Lorenz Adrian Johann Heider Jürgen Moser Carola Schulzke
Matthias Boll Gunhild Layer Constanze Pinske Günter Schwarz
Holger Dobbek Silke Leimkühler Antonio J. Pierik Seigo Shima
Oliver Einsle Roland Lill Gary Sawers Basem Soboh
Thorsten Friedrich Ralf R. Mendel Raffael Schaffrath Matthias Ullmann
Ute Krämer Franc Meyer Volker Schünemann
Günter Fritz /
Julia Fritz-Steuber
Serena DeBeer /
Frank Neese
Carsten Berndt /
Christopher Lillig
Oliver Lenz /
Ingo Zebger

Comparative membrane metalloproteomics of respiring anaerobes

Lorenz Adrian
Helmholtz-Zentrum für Umweltforschung, Leipzig

We will study the occurrence and distribution of membrane-bound metalloprotein complexes in diverse prokaryotes by ICP-MS (inductively coupled plasma mass spectrometry) and LC-MS/MS (liquid chromatography mass spectrometry) to quantify metals and protein subunits, respectively. The study will consist of three work packages: First, the comparative analysis of metals and proteins in membrane preparations of whole cells, i.e. shotgun membrane metalloproteomics; second, the comparative analysis of metals and proteins in metalloprotein complexes after separation of the complexes by native gel electrophoresis; and third, the mass spectrometric analysis of metal-containing organic cofactors associated with metalloproteins. We will focus on protein complexes containing the metals Fe, Ni, Co and Mo and the metal-containing cofactors F430, cobalamin and Mo-pterins, all involved in key reactions of anaerobic energy-conserving processes.

Planned cooperation

We plan to use microorganisms studied in other groups also for our metalloproteome analyses. We are happy about further suggestions of microorganisms to include in our study and will share the data for joint use. In addition we offer scientific cooperation on our instruments for protein/metabolite/cofactor identification (or identification of their modifications) by nLC-MS/MS (Orbitrap Fusion), metal analysis (ICP-MS), electron microscopy, Tof-SIMS, FT-ICR or nanoSIMS.

Projected cooperations include:
AG Shima (Strains)
AG Sawers (Ni/Fe hydrogenases in E. coli)
AG Layer (Proteins in F430 biosynthesis)
AG Leimkühler (CISM-complexes in respiratory chains [Mo])
AG Dobbek (protein mass spectrometry)
AG Lill (nanoSIMS)

Interconnections of metallo enzyme synthesis machineries in Geobacter metallireducens: cofactor assembly and function of W/FeS-containing BamB

Matthias Boll
University of Freiburg

Aromatic compounds can be fully degraded by anaerobic bacteria using oxygenase-independent pathways. Here, most monocyclic aromatic growth substrates are converted to the central intermediate benzoyl-CoA, that is dearomatized by reductases to cyclic 1,5-dienoyl-CoA at an extremely low redox potential (E°’ = –622 mV). Obligate anaerobes such as the Fe(III) respiring model organism Geobacter metallireducens employ a W-cofactor containing class II benzoyl-CoA reductase. The active site component BamB belongs to the W-containing family of aldehyde:ferredoxin oxidoreductases. A Bam(BC)2 complex with four [4Fe-4S] clusters and a catalytic W-bispterin (bisWPT)-cofactor has initially been characterized. The W-atom is coordinated by five S-atoms from the pterin and the protein and by a yet non-identified 6th, probably cyano-ligand. The 6th ligand mediates electron transfer from the reduced W to the benzene ring representing an unprecedented mode of catalysis among Mo/W-cofactor containing enzymes. The project aims (i) to unravel the nature and function of the BamB active site by a number of spectroscopic and structural methodologies with a special focus on the crucial 6th W-ligand; (ii) to elucidate the synthesis and assembly of the unusual bisWPT-cofactor of BamB, in particular the function of the bamL-encoded chaperone; and (iii) to shed light into the interconnections of metallo enzyme synthesis in G. metallireducens that is capable of producing Mo-/W-/NiFe/FeS-enzymes in parallel. The whole project is based on intensive methodical and topic-related collaborations with numerous members of SPP1927 and international experts.


Hans Heider (Marburg): Function of Mo/W-enzymes from anaerobic bacteria
Thorsten Friedrich (Freiburg): EPR spectroscopy of NADH-oxidoredcutase modules
Gary Sawers (Halle): CN-cofactor assembly and function
Oliver Lenz (Berlin): Spectroscopic methodthodologies
Silke Leimkühler (Potsdam): Regulatory network of Mo/W-pterin-cofactor synthesis
Serena DeBeer/Frank Neese (BN): X-ray spectroscopy methods, DFT based approaches
Antonio Pierik/V. Schünemann (KL): EPR / Mössbauer spectroscopy [FeS], W
Carola Schulzke (Greifswald): Synthetic W-cofactors

The role of FeS-glutaredoxins and their cofactors in diseases

Carsten Berndt                                                                            Christopher Lillig
Universität Düsseldorf                                                                 Ernst-Moritz-Arndt Universität Greifswald

Glutaredoxins (Grxs) are unique FeS-proteins. They coordinate a FeS cluster between two monomers using two molecules of non-covalently bound glutathione and two protein thiols in the active site as ligands. FeS binding thus inhibits enzymatic activity as oxidoreductase. Following our identification of human Grx2 as the first FeS-Grx, numerous other Grxs in all kingdoms of life have been characterized as FeS coordinating proteins. Although several essential functions of FeS-Grxs have been identified, e.g. during vertebrate development, FeS cluster biosynthesis, and iron trafficking, the distinct molecular role of the coordinated cofactor remains largely unknown. In our project we will investigate the role of Grx coordinated FeS clusters in two different diseases connected to disturbed iron homeostasis: multiple sclerosis and cardiac hypertrophy. We will combine in vitro techniques (biochemistry and a variety of spectroscopic methods) with ex vivo (primary cell cultures, organotypic slice cultures) and in vivo models (animal models for multiple sclerosis, cardiac hypertrophy, and iron overload) as well as patient material. To reach our goals, we can refer to our well documented expertise in Grxs in general and structure/function relationship of FeS-Grxs in particular. This knowledge provided the base for the construction of specific mutated forms of Grxs that differ in their ability to coordinate the FeS cluster and their enzymatic activity. Using these mutated Grxs we were already able to demonstrate nitric oxide detoxification as new function of Grx coordinated FeS clusters. The in-depth characterization of the nitric oxide dependent disassembly of holo-Grxs will be one of the emphases of our project, the first comprehensive investigation of the role of FeS cluster coordinated by mammalian Grxs.


Iron-sulfur cluster electronic structural evolution and its contribution to diverse functionality

Serena DeBeer and Frank Neese
Max Planck Institute for Chemical Energy Conversion, Mülheim an der Ruhr

This project represents a dedicated effort to combine the results of advanced X-ray spectroscopic methods with state-of-the-art quantum chemical calculations in order to obtain insight into the electronic structural evolution through a series of iron-sulfur clusters ranging from relatively straightforward Fe-S monomers all the way to the intimidatingly complex FeMoco active site of nitrogenase. The prime target for the experimental part of this proposal is to develop 2p3d resonant X-ray emission (RXES) spectroscopy, a two-dimensional form of inelastic X-ray scattering, into a major tool for the investigation of complex systems, such as Fe/S clusters. The small amount of data that has been collected so far indicates that the information content of this method is very high, but the complexity of the underlying theory has so far prevented very detailed assignments to be made and electronic structure related conclusions to be drawn. Hence, we plan to closely combine RXES experiments (as well as other, more established techniques) in conjunction with high-level ab initio electronic structure methods. The calculations will largely be based on the density matrix renormalization group (DMRG) formalism, the only available method that is able to deal with the complexity of oligonuclear open-shell, mixed-valence transition metal complexes such as that presented by Fe/S clusters. The validity and scope of applicability of model Hamiltonians such as the widely used Heisenberg-Double Exchange (HDE) Hamiltonian will be established alongside the analysis of the experimental data. Taken together, we believe that the present project will arrive at experimentally validated electronic structure descriptions of Fe/S clusters at an unprecedented level of detail. The consequences of these electronic structure intricacies on the reactivity of these clusters will be discussed.


