce the Ni(II)F430 in MCR to the Ni(I) oxidation state. Mechanism 2 is backed up by our experimental finding that in active MCR coenzyme M reversibly coordinates with its thiol sulfur to Ni(I) of F430 when coenzyme B is present (see Report 2002-2003).
Considering an involvement of MCR in AOM (see below), both mechanisms are not very likely. Assuming mechanism 1 methane oxidation would start by a nucleophilic attack of methane to Ni(II) of F430. This can be excluded since Ni(II) of F430 is not electrophilic enough to be able to attack methane with a pKa of above 40. The low electrophilicity of F430(Ni II) is reflected by the low redox potential Eo´ < - 600 mV of the Ni(II)F430/Ni(I)F430 couple. Mechanism 2 is likewise problematic. Methane oxidation would start by the reaction of methane with the CoB-S. thiyl radical. The bond dissociation energy of the C-H bond in methane is 439 kJ/mol as compared to that of the S-H bond of only 365 kJ/mol, which makes a reaction of methane with a thiyl radical yielding a methyl radical and a thiol thermodynamically very unfavorable.
A third mechanism is therefore proposed which is a modification of mechanism 1. Methyl-Ni(III)F430 generated from Ni(I)F430 and methyl-coenzyme M reacts with a proton yielding methane and Ni(III)F430. Free Ni(III)F430 is a very strong electrophile (Eo’ > + 1 V) and this is probably also true for enzyme bound Ni(III)F430 although the EPR spectrum and the UV/visible spectrum of free Ni(III)F430 and those of Ni(III)F430 in MCRox (see above) are very different indicating major differences in the coordination sphere (Duin et al., 2004). The back reaction, the oxidation of methane, would therefore start with the electrophilic metalation of methane by reaction of methane either end-on or side-on with the high-valent Ni(III) complex in MCR as described for the activation of C-H bonds by other high-valent metal complexes (Shima and Thauer 2005).
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Methyl-coenzyme M reductase from methanotrophic archaea
The archaea catalyzing the anaerobic oxidation of methane (AOM) have been shown to contain gene homologues of mcrBGA and of most of the other genes involved in CO2 reduction to methane in methanogenic archaea. Only a gene homologue of mer coding for methylenetetrahydromethanopterin reductase was not found. Methanotrophic archaea present in the microbial mats catalyzing AOM in the Black Sea were found to contain at least two different MCR designated Ni-protein I and Ni-protein II, which could be separated by anion exchange chromatography (Krüger et al., 2003). Ni-protein II contained normal F430 with a molecular mass of 905 Da, whereas Ni-protein I contains a modified F430 with a molecular mass of 951 Da. Ni-protein I is present in a concentration of 7% of the extracted soluble proteins and Ni-protein II in a concentration of up to 3%. The N-terminal amino acid sequences of the three subunits of Ni-protein I were determined by Edman degradation and used to identify the encoding genes in a metagenome library of the mats. The codon usage and tetranucleotide signature of the three genes in the cluster mcrBGA revealed that the three genes are located on the genome of the dominant ANME-1 archaeon present in the mats. The deduced amino acid sequences show a high degree of sequence similarity to MCR from methanogenic archaea but with some distinct differences: in the α-subunit the glutamine, which in MCR from methanogenic archaea is post-translationally methylated at C2, is substituted by a valine. Two amino acids downstream of the valine there is a cysteine rich sequence CCX4CX5C not present in MCR from methanogenic archaea (Figure 3). This cysteine rich stretch in the α-subunit of MCR is also found in the DNA sequence of the gene homologues present in the metagenomic library of other microbial consortia catalyzing AOM. Due to these differences and the presence of a modified F430 the catalytic properties of the enzyme from methanotrophic archaea could differ significantly from those of MCR from methanogenic archaea. Thus, the catalytic efficiency could be higher (Shima and Thauer 2005).
As indicated above, the most abundant MCR in microbial mats catalyzing AOM in the Black Sea contains a modified F430 with a molecular mass of 951 Da (Krüger et al., 2003). The structure of the 951 Da cofactor has recently been determined to be 172-methylthio F430. This modified F430, which can easily be identified by its characteristic MALDI-TOF mass spectrum, was not found in any of the methanogenic archaea analyzed in this respect nor in microbial cells present in the anaerobic digesters of the waste treatment plant in Marburg. The modified cofactor was found, however, in all habitats with AOM. But besides the modified cofactor the normal F430 with a mass of 905 Da is always present. It has already been mentioned that Ni-protein II, isolated from the microbial mats catalyzing AOM in the Black Sea, contained only the 905 Da cofactor (Krüger et al., 2003) indicating that AOM is not restricted to MCR containing 172-methylthio F430.