F. Meyer M. Boll   G. Fritz
O. Einsle O. Lenz S. Shima
C. Schulzke R. Lill   C. Berndt                       

CooC2/AcsF and Cfd1/Nbp35: maturation of complex Fe/S-clusters by MinD-type ATPases

Holger Dobbek
Humboldt Universität Berlin

Anaerobic bacteria and archaea use homologous enzymes to convert CO2 and H2 to acetyl-CoA and methane, which determine since the dawn of life the global carbon cycle on earth.

In recent years, we have discovered and investigated a Ni insertase for the central enzyme acetyl-CoA synthase (ACS). While one or more maturation enzymes are known for all other central Ni enzymes; so far no enzyme supporting the maturation of Ni,Ni-[4Fe4S] cluster, called Cluster A, has been described. We could show that the maturation protein CooC2, a MinD-family ATPase, forms a specific complex with the Ni-deficient ACS, that this protein complex binds two Ni ions with high affinity and finally that the active ACS is liberated by the addition of Mg-ATP to the complex. Bioinformatic analyzes suggest that the CooC ATPases can be divided into three subclasses: one subclass contains ATPases for the maturation of the [NiFe4S4] center of the carbon monoxide dehydrogenases; one subclass, with CooC2 as the first example described functionally, consists of ATPases responsible for the maturation of the ACS; the role of the third subclass is unknown.

In the first funding period we want to investigate: (I) whether the homologous enzymes from archaea have the same function as the bacterial CooC2 protein. This is particularly interesting since the ACS from Archaea differs in size and association with CODH and CoFeSP from the bacterial enzyme: compared to the bacterial enzymes it misses the N-terminal domain, but is present in a multi-enzyme complex of 2.5 MDa. We want to investigate (II), how the high affinity for Ni is generated in the CooC2-apoACS complex and which role ATP plays in the maturation. In addition to apo- and holo-ACS, we can produce two stable maturation intermediates giving us the rare opportunity to “watch” the stepwise assembly of a complex Ni, Fe, S-cluster. Furthermore, (III) we want to compare the insertion strategy of CooC2 with a different type MinD metal-insertase, Cfd1/Nbp35, a key complex of cytosolic Fe/S cluster maturation machinery. In order to achieve the three objectives, integration in the SPP and the interaction with other groups of the SPP are indispensable.


AG Pierik (EPR spectroscopy)
AG Schünemann (Mössbauer spectroscopy)
AG Zebger (Vibrational spectroscopy)
AG Leimkühler (Metal analysis)
AG Lill (Cfd1-Nbp35)
AG Adrian (Proteomics).

Complex Iron-Sulfur Cluster Assembly for Biological Nitrogen Fixation

Oliver Einsle
Albert-Ludwigs-Universität Freiburg

The [Mo:7Fe:9S:C]:homocitrate cluster known as FeMo cofactor is the catalytic center of nitrogenase and thus the active principle of biological nitrogen fixation. It is synthesized ex situ, in parallel with the maturation of the protein component NifDK, requiring the interplay of a large number of maturation factors that assemble the unique and intricate structure of the cluster from simple [2Fe:2S] fragments. The biogenesis of nitrogenase largely does not rely on the constitutive enzymes of [Fe:S] assembly, but employs a dedicated system consisting of the cysteine desulfurase NifS, the scaffold protein NifU and the radical/SAM enzyme NifB that assembles the cluster core while inserting an unusual, interstitial carbide ion into its center. In order to better understand the molecular basis of FeMo cofactor biogenesis, this project focuses on the recombinant production and analysis of the proteins NifS, NifU and NifB from a variety of organisms, aiming at a crystallographic analysis as well as a spectroscopic and functional characterization within the Priority Programme 1927.

We will furthermore investigate the recently presented complex of FeMo cofactor with the inhibitor carbon monoxide. From this, we on one hand expect important insights into the general reaction mechanism of nitrogenase, and on the other hand a deeper analysis of the reduction of CO itself. While CO acts primarily as an inhibitor of Mo-dependent nitrogenase, the alternative, V-dependent enzyme can break the triple bond of the molecule, reducing the carbon portion to a spectrum of short-chain and partially unsaturated hydrocarbons. The mechanistic implications of these activities are only partly understood and will be addressed by a concerted study employing a broad range of methodologies.

A further part of the project aims at the application and development of the SpReAD method that allows for a spatially-resolved analysis of the X-ray absorption properties of individual atoms within a crystalline sample. This provides information on the individual oxidation states of atoms and thereby on the properties and reactivities of entire metal enters. The method is established and will be used in a series of collaborations within priority program 2917, including systems such as nitrogenase, hydrogenase and CO dehydrogenase.


Thorsten Friedrich                                          Gunhild Layer/Jürgen Moser
Serena DeBeer / Frank Neese                       Oliver Lenz
Ingo Zebger                                                    Matthias Ullmann
Holger Dobbek                                               Antonio Pierik
Volker Schünemann                                       Matthias Boll
Franc Meyer

Assembly of Iron-Sulfur Clusters in the NADH:Ubiquinone Oxidoreductase

Thorsten Friedrich
University Freiburg

Energy-converting NADH:ubiquinone oxidoreductase, complex I, couples the transfer of two electrons from NADH to ubiquinone with the translocation of four protons across the membrane. The Escherichia coli complex consists of 13 different subunits and one flavin mononucleotide and nine different iron-sulfur (Fe/S) clusters as cofactors for electron transfer. The assembly of the Fe/S clusters in bacterial complex I has not been investigated so far. Their biogenesis is assumed to be catalyzed by the ISC and SUF systems, but this has never been challenged experimentally. One aim of the project is to identify whether one or both maturation systems are involved in the biogenesis of the Fe/S clusters of complex I, taking into account different growth conditions. Other assembly factors possibly involved in their biogenesis such as GrxD, BolA, NfuA and others have not yet been identified. Especially, it is not known whether the biogenesis of individual clusters of the complex depend on individual assembly factors. In the project we will characterize factors needed for the assembly of the individual clusters under normal and stress conditions such as the presence of reactive oxygen species (ROS). A special focus is put on the role of the inducible lysine decarboxylase, known to be involved in bacterial acid stress response, for the assembly of the Fe/S cluster called N2 that is coordinated by a unique arrangement of cysteine residues. The possible involvement of a putative triad complex comprising the inducible lysine decarboxylase, the AAA+ enzyme RavA and ViaA containing a von-Willebrandt factor in the assembly of cluster N2 is the main focus of this project. The complicated assembly of N2 might be due to its proposed function in energy-conservation. Consequently, we will investigate whether cluster N2 is directly involved in proton translocation.

Planned cooperations:

Adrian:  Complex I in Dehalococcoides McCartyi
Berndt/Lillig:  Role of GrxD in the assembly of complex I iron-sulfur clusters
Einsle:  SpReAD of complex I and variants
Leimkühler:  Comparative analysis with nitrate/DMSO reductases
Lill:  Comparative analysis of the role GrxD, BolA and NfuA
Mendel:  Metal binding to LdcI (CadA)
Ullmann:  Protonation of Fe/S cluster
Zebger:  Resonance Raman spectroscopy of complex I iron-sulfur clusters

Analysis of the properties and the assembly of the intramembranous FeS cluster of the Na+ -NQR and the RNF complex

Günter Fritz                                                                                Julia Fritz-Steuber
Uniklinik Freiburg                                                                       University of Hohenheim