Publications 2004-2005
Krüger, M., Meyerdierks, A., Glöckner, F.O., Amann, R., Widdel, F., Kube, M., Reinhardt, R., Kahnt, J., Böcher, R., Thauer, R.K. & Shima, S. (2003) A conspicuous nickel protein in microbial mats that oxidise methane anaerobically. Nature 426, 878-881.
Lyon, E., Shima, S., Buurman, G., Chowdhuri, S., Batschauer, A., Steinbach, K. & Thauer, R.K. (2004) UV-A/blue-light inactivation of the “metal-free” hydrogenase (Hmd) from methanogenic archaea. The enzyme contains functional iron after all. Eur. J. Biochem. 271, 195-204.
Aufhammer, S., Warkentin, E., Berk, H., Shima, S., Thauer, R.K. & Ermler, U. (2004) Coenzyme binding in F420-dependent secondary alcohol dehydrogenase, a member of the bacterial luciferase family. Structure 12, 361-370.
Duin, E.C., Signor, L., Piskorski, R., Mahlert, F., Clay, M.D., Goenrich, M., Thauer, R.K., Jaun, B. & Johnson, M.K. (2004) Spectroscopic investigation of the nickel-containing porphinoid cofactor F430. Comparison of the free cofactor in the +1, +2 and +3 oxidation states with the cofactor bound to methyl-coenzyme M reductase in the silent, red and ox forms. J. Biol. Inorg. Chem. 9, 563-576.
Sakasegawa, S-I., Hagemeier, C.H., Thauer, R.K., Essen, L. & Shima, S. (2004) Structural and functional analysis of gpsA gene product of Archaeoglobus fulgidus: A glycerol-3-phosphate dehydrogenase with an unusual NADP+ preference. Protein Science 13, 3161-3171.
Seedorf, H., Dreisbach, A., Hedderich, R., Shima, S. & Thauer, R.K. (2004) F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification. Arch. Microbiol. 182, 126-137.
Goenrich, M., Mahlert, F., Duin, E.C., Bauer, C., Jaun, B. & Thauer, R.K. (2004) Probing the reactivity of Ni in the active site of methyl-coenzyme M reductase with substrate analogues. J. Biol. Inorg. Chem. 9, 691-705.
Shima, S. Lyon, E., Sordel-Klippert, M., Kauß, M., Kahnt, J., Thauer, R.K. Steinbach, K., Xie, X., Verdier, L. & Griesinger, C. (2004) The cofactor of the iron-sulfur cluster free hydrogenase Hmd: Structure of the light-inactivation product. Angew. Chem. Int. Ed. Engl. 43, 2547-2551 (deutsche Ausgabe: 116, 2601-2605).
Lyon, E.J., Shima, S., Böcher, R., Thauer, R.K., Grevels, F.-W., Bill, E., Woseboom, W. & Albracht, S.P.J. (2004) Carbon monoxide as an intrinsic ligand to iron in the active site of the iron-sulfur cluster free hydrogenase (Hmd) as revealed by infrared spectroscopy. J. Am. Chem. Soc. 126, 14239-14248.
Buchenau, B. & Thauer R.K. (2004) Tetrahydrofolate-specific enzymes in Methanosarcina barkeri and growth dependence of this methanogenic archaeon on folic acid or p-aminobenzoic acid. Arch. Microbiol. 182, 313-325.
Acharya, P., Goenrich, M., Hagemeier, C.H., Demmer, U., Vorholt, J.A., Thauer, R.K. & Ermler, U. (2005) How an enzyme binds the C1 carrier tetrahydromethanopterin: Structure of the tetrahydromethanopterin-dependent formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1. J. Biol. Chem. 280, 13712-13719.
Goenrich, M., Duin, E.C., Mahlert, F. & Thauer, R.K. (2005) Temperature dependence of methyl-coenzyme M reductase activity and of the formation of the methyl-coenzyme M red2 state induced by coenzyme B. J. Biol. Inorg. Chem. 10, 333-342.