Respiratory complexes may contain FeS clusters that are involved in electron transfer coupled to ion translocation. The so-called “Rhodobacter nitrogen fixation” (Rnf) respiratory complex was identified as an essential part of the nitrogen fixation machinery. Deletion of the rnf genes causes a defect in FeS cluster assembly characterized by an accumulation of inactive and iron deficient NifH protein. The Rnf complex consists of six subunits and catalyzes the oxidation of ferredoxin using NAD+ as an electron acceptor to drive transport of Na+ across the membrane. Rnf complex is closely related to another Na+-pumping respiratory enzyme, the Na+-translocating NADH:quinone oxido-reductase (Na+-NQR). Both membrane protein complexes share the same basic architecture and mechanism of redox-driven ion translocation. The X-ray structure of Na+-NQR revealed an unusual and hitherto unknown FeS center in the midst of the membrane that is part of a transmembrane electron transfer pathway. The FeS binding site is strictly conserved in the corresponding subunits RnfA and RnfE of the Rnf complex. This FeS center is so far unique with respect to its unprecedented localization, its coordination and its unusual spectroscopic properties. The localization in the membrane raises the question how this FeS is inserted into these integral membrane proteins, and we will study its maturation by genetic and biochemical approaches. We will characterize the intramembranous FeS cluster by Mössbauer, EXAFS and quantum chemical calculations in collaboration with S. deBeer and F. Neese to resolve the electronic structure of the cluster. We want to explore whether the Fe-Fe distances change during electron transfer. Such redox-dependent changes might influence the structure of the redox-driven pump. The redox properties of the FeS cluster will be addressed using EPR spectroscopy in collaboration with O. Einsle


With S. DeBeer and F. Neese:
Mössbauer, EXAFS and quantum chemical calculations to resolve the electronic structure of the intramembranous FeS cluster of the NQR

With O. Einsle:
EPR spectroscopy to determine the redox potential of the intramembranous FeS cluster at varying Na+ concentrations

Diurnal Dynamics in the Biogenesis of Iron-Sulfur Cluster and other Metalloproteins of Plants in Dependence on Cluster Assembly

Ute Krämer
Ruhr-Universität Bochum

The genome of the genetic model plant Arabidopsis thaliana encodes more than 100 proteins that require iron-sulfur (FeS) cluster cofactors for their biological function, for example in photosynthesis, respiration and nucleic acid metabolism. Plants possess three cellular pathways of FeS cluster assembly in chloroplasts, mitochondria and the cytosol, respectively. As sessile organisms, they must efficiently protect the biogenesis of FeS proteins under fluctuating and non-optimal environmental conditions, for example in the presence of reactive oxygen species or an excess of interfering metals. To examine how this is accomplished, we are analyzing the diurnal dynamics in the biogenesis of FeS-dependent and other metalloproteins in different cellular compartments by using genome-wide translatome sequencing (ribo-seq) as a proxy. This is combined with an analysis of the diurnal dynamics of metal and low-molecular-weight chelator concentrations, sub-cellular metal distribution and enzyme activities of major molybdenum cofactor(Moco-) and FeS-dependent proteins. We compare wild-type plants with plants exhibiting defects in each of the three cellular FeS cluster assembly pathways to reveal a possible dependence on, and regulatory roles of, subcellular FeS cluster assembly for metal homeostasis and metalloprotein biosynthesis. Our results can serve as a basis for future strategies to optimize bioenergy production and agricultural fertilization schemes.


Silke Leimkühler
Julia Bailey-Serres (outside SPP, UC Riverside, CA, USA)
Thomas Happe (ouside SPP, Ruhr University Bochum, Germany)
Andreas Meyer (outside SPP, University of Bonn, Germany)

Tungsten-dependent aldehyde oxidoreductases: specific W-cofactor insertion and interactions with Fe-S clusters in catalysis and maturation

Johann Heider
Universität Marburg

The aim of this project is to characterize the biochemistry, structure and maturation of AOR from the betaproteobacterium Aromatoleum aromaticum, a recently detected tungsten-containing enzyme of the aldehyde oxidoreductase (AOR) family. The enzyme was purified from phenylalanine-grown cells of an overexpressing mutant and found to consist of a complex of three subunits (two AOR-type subunits carrying a W-bis-MPT cofactor and an Fe-S cluster, two electron transfer subunits carrying four Fe-S clusters and one FAD-containing subunit), which is different from the well-characterized homodimeric structure of AOR enzymes from hyperthermophilc Archaea, but resembles the situation of a previously reported orthologue from Moorella thermoacetica. The enzyme is surprisingly resistant against oxygen and turns over various aldehydes with very high specific activities, although it appears to lack stoichiometric amounts of W and the associated MPT molecules of the W‑bis‑MPT cofactors in the AOR-type subunits. The project will be centered around two major questions: (i) the biochemistry of AOR itself, including the catalytic properties, structure-function relations, functional relevance of the additional subunits and Fe-S-clusters and the reason for oxygen resistance, and (ii) the maturation pathway of the W-cofactor in AOR-type enzymes, including the identification and characterization of additional synthetic enzymes involved, the basis of discrimination from Mo-cofactor assembly and the necessity and interplay of W-cofactor maturation with Fe-S clusters and their synthesis.


Antonio Pierik, Technische Universität Kaiserslautern: EPR-spectroscopic characterization of AOR and maturation factors

Volker Schünemann, Technische Universität Kaiserslautern: Mössbauer spectroscopic characterization of AOR

Silke Leimkühler, Potsdam: W-cofactor synthesis, analytics of W-cofactor and biosynthetic precursors, in vitro system for W-cofactor synthesis and incorporation

Günter Schwarz, Köln: similarity and differences in W-cofactor synthesis in hyperthermophilic Archaea, in-vitro conversion of cPMP into metal-binding pterin (MPT) and in-vitro Synthesis of W-cofactor using Pyrococcus furiosus MoaB and MoeA1/2 proteins.

Carola Schulzke, Greifswald: biological effects of chemically synthesized cofactors and analogs

Matthias Boll, Freiburg: W-enzyme benzoyl-CoA reductase, cooperation and feedback between the AGs on cofactor maturation pathways

Roland Lill, Marburg: analysis of FeS clusters synthesis and function in AOR, spectroscopic methods

Seigo Shima, Marburg: crystallization of AOR and cofactor maturation proteins

Characterization and reaction mechanism of the nitrogenase-like reductase NflH/NflD involved in cofactor F430 biosynthesis

Gunhild Layer
Universität Leipzig

The complex metallo-enzyme nitrogenase and several simpler, iron-sulfur cluster containing nitrogenase-like enzyme systems are involved in three key metabolic processes necessary to sustain life on earth, namely nitrogen fixation, photosynthesis and biological methane formation. Whereas nitrogenase catalyzes the reduction of dinitrogen to ammonia, the nitrogenase-like enzymes dark operative protochlorophyllide oxidoreductase (DPOR) and chlorophyllide oxidoreductase (COR) catalyze specific C-C double bond reduction reactions during the biosynthesis of chlorophylls and bacteriochlorophylls, respectively. The third nitrogenase-like enzyme system is termed Nfl (nitrogen fixation like), is found in all methanogenic archaea and catalyzes several C-C double bond reduction reactions during the biosynthesis of the nickel-containing tetrapyrrole cofactor F430. The Nfl system is only scarcely characterized. Therefore, the aim of this project is the characterization and reaction mechanism of this nitrogenase-like reductase.

As is the case for nitrogenase, DPOR and COR, the Nfl system consists of two components: a reductase termed NflH and a catalytic component named NflD. However, the Nfl system seems to exhibit a simpler architecture than nitrogenase, DPOR and COR. Based on our preliminary work, NflH and NflD are homodimeric proteins containing an intersubunit [4Fe-4S] cluster each. NflH is proposed to transfer electrons to NflD where the reduction of the actual substrate takes place. In this project within the SPP 1927 we plan to characterize the iron-sulfur clusters of NflH and NflD in detail by different spectroscopic techniques and identify the cluster ligands. The potential ATP-induced conformational changes within NflH and the interaction between the two proteins will be studied. The substrate specificity of NflH/NflD will be investigated in order to obtain insights into the NflD active site architecture and plasticity. We will study the possibility that the reductase components of nitrogenase, DPOR and COR might be exchangeable with the reductase of the Nfl system. Another aim will be the identification of potential interaction partners of NflH/NflD in vivo and in vitro. Finally, we will try to crystallize NflH and NflD in order to obtain the crystal structures of these proteins.



Lorenz Adrian (UFZ Leipzig)  –  Mass-spectrometry

Oliver Einsle (Universität Freiburg)  –  Small artificial substrates, X-ray crystallography

Jürgen Moser (TU Braunschweig)  –  Artificial tetrapyrrole substrates, Anaerobic protein crystallization

Antonio Pierik (TU Kaiserslautern)  –  EPR spectroscopy, Dye-mediated redox titration

Volker Schünemann (TU Kaiserslautern)  –  Mössbauer spectroscopy

Ingo Zebger (TU Berlin)  –  Resonance Raman spectroscopy, Conformational changes in NflH and NflD,
FT-IR difference spectroscopy

Iron-sulphur cofactors involved in metal centre assembly and catalysis of hydrogenase

Oliver Lenz, Ingo Zebger
Technische Universität  Berlin

This collaborative research project addresses two fundamental areas of the Priority Programme (PP) 1927, namely the FeS protein-mediated biosynthesis of complex metal cofactors and the catalytic function and application of an intricate metalloenzyme. Combfined biochemical and spectroscopic studies will concentrate on a thermostable NAD+-reducing [NiFe] hydrogenase, which represents an evolutionary ancestor of Complex I. This enzyme harbours a H2/H+-cycling active site with a (CysS)2Ni(μ-CysS)2Fe(CN)2(CO) architecture, a multitude of FeS clusters involved in electron transfer, and a flavin-containing centre, at which catalytic NAD+-/NADH conversion takes place. Due to its capability to recycle the cofactor NADH in the presence of O2 and at high temperature, the enzyme holds great potential for biotechnological applications. The molecular details of the underlying catalytic processes will be investigated in a multidisciplinary approach, which, among biochemical and other physical techniques, involves resonance Raman (RR) and infrared (IR) spectroscopy to identify and characterise catalytically relevant redox states both in vitro and in vivo. In this context, we will apply a recently established IR setup, which allows both gas exchange and spectro-electrochemical analysis of the samples. In the area of metal centre biosynthesis, we will focus on a recently discovered CO synthase that provides the diatomic CO ligand, which is part of the unique (CysS)2Ni(μ-CysS)2Fe(CN)2(CO) cofactor, by using formyltetrahydrofolate of the central one-carbon metabolism as the substrate. The enzyme reaction seems to involve iron-based chemistry, which will be investigated thoroughly using biochemical, spectroscopic, and computational methods available in PP 1927. Additionally, vibrational spectroscopy will be used to investigate the role of FeS clusters and multinuclear metal centres in maturation and catalysis of [Fe] and [NiFe] hydrogenase, nitrogenase, nitrogenase-like enzymes, Complex I and other PP-relevant FeS-proteins. The experimental work and the corresponding data evaluation will be performed in close collaboration with researchers from the PP 1927 as well as external experts. The corresponding results are expected to provide a detailed picture of unifying principles and individual aspects of iron(sulphur)-based biosynthetic and catalytic processes for a variety of complex metalloproteins, especially hydrogenases.


– G. Sawers:  Function of the accessory HypD protein involved in [NiFe]-hydrogenase active site assembly, biosynthesis of the active site carbonyl ligand under anaerobic conditions.

– S. Shima: Spectroscopic characterisation in vitro biosynthesis of the catalytic centre of [Fe]-hydrogenase.

– O. Einsle: Spectroscopic characterisation of the P-cluster and the catalytic centre of nitrogenase.

– S. DeBeer / F. Neese: Analysis of metal-ligand interactions by X-ray absorption/emission spectroscopy and DFT calculations on metal cofactors.

– G. Layer & J. Moser: Spectroscopic characterisation, substrate interaction, and conformational changes in nitrogenase-like enzymes.

– S. Leimkühler:  Formate dehydrogenase- hydrogenase comparison, metal content determinations.

– T. Friedrich: Analysis of pronation events in the vicinity of the FeS cluster, N2, of Complex I. Complex I-hydrogenase comparison

– H. Dobbek: Spectroscopic analysis of Ni insertion into and CO binding to acetyl-CoA synthase.

– C. Berndt /C. H. Lillig:  NO detoxification process and disassembly of the FeS cluster in glutaredoxin.

– F. Meyer / C.Schulzke: Spectroscopic characterisation of FeS and Moco model compounds.

Cooperative function of Iron-Sulfur clusters and metal homeostasis on the biosynthesis of molybdoenzymes

Silke Leimkühler
Universität Potsdam

Recently, a link between the biosynthesis of the molybdenum cofactor (Moco) and FeS cluster biosynthesis has been discovered. The biosynthesis of Moco is a highly conserved and complex pathway, in which the MoaA protein presents a conserved protein which catalyzes the first and essential step in Moco biosynthesis in bacteria. MoaA belongs to the class of radical/SAM enzymes which contain the SAM-binding [4Fe4S] cluster at their N-terminus and, in addition, the MoaA proteins contain a [4Fe4S] cluster at its C-terminus, which binds the substrate GTP. The further steps of Moco biosynthesis involve the IscS protein as a major player. IscS was initially described as a housekeeping L-cysteine desulfurase which mobilizes the sulfur for FeS cluster biosynthesis. IscS was shown to be additionally involved in the mobilization of sulfur for the synthesis of the dithiolene group of MPT. Recently it was shown that IscS is also required for the formation of the terminal sulfido ligand present at the molybdenum active site of molybdoenzymes like formate dehydrogenases and the periplasmic aldehyde oxidoreductase in Escherichia coli. In addition, many molybdoenzymes coordinate FeS clusters and, thus, directly depend on FeS cluster synthesis and their insertion to produce active holo-enzymes.

Our goal is to study the crosstalk of FeS cluster biosynthesis and Moco biosynthesis in E. coli. A special link is presented between both pathways on the level of availability of the L-cysteine desulfurase IscS, and the so far unidentified FeS cluster inserting proteins. We will study the biosynthesis and insertion of FeS clusters into MoaA and molybdoenzymes like nitrate reductase, DMSO reductase and the periplasmic aldehyde oxidoreductase. Total metal homoeostasis (here the availability of Fe, Mo and Zn) will be analyzed on its effect on Moco biosynthesis, the synthesis of cPMP, the conversion of cPMP to MPT and the insertion of Moco and FeS clusters into molybdoenzymes. In total, our analyses will shed light into the complex FeS cluster network for molybdoenzyme maturation in E. coli.


Lorenz Adrian (UFZ Leipzig): blue native 2D nLC MS/MS

Matthias Boll (Universität Freiburg): Wco insertion and biosynthesis

Thorsten Friedrich (Universität Freiburg): comparative studies of the FeS cluster insertion

Johann Heider (Universität Marburg): Moco and Wco biosynthesis

Ute Krämer (Ruhr-Universität Bochum): RNAseq

Roland Lill (Universität Marburg): FeS clusters insertion

Ralf Mendel (TU Braunschweig): FeS cluster insertion into Nit7

Antonio Pierik (Universität Kaiserslautern): identification of common motifs for FeS clusters

Constanze Pinske (Universität Halle): studies on the assembly of formate:hydrogen lyase

Gary Sawers (Universität Halle): influence on hydrogenase activity in E. coli k.o. strains

Volker Schünemann (Universität Kaiserslautern): Mössbauer spectroscopy on whole cells,

Carola Schulzke (Universität Greifswald): Moco model compounds

Günter Schwarz (Universität Köln): comparative studies of FeS insertion into MoaA/MOCS1A

Hans Richnow (UFZ Leipzig): NanoSIMS on Fe and Mo distribution in bacterial cells

Role of the mitochondrial Bol1 and Bol3 proteins in iron-sulfur cluster delivery to diverse recipient proteins