Aufhammer, S.W., Warkentin, E., Ermler, U., Hagemeier, C.H., Thauer, R.K. & Shima, S. (2005) Crystal structure of methylenetetrahydromethanopterin reductase (Mer) in complex with coenzyme F420: Architecture of F420/FMN binding site of enzymes within the nonprolyl cis-peptide containing bacterial luciferase family. Protein Science 14, 1840-1849.
Warkentin, E., Hagemeier, C.H., Shima, S., Thauer, R.K. & Ermler, U. (2005) The structure of F420-dependent methylenetetrahydromethanopterin dehydrogenase: a crystallographic ‘superstructure’ of the selenomethionine-labelled protein crystal structure. Act. Cryst. D 61, 198-202.
Shima, S., Lyon, E.J., Thauer, R.K., Mienert, B. & Bill, E. (2005) Mössbauer studies of the iron-sulfur cluster-free hydrogenase (Hmd): The electronic state of the mononuclear Fe active site. J. Am. Chem. Soc. 127, 10430-10435.
Goenrich, M., Thauer, R.K., Yurimoto, H., Kato, N. (2005) Formaldehyde activating enzyme (Fae) and hexulose-6-phosphate synthase (Hps) in Methanosarcina barkeri : a possible function in ribose-5-phosphate biosynthesis. Arch. Microbiol. 184, 41-48.
Seedorf,H., Kahnt, J., Pierik, A.J. & Thauer, R.K. (2005) Si-face stereospecificity at C5 of coenzyme F420 for F420H2 oxidase from methanogenic archaea as determined by mass spectrometry. FEBS J. 272, 5337-5342.
Harmer, J., Finazzo, C., Piskorski, R., Bauer, C., Jaun, B., Duin, E.C., Goenrich, M., Thauer, R.K., van Doorslaer, S. & Schweiger, A. (2005) Spin density and coenzyme M coordination geometry of the ox1 form of methyl-coenzyme M reductase: A pulse EPR study. J. Am. Chem Soc. in press
Fricke, F., Seedorf, H., Henne, A., Krüer, M., Liesegang, H., Hedderich, R., Gottschalk, G. & Thauer, R.K. (2005) The genome sequence of Methanosphaera stadtmanae reveals why this human intestinal archaeon is restricted to methanol and H2 for methane formation and ATP synthesis. J. Bacteriol. in press
Pilak, O., Mamat, B., Vogt, S., Hagemeier, C.H., Thauer, R.K., Shima, S., Vonrhein, C., Warkentin, E. & Ermler, U. (2005) The crystal structure of the apoenzyme of the iron-sulfur cluster free hydrogenase (Hmd). FEBS J. submitted
Acharya, P., Warkentin, E., Ermler, U., Thauer, R.K. & Shima, S. (2005) The structure of formylmethanofuran:tetrahydromethanopterin formyltransferase in complex with its coenzymes. J. Mol. Biol. submitted
Hinderberger, D., Piskorski, R., Goenrich, M., Harmer, J., Thauer, R.K., Schweiger, A. & Jaun, B. (2006) A nickel-alkyl bond in an inactivated state of the enzyme catalyzing methane formation.
Reviews
Shima, S., Thauer, R.K. & Ermler, U. (2004) Hyperthermophilic and salt-dependent formyltransferase from Methanopyrus kandleri. Biochem. Soc. Trans. 32, 269-272
Shima, S. & Thauer, R.K. (2005) Methyl-coenzyme M reductase (MCR) and the anaerobic oxidation of methane (AOM) in methanotrophic archaea. Curr. Opin. Microbiol. in press
Thauer, R.K. & Shima S. (2006) Methyl-coenzyme M reductase in methanogenic and methanotrophic archaea. In Archaea Biology (Garrett, R. & Klenk, H.-P., eds) Blackwell Publishing, Inc. Malden, USA, pp
Thauer, R.K. (2005) 58 Semester Hochschullehrer der Mikrobiologie am Fachbereich Biologie der Philipps-Universität Marburg. Ergebnisbericht zur Abschiedsvorlesung am 11. Februar 2005. Eigenverlag, pp 1-106.