Roland Lill
Phillips-Universität Marburg

The biogenesis of mitochondrial iron-sulfur (Fe/S) proteins is catalyzed by the ISC assembly machinery and can be dissected into three major steps. First, a [2Fe-2S] cluster is synthesized de novo on the scaffold protein Isu1 by the sulfur donor Nfs1-Isd11 and other ISC factors. Second, the Fe/S cluster is released from Isu1 by a dedicated Hsp70 chaperone system, and transferred to the monothiol glutaredoxin Grx5. Finally, the transiently Grx5-bound [2Fe-2S] cluster is converted into a [4Fe-4S] cluster by the Isa1-Isa2-Iba57 proteins and inserted into recipient apoproteins by specific ISC targeting factors. Especially the last step of this complicated and essential biosynthetic pathway is poorly understood. The ISC targeting factors Nfu1 and Ind1 assist Fe/S cluster insertion into dedicated apoproteins by transiently binding the [4Fe-4S] cluster. Two other proteins (Bol1 and Bol3) have been suspected as additional specific ISC targeting factors based on the fact that functional loss of human BOLA3 (the homolog of yeast Bol3) leads to a similar disease phenotype as mutations in human NFU1. We have recently verified this idea in yeast and were able to show that Bol1 and Bol3 perform largely overlapping functions in this late step of Fe/S protein assembly (Uzarska et al. eLife, 2016; Melber et al. eLife, 2016). They act as specific late-acting ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a small subset of mitochondrial [4Fe-4S] proteins such as lipoate synthase and succinate dehydrogenase. They cannot replace yeast Nfu1 that also participates in this maturation step. Bol1 and Bol3 form dimeric complexes with monothiol glutaredoxin Grx5 which differentially influences the stability of the Grx5-bound [2Fe-2S] cluster, but the physiological meaning is unclear. To analyze whether Bol proteins directly bind to the [2Fe-2S] cluster of Grx5, we are analyzing the human BOLA1 protein with mutations in potential Fe/S cluster ligands in its contact with human holo-GLRX5. We also investigate the role of BOLA1 and BOLA3 in human cell culture to understand the relative physiological function of these assembly factors and compare their functions to yeast. The general aim of this project is a comprehensive understanding of the molecular function of the Bol proteins.



Thorsten Friedrich: Function of the bacterial glutaredoxin GrxD, BolA and NfuA proteins in respiratory complex I assembly.

Carsten Berndt / Christopher H. Lillig: Vertebrate glutaredoxins in regulatory processes.

Antonio J. Pierik: Function of the conserved tryptophan in cytosolic-nuclear Fe/S proteins. EPR spectroscopy 

Silke Leimkühler: ISC protein function during the assembly of MoCo in bacteria.

Ralf R. Mendel: Role of the mitochondrial Fe/S protein Nit-7A in fungal Moco biosynthesis.

Günter Schwarz: Maturation of mitochondrial MOCS1A and MOCS1AB proteins.

Holger Dobbek: Function of metal-dependent MinD-type ATPases.

Franc Meyer: Chemistry of Fe/S cluster coordination, model compounds mimicking Fe/S cluster coordination.

Sandra DeBeer: X-ray spectroscopy on Grx-Bol model systems.

Seigo Shima – Hans Heider: Local technical collaboration in the use of equeipment.

Lorenz Adrian – Hans H. Richnow: Use of NanoSIMS and ToFSIMS.

Molybdenum cofactor-biosynthesis and crosstalk to FeS metabolism in Neurospora crassa

Ralf R. Mendel
TU Braunschweig

Molybdenum cofactor (Moco) is the catalytically active prosthetic group in Mo-containing enyzmes. In all eukaryotes studied so far, the first step of Moco biosynthesis resides in the mitochondria while all subsequent steps proceed in the cytosol. This step is catalyzed by two proteins where the first one (Nit-7A) harbors two FeS clusters of the [4Fe-4S] type. But GTP as starting compound is also available in the cytosol. And the cytosol harbors also a FeS cluster synthesis machinery. So the question arises why Moco biosynthesis step 1 is located in the mitochondria. Using the filamentous fungus Neurospora crassa as yeast-like model system we wish to analyse the crosstalk between Moco-biosynthesis and FeS metabolism. In yeast (S. cerevisiae) all principles of eukaryotic FeS cluster biogenesis have been successfully worked out, but yeast has no Mo metabolism. In a first project line, we plan to express the N. crassa nit-7 gene in yeast thus establishing Moco biosynthesis step 1 in yeast. A successful expression of nit-7 in yeast would open up a whole new perspective because the complete arsenal of yeast FeS biogenesis research tools would be at our hands. Clearly, this attempt would also allow to uncover whether the yeast mitochondrial exporter Atm1p which facilitates the export of FeS cluster equivalents, would be able to export also the product of Moco biosynthesis step 1, which we have postulated for its plant homolog ATM3. For a second project line, in preliminary experiments we have already expressed Nit-7A ectopically and stably in the cytosol of N. crassa in a nit-7 knock out-background. We plan to characterize this strain (i) phenotypically (growth on nitrate-media creates a strong demand for Moco and thus also for FeS), (ii) biochemically (re-isolation of ectopically expressed Nit-7A and Nit-7AB and analysis of FeS-clusters), and (iii) via transcriptome analysis (nitrate-induced transcriptional changes in FeS cluster assembly and Moco biosynthesis) in order to address the following questions: What is the donor of FeS clusters for ectopically expressed Nit-7A in the cytosol? Is this process co-regulated with Moco- and FeS-biosynthesis? Are there differences between fungal and human step 1 Moco biosynthsis?


  • Roland Lill
  • Günter Schwarz
  • Silke Leimkühler
  • Antonio Pierik
  • Carola Schulzke

Deciphering the function of Fe/S cofactors with alternative cluster ligands: model studies using synthetic analogues

Franc Meyer
Georg-August-Universität Göttingen

Building upon our recent work on the development of high fidelity functional models for the {Cys2His2} ligated Rieske center and the isolation of [2Fe-2S] clusters in unusual oxidation states, this projects aims at deciphering the function of Fe/S cofactors with alternative cluster ligands in a synthetic analogue approach. Specific objectives are: (A) Elucidating intrinsic trends for the thermodynamic and kinetic parameters of proton coupled electron transfer (PCET) reactions at Fe/S clusters, mainly [2Fe-2S] clusters with His-like terminal ligands, to demonstrate a potential PCET role of His-ligation beyond the Rieske centers. (B) Understanding intrinsic ligand rearrangement and exchange processes at specifically designed [2Fe-2S] clusters, which is relevant for understanding ligand exchange during biological Fe/S cluster maturation and transfer. (C) Characterizing the species that form upon reaction of mixed S/N ligated [2Fe-2S] clusters with nitric oxide in order to unravel the effect of cluster oxidation state and His protonation state on NO reactivity pathways, and on potential H2S generation. (D) Emulating radical reactivity at auxiliary Fe/S cofactors in radical SAM enzymes and elucidating factors that control sulfur donation from the Fe/S cluster to a substrate, as seen in biotin synthase (BioB) or lipoyl synthase (LipA). (E) Providing suitable chemically synthesized Fe/S model systems for various other groups in the SPP network; these models either will serve as benchmark systems for new spectroscopic or analytical methods, or will be used for reconstituting Fe/S proteins via integration into the apoforms. In addition, some exploratory attempts will be made at synthesizing a first Fe/S cluster that contains a carbide (C) or carbyne unit (CR) within the cluster core.


  • Serena DeBeer/Frank Neese: we provide model systems that are crucial for benchmarking X-ray spectroscopy signatures of Fe/S clusters. On the other hand, the X-ray spectroscopy and theory results will provide important electronic structure information that may help to rationalize Fe/S cluster reactivity patterns observed in this project. Furthermore, DFT support from the Neese group will help to establish the thermodynamics of the radical reactions.
  • Volker Schünemann: NRVS studies of intermediates and products of the reaction of synthetic [2Fe-2S] complexes to identify characteristic NO-iron-sulfur modes may provide valuable reference data for the NO-mediated Grx iron-sulfur disassembly that the Schünemann groups studies in cooperation with Carsten Berndt (HHU Düsseldorf) and Christopher H. Lillig (U Greifswald). Furthermore, magnetic field Mössbauer measurements on our synthetic Fe/S models will be performed in the Schünemann lab.
  • Carola Schulzke: we will perform magnetic measurements (SQUID) for the molybdenum dithiolene complexes prepared in the Schulzke lab as a contribution to deciphering electronic structures, oxidation and spin states of those molybdopterin model systems. On the other hand, we plan to perform temperature dependent electrochemical measurements in collaboration with the Schulzke group to assess the thermodynamics (entropy and enthalpy) of the redox processes relevant for the PCET studies in our group.
  • Matthias Ullmann: we will provide data for redox potentials and pKa values of model complexes that are needed for his simulations of the coupling of catalysis and electron transfer along chains of Fe/S clusters
  • Oliver Einsle: a series of [2Fe-2S] (and perhaps also [4Fe-4S]) clusters in varied oxidation states will be made available to the Einsle group to collect reference and benchmarking data for their SpReAD analysis (see section 2.3.E).
  • Ingo Zegber/ Peter Hildebrandt: it was envisaged to provide model complexes as reference compounds for their resonance Raman measurements.

Nitrogenase-like biosynthesis of bacteriochlorophylls

Jürgen Moser
Technische Universität Braunschweig

The capturing of the global energy demand vitally depends on the biosynthesis of (bacterio)chlorophylls. Also the reduction of atmospheric dinitrogen to ammonia is essential for the fuelling of ecosystems. The sophisticated biochemistry of nitrogenase plays a fundamental role for the formation of chlorophylls (Chls) and bacteriochlorophylls (Bchls). The biosynthesis of both, Chls and Bchls requires the stereospecific reduction of the C17-C18 double bond of ring D of protochlorophyllide (Pchlide) which can be catalyzed by the nitrogenase-like enzyme dark-operative protochlorophyllide oxidoreductase (DPOR). Only recently, the X-ray crystallographic structure of the substrate-bound, transition state complex between DPOR subcomplexes L2 and (NB)2 revealed the details of the dynamic interaction of L2 and (NB)2.

The biosynthesis of all Bchls requires a second nitrogenase-like enzyme termed chlorophyllide oxidoreductase (COR) for the stereospecific reduction of the C7-C8 position of chlorophyllide. In the initial phase of the project we want to establish the recombinant production and purification of the COR subcomplexes X2 and (YZ)2 for subsequent X-ray crystallographic experiments. Furthermore, we are constructing a chimeric L2 protein with the ability to interact with the catalytic complex of nitrogenase. All proteins will be characterized in protein-protein interaction experiments and spectroscopic approaches.


Planned collaborations

Oliver Einsle  –  Comparative analyses of COR, DPOR and nitrogenase, chimeric, COR/nitrogenase experiments
Antonio Pierik  –  EPR
Ingo Zebger  –  EPR, redox titration, resonance Raman spectroscopy
Gunhild Layer  – Chimeric COR, NflD/NflH experiments, protein-protein interaction experiments,
artificial substrate spectrum of NflD/NflH

Spatiotemporal assembly and function of the modular formate hydrogenlyase complex

Constanze Pinske
Martin-Luther Universität Halle

Escherichia coli is a facultative anaerobic bacterium that is able to produce hydrogen through the oxidation of formate when grown on glucose and in the absence of external electron acceptors like oxygen. This reaction is catalysed by the membrane-associated formate hydrogenlyase (FHL) complex, which contains seven subunits. One of the two catalytic subunits within the cytoplasmic domain is formate dehydrogenase H (formate DH). The protein harbours a molybdenum cofactor where formate is oxidised to CO2 and electrons. The second catalytic subunit is a [NiFe]-hydrogenase that uses these electrons to reduce protons and thus generate hydrogen. The electrons are transferred between both subunits by three iron sulfur cluster proteins. The cytoplasmic domain of the FHL complex is attached to the inside of the membrane by two further integral membrane subunits.

Previous work has established that a pool of accessory proteins (called Hyp proteins) are needed for the synthesis and insertion of the NiFe(CN)2CO cofactor during maturation of the FHL complex. It is possible to purify the whole complex or individual sub-complexes, but the order of events during assembly is not understood. For example, the function of the additional accessory protein HycH remains unknown. HycH interacts with the large hydrogenase subunit HycE during assembly of the complex, but it is not a structural subunit of the FHL complex. Also, the interactions of the formate DH with the hydrogenase and the iron sulfur cluster proteins are still uncharacterized.

Understanding FHL assembly will be of great value for the biotechnological exploitation of its hydrogen-producing capability. For this reason we plan to characterise the interaction of HycH with HycE. We also want to characterize the interplay between the other subunits. Therefore, we plan to make structural investigations e.g. crystallisation and interactions studies and will determine the influence on activity and stability of the FHL complex.


Silke Leimkühler, Potsdam
Seigo Shima, Marburg
Thorsten Friedrich, Freiburg
Gary Sawers, Halle

Molecular determinants for cytosolic iron-sulfur cluster insertion

Antonio J. Pierik
University of Kaiserslautern

Cytosolic and nuclear iron-sulfur (FeS) proteins act in a broad range of cellular processes in eukaryotes. These proteins are of crucial importance for tRNA modification, ribosomal maturation, DNA replication and repair, and are therefore found in all eukaryotes, including man. The so-called cytosolic iron-sulfur protein assembly (CIA) machinery composed of 11 proteins takes care of FeS protein biogenesis in this cell compartment. In contrast to the rapid increase of knowledge on this machinery, determinants for recognition of the more than forty FeS proteins among the thousands of cytosolic proteins have remained completely obscure. A path driven by FeS cluster transfer to a thermodynamically favoured binding site at the target is tacitly assumed. However, our bioinformatic analysis and preliminary data suggest that a C-terminal tryptophan residue is one of the determinants for the recognition of FeS apoproteins by the Cia1-Cia2-Mms19 targeting complex of the CIA machinery.

In this project we will identify the role and extent of interaction of the C-terminal tryptophan-containing peptides and potential other amino acid sequence determinants with the targeting complex. Mutagenesis experiments, yeast two-hybrid screens, EPR spectroscopy and bioinformatic analysis is used to define surfaces of interaction important for cluster insertion. By training and cooperation the findings will be extended with 3D-structural characterization and compared with assembly in complex FeS-containing enzymes. The interdisciplinary project will yield insight into the molecular determinants for FeS cluster insertion into eukaryotic cytosolic and nuclear proteins. Moreover, this knowledge can be applied to more efficiently assemble complex FeS proteins in the biotechnologically favourable organism yeast.



55Fe-incorporation in the laboratory of Lill.
Peptide binding studies in the laboratory of Leimkühler.
NMR-titration at the CERM in Florence.
Cocrystallization trials in the laboratory of Dobbek.
Training of a PhD student of the Dobbek group (together with Schünemann group).
Training of a PhD student of the Layer group (EPR-based redoxtitration).


EPR (and Mössbauer) spectroscopy (Berndt/Lillig, Heider, Shima, Lill).
EPR spectroscopy of molybdenum (V) in model compounds (Schulzke).
Spectroscopic characterization of maturation factors (Sawers, Schwarz).