Shima, S & Thauer, R.K. (2006) Anaerobic methane oxidation by archaea: a biochemical approach. Bioscience and Industry, in press
Thauer, R.K. & Hamilton, W.A. (2006) Energy Metabolism of Sulphate Reducing Bacteria. In Sulphate-Reducing Bacteria: Environmental and Engineered Systems (Barton, L.L. & Hamilton, W.A., eds) Cambridge University Press, in press
Diploma theses
Pilak, Oliver (2004) Synthese von Hydrogenase Hmd und von zwei vermuteten Isoenzymen in Methanothermobacter marburgensis bei Wachstum unter Fe-, NH3- oder Ni-Limitierung
Vogt, Sonja (2004) Die Rolle von Cystein 176 im aktiven Zentrum der Eisen-Schwefel-Cluster-freien Hydrogenase Hmd von Methanococcus jannaschii
Doctoral theses
Aufhammer, Stephan (2004) Kristallstrukturen von F420-abhängiger Alkohol-Dehydrogenase (Adf) und F420-abhängiger Methylentetrahydromethanopterin-Reduktase (Mer)
Buchenau, Bärbel (2004) Tetrahydrofolat-spezifische Enzyme im Baustoffwechsel von Methanosarcina barkeri sowie die Rolle von Folsäure und p-Aminobenzoesäure als Wachstumsfaktoren in diesem Archaeon
Goenrich, Meike (2004) Untersuchungen zum Katalysemechanismus von Methyl-Coenzym-M-Reduktase in methanogenen Archaeen
Guest scientists
Hamilton, Allan (United Kingdom)*
Kato, Nobuo (Japan)*
Li, Fuli (China)*
Lyon, Erica (USA)*
Netrusov, Alexander (Russia)*
*part of the time reported
Structure of the group (11/2005)
Group leader
Prof. Dr. Rolf Thauer
Secretary
Monika Schmidt
Postdoctoral fellows
Dr. Bärbel Buchenau
Dr. Meike Goenrich
Dr. Christoph Hagemeier
Dr. Fuli Li
PhD/Diploma students
Julia Hinderberger
Henning Seedorf
Technical assistants
Reinhard Böcher
Johanna Moll
Invited lectures abroad
Advisory Board Meeting of the Southeast Collaboratory for Structural Geneomics, Athens/Georgia/USA (18-22/05/04);
● Shell Global Solutions International, Amsterdam/Netherlands (15-16/06/04);
● Gordon Research Conference “Environmental Bioinorganic Chemistry”, Bates College, Lewistone, Main/USA (20-25/06/04);
● Gordon Research Conference “Molecular Basis of Microbial One Carbon Metabolism”,Mamit Holyoke College, South Hadley, Ma/USA (01-06/08/04);
● 7th International Hydrogenase Conference, University of Reading/UK (24-29/08/04);
● 19th Enzyme Mechanisms Conference, Asilomar/USA (05-09/01/05);
● Symposium “Biotechnology and its future on C1 microorganism”, Sapporo/Japan (25/03-01/04/05);
● ASM 105th General Meeting, Division K Lecture, Atlanta/USA (05-09/06/05);
● Int. Congress of Bacteriology and Appl. Microbiology (IUMS), San Francisco/USA (24-28/07/05);
● ICBIC-12, Ann Arbor/USA (31/07-06/08/05);
● Gordon Research Conference “Vitamin B12 & Corphins”, Oxford/UK (18-23/09/05);
● Roger Y. Stanier Memorial Lecture, University of California, Berkeley/USA (03/11/05);
● Seminar at the University of California, Davis/USA (4/11/05);
● Pacifichem, Honolulu/Hawaii/USA (15-20/12/05)
Grants
DFG Graduiertenkolleg „Proteinfunktion auf atomarer Ebene“ – Support for 1 graduate student*
Krupp Foundation – Scholarship for one postdoc*
Peter und Traudel Engelhorn Stiftung: Support for 1 graduate student*
BMBF: Support of the project “Structure and function of the iron-sulfur-cluster free hydrogenase from methanogenic Archaea”
Dow Chemical Company
Fonds der Chemischen Industrie
*part of the time reported
Address
Prof. Dr. Rolf Thauer
Max-Planck-Institut
für terrestrische Mikrobiologie
Karl-von-Frisch-Straße
D-35043 Marburg/Germany
Phone +49 6421 178-101
Fax +49 6421 178-109
E-mail thauer@mpi-marburg.mpg.de