Towards a mechanistic understanding of the role of the iron-sulphur cluster-containing HypD protein in diatomic ligand biosynthesis of NiFe-hydrogenases

Gary Sawers
Martin-Luther Universität Halle

[NiFe]-hydrogenases have an unusual active-site cofactor whereby the iron atom carries a carbon monoxide and two cyanides as diatomic ligands. Six conserved Hyp proteins are required to synthesize this NiFe(CN)2CO cofactor. The Fe(CN)2CO portion of the cofactor is synthesized on a scaffold comprising HypCDEF. It is inserted into the apo-catalytic subunit and then nickel is added by the HypA, HypB and SlyD. The synthesis of the Fe(CN)2CO group is not fully understood but it is known that the HypE2F2 heterotetramer generates the cyanide ligands from the precursor carbamoylphosphate. HypD is an iron-sulphur enzyme that, in complex with the small ‘chaperone-like’ protein HypC, has a key function in Fe(CN)2CO synthesis. We have strong circumstantial evidence that the CO ligand is generated from endogenous CO2 bound to an iron atom by HypCD. The HypCD complex functions as a scaffold with which the other Hyp proteins interact. Moreover, HypD is the only Hyp protein with a redox function. We will focus primarily on elucidating the biochemical function of HypD and how it generates the Fe(CN)2CO moiety. HypC and HypD form a tight complex and HypC is responsible for recognition of the apo-catalytic subunit of the hydrogenase by the complex. However, HypC also binds iron with CO2 attached to it, but so far, it has not been possible to isolate HypC with a bound Fe(CN)2CO moiety. Therefore, our current working hypothesis is that HypC delivers the iron bound by CO2 to HypD where reduction to an Fe(I)-CO intermediate occurs. Subsequently, the cyanide groups are transferred from the HypEF complex to the Fe(I)-CO. We have recently demonstrated that HypD undergoes disulphide-thiol redox chemistry, explaining how CO2 reduction might proceed. Therefore, the aims of this project are: to determine the mechanism by which HypD catalyzes reduction of CO2; to analyze whether ATP is involved in CO2 reduction; to show how the HypCD complex transfers the Fe(CN)2CO group into the active site of the hydrogenase; and to identify the physiological electron donor to HypD.



Lorenz Adrian, Leipzig
Antonio Pierik, Kaiserslautern
Constanze Pinske, Halle
Seigo Shima, Marburg
Ingo Zebger and Oliver Lenz, Berlin

Fe/S cluster regulation & crosstalk of two radical SAM modifiers by one electron donor in yeast?

Raffael Schaffrath
Universität Kassel

Iron-sulfur (FeS) clusters in radical SAM enzymes use electrons for radical formation in many biochemical reactions and crucial for all cells (including our own). Accordingly, radical SAM enzyme defects can cause disease in humans. For instance, ovarian cancer is linked to mutations in a radical SAM enzyme (Dph1-Dph2) that modifies the essential mRNA translation factor (EF2) by diphthamide, and defects in Elongator, a tRNA modifying complex with a radical SAM catalytic subunit (Elp3), associate with neuropathies. Intriguingly, both enzymes share one electron transfer protein, Kti11 (aka Dph3), which donates electrons to Dph1-Dph2 and in complex with Kti13, has been implicated in electron flow to Elp3. This suggests regulation of two radical SAM enzymes via electron flow to (and from) their FeS clusterss. In principle, the Kti11-Kti13 complex may feed electrons into both modification pathways or restrict them to Elongator (Elp3). Although there is evidence to support either option, the relevance of Kti13 in the diphthamide pathway is still moot, and a combination of both models, where Kti13 supports electron flow to the two radical SAM enzymes but with a differential contribution to each, cannot be excluded yet.

Hence, we aim to study the precise role of Kti13 and its potential to guide electron flow from Kti11/Dph3 to FeS clusters in Dph1-Dph2 or Elongator (Elp3). Using yeast as a model eukaryote, this will provide insights into proper regulation of radical SAM catalysis and prevention of non-biological electron flow to random, potentially harmful acceptors. Moreover, with the tRNA and EF2 modifiers being linked to mRNA translation, elucidation of the mode of electron flow to their FeS clusters may inform about functional cross-talk among both radical SAM enzymes. To achieve these aims we will answer the following questions.


  • can Kti13 guide electrons from Kti11/Dph3 to FeS clusters in Elp3 and/or Dph1-Dph2 ?
  • do Kti11/Dph3 separation of function mutations reveal differential FeS cluster regulation ?
  • is there cross-talk among both radical SAM enzymes in mRNA translation via Kti11/Dph3?

With radical SAM chemistry being intimately linked to S-transfer for molybdenum cofactor (MOCO) synthesis and inappropriate levels of MOCO related to disease, our project has biomedical relevance in the long term. Moreover, by addressing two radical SAM modifiers involved in mRNA translation, a step in protein synthesis vital for all cells, the project neatly fits the SPP1927 focus Iron-Sulfur for Life.


  • Rolland Lill, Marburg
  • Silke Leimkühler, Potsdam
  • Antonio Pierik, Kaiserslautern
  • Holger Dobbek, Berlin
  • Gary Sawers, Halle/S.

Mössbauer spectroscopic methods to study iron-sulfur assembly, disassembly and catalysis 

Volker Schünemann
University of Kaiserslautern

The aim of this project is to contribute to the understanding of the assembly, the biosynthesis and the catalysis of iron-sulfur centers with experimental methods based on the Mössbauer effect. Thereby we want to get more insight into functional electronic, vibrational and structural properties of the iron sites in polynuclear iron-sulfur centers.

Using laboratory based Mössbauer spectroscopy in high fields up to 5 T we will identify ligand binding sites in a variety of iron-sulfur enzymes provided by collaboration partners within the SPP1927. These proteins have 2Fe-2S, 4Fe-4S centers but also more complex iron-sulfur centers like those in nitrogenase.

In addition, we will study the Fe-S cluster assembly within cells. Here we envisage temperature and field dependent Mössbauer spectroscopy in order to identify the iron-sulfur centers within the cells and quantify the corresponding iron-sulfur contribution in different cell lines.

Also the interaction of iron-sulfur centers with small signaling molecules like NO and CO will be investigated and synchrotron based nuclear resonance vibrational spectroscopy (NRVS) experiments will be performed in order to identify iron ligand vibrations.


  • Pierik, TU Kaiserslautern: EPR-investigations of Fe-S centers
  • DeBeer, MPI for Chemical Energy Conversion, Mülheim: FeMoCo
  • Boll, U. Freiburg: Mössbauer Spectroscopy on class II Benzoyl CoA reductase
  • Berndt, U Düsseldorf: Mössbauer spectroscopy and NRVS of human Grx2
  • Dobbek, HU Berlin: ACS maturation
  • Einsle, U Freiburg: Mössbauer spectroscopy and NRVS on CO nitrogenase
  • Layer, U Leipzig: Nitrogenase-like enzyme system NflH/NflD
  • Lill, U Marburg: 4Fe-4S insertion into mitochondrial apoproteins
  • H. Lillig, U Greifswald: Mössbauer spectroscopy and NRVS of human Grx2
  • Leimkühler, U Potsdam: Fe-S assembly in E. coli cells
  • Meyer, U Göttingen: NO-iron-sulfur model compounds
  • Neese, MPI for Chemical Energy Conversion, Mülheim: Theoretical studies on FeMoCo
  • Schwarz, U Köln: Iron-sulfur binding in MOCS1A and MOCS1AB
  • S. Shima, MPI for Terrestrial Microbiology, Marburg: Hydrogenases

Sulfido dithiolene complexes modeling molybdenum and tungsten-dependent oxidoreductases

Carola Schulzke
Ernst-Moritz-Arndt Universität Greifswald

Iron-sulfur cluster are very important for the maturation/biosynthesis and the function of essential oxidoreductases. We aim to investigate several aspects of the FeS dependent biosynthesis and catalytic activity of molybdenum and tungsten cofactors and their respective enzymes by means of synthesizing, characterizing and evaluating model compounds regarding catalytic activity, kinetic properties and interactions with proteins (chaperones, apoenzymes). Important questions to be addressed include particularly the role of the sulfido-ligand (inserted by IscS in vivo) at the active site of molybdenum and tungsten enzymes. It is planned to understand at the chemical level the mode of action of sulfide insertion and sulfido function for the reactivity of enzymes in detail, to elucidate the catalytic reaction mechanisms and to help distinguishing between actual and artifactual presence of sulfur in the active sites. The possibility to specifically label distinct atoms of the model complexes with isotopes will significantly expedite the respective investigations regarding the latter. The findings are envisioned to support the understanding of cellular processes and eventually medical aspects for the treatment of MoCo (molybdenum cofactor) related diseases. Known mono-oxo bis-dithiolene complexes of molybdenum and tungsten with dithiolene ligands mimicking aspects of the natural ligand system molybdopterin (MPT) will be tested for binding an additional sulfide ligand giving MOS species and for changing the MO moiety to an MS moiety. In a second approach metal precursors will be used in procedures adapted from methods for oxo complexes, which already carry the sulfide ligand as well as those with readily replaceable ligands as halides or CO. In addition the development of new metal precursors and new ligand systems facilitating binding tom proteins is planned. Besides a thorough characterization by various methods, the synthetic complexes will be tested for their catalytic potential, kinetic properties and biological activity (specific binding with proteins involved in the maturation of molybdenum and tungsten cofactors and the respective apoenzymes; catalytic activity of the semi-synthetic enzymes).


  • Silke Leimkühler: protein binding, incorporation experiments, catalytic assays, determining oxo versus sulfido coordination in the active sites; outgoing lab rotations
  • Matthias Boll: catalytic transformations of aromatic substrates with models and enzymes, identifying the sixth ligand in W‐dependent benzoylcoenzyme A reductase
  • Matthias Ullmann: providing electrochemical and pKa data of model compounds to be used for optimizing calculations of the active site properties of the enzymes
  • Johann Heider: investigating the oxidation state requirements for an incubation of the mature cofactor into the apo‐enzymes, specifically aimed at tungsten‐dependent AOR
  • Serena DeBeer/Frank Neese: X‐ray absorption and emission spectroscopy on our model compounds and combined enzyme‐model species, theoretical evaluation of the data; producing MoFe3‐clusters following procedures developed by the Holm group
  • Ingo Zebger/Oliver Lenz: Resonance Raman spectroscopy of model complexes to complement IR studies)
  • Ralf Mendel: interactions of our models with fungal proteins to determine their protein binding properties/potential
  • Antonio J. Pierik: EPR of our model compounds in oxidation state +5 and comparison with protein data
  • Franc Meyer: magnetic measurements (SQUID) of our complexes for deciphering electronic structures, oxidation and spin states; temperature dependent electrochemical measurements to assess the thermodynamics (entropy and enthalpy) of the redox processes relevant for the studies

Biogenesis, mitochondrial assembly and function of human FeS-dependent molybdenum cofactor synthesis MOCS1 proteins

Günter Schwarz
Universität Köln

The molybdenum cofactor (Moco) is synthesized in a highly conserved multi-step pathway which is strictly dependent on the biosynthesis of [4Fe-4S] clusters. Human MOCS1 proteins are expressed by alternative splicing yielding two active proteins, MOCS1A and MOCS1AB. MOCS1A harbours two [4Fe-4S] clusters and is suggested to catalyse the radical SAM-dependent conversion of GTP into 3´,8-cylo-dihydro-GTP based on findings in E. coli. MOCS1AB represents a fusion protein of the MOCS1A and MOCS1B domains with only the MOCS1B domain exhibiting activity. Even though the overall biosynthesis of Moco is highly conserved, the fusion of MOCS1A and MOCS1B can only be found in animals and fungi. Whether this fusion has any effect on protein function or maturation has yet to be answered.

After having identified both MOCS1A and MOCS1AB as mitochondrial proteins it remains to be elucidated, what functions are retained within the MOCS1A domain, how are the [4Fe-4S] clusters incorporated, how does this domain interact with the MOCS1A protein and how does the overall machinery function and assemble within mitochondria. Additionally functional aspects of the reaction mechanism and parameters of MOCS1 proteins are yet to be determined. This is of particular interest in order to understand Moco deficiencies (MoCD) caused by single amino acid exchanges resulting in severe neurodegeneration.

Furthermore the interplay of [FeS] cluster biogenesis and MOCS1 activity still remains unclear. Therefore we are interested in investigating the influence of diseases linked to [4Fe-4S] cluster biosynthesis to MoCD, since diseases linked to FeS protein assembly often also manifest with sever neurodegeneration. In this context we are interested in the [4Fe-4S] cluster maturation and insertion in MOCS1A proteins within the mitochondrial matrix.



Roland Lill (Marburg): Determination of the influence of different FeS protein maturation defects on Moco synthesis by investigating the function of MOCS1A. We will focus on SiRNA based depletion of either core ISC components, ABCB7, or ISCA-IBA57 proteins to analyse where MOCS1A receives its [4Fe-4S] cluster. Furthermore, patient fibroblast and body fluids will be provided to determine Mo enzyme activities and Mo enzyme biomarkers.

Silke Leimkühler (Potsdam): Bacterial Moco synthesis and comparison of our MOCS1 data to MoaA and MoaC. Determination of Fe content by ICP-MS.

Ralf R. Mendel (Braunschweig): The Mendel lab aims to relocate the step 1 in Moco synthesis from mitochondria into the cytosol. Comparison of our results with the Nit7A/AB proteins.

Antonio J. Pierik (Kaiserslautern): EPR spectroscopy of MOCS1A and MOCS1AB proteins.

Volker Schünemann (Kaiserslautern): Mössbauer spectroscopy of MOCS1A and MOCS1AB proteins.

Carsten Berndt (Düsseldorf): Investigation of the potential molecular link between glutaredoxins (Grxs), Moco biosynthesis, and activities of Moco enzymes. We will measure activity of Moco proteins in cell lines with modulated expression of Grx2 (redox regulation), Grx3 (iron trafficking) or Grx5 (FeS cluster biosynthesis). We will also investigate sulfite oxidase related biomarker status in Grx deficient mice.

Johann Heider (Marburg) and Matthias Boll (Freiburg): Support in the in vitro insertion of tungsten cofactor (Wco) via Wco synthesizing proteins (MoaB and MoeA1/A2) that convert metal-binding pterin (MPT) into Wco.

[Fe]-hydrogenase: Role of Iron-sulfur bonding in holoenzyme assembly and in FeGP-cofactor biosynthesis

Seigo Shima
Max Planck Institute for Terrestrial Microbiology, Marburg

[Fe]-hydrogenase catalyzes the reversible transfer of a hydride from H2 to methenyl-tetrahydromethanopterin, which is an intermediary step in methanogenesis from H2 and CO2. The enzyme contains one iron per active site. The iron is associated with a unique iron-guanylylpyridinol (FeGP) cofactor, in which a low spin Fe(II) is complexed with two CO, one pyridinol-nitrogen, one acyl-carbon and, when the cofactor is bound to the holoenzyme, one thiolate from Cys176. The iron-sulfur bond is essential for hydrogenase activity. Apparently, iron-sulfur bonding is involved in assembly and catalysis of [Fe]-hydrogenase and the biosynthesis of its FeGP cofactor. In the proposed project, we want to study the biosynthesis of the FeGP cofactor. In many methanogens, the [Fe]-hydrogenase structural gene (hmd) is clustered with hmd-co-occurring genes (hcgA-G), which have been shown to be involved in FeGP cofactor biosynthesis. In previous studies, we have identified the function of five of the hcg gene-products (HcgB, HcgC, HcgD, HcgE and HcgF) using structure to function strategies and biochemical assays. HcgB catalyzed guanylyl-transfer from GTP to the 4-hydroxypyridinol. HcgC is a SAM-dependent methyltransferase; HcgD is a putative iron chaperone. HcgE catalyzes the adenylylation of the carboxy group of a 6-carboxymethyl-guanylylpyridinol precursor. Subsequently the product of HcgE reacts with Cys9 of HcgF yielding a thioester and AMP. Hypothesis is, based on chemical precedents that the thioester now reacts with an iron species forming the acyl and thiolate ligands. In the next three years, we would like to prove or disprove this hypothesis and identify the function of the two remaining Hcg proteins (HcgA and HcgG), both of which are predicted to be iron-sulfur proteins. For this purpose, we will – as before – employ structure to function strategies, in vitro biosynthesis and metabolite analysis of hcg knock-out mutants.


The collaborations we plan within the SPP

– Peter Hildebrandt and Ingo Zebger, Technische Universität Berlin, Institut für Chemie; Resonance Raman spectroscopic analysis of the biosynthetic intermediates.

– Antonio J. Pierik and Volker Schünemann, Technische Universität Kaiserslautern, Fachbereich Chemie; Electron paramagnetic resonance and Mössbauer spectroscopic analysis of the biosynthetic intermediate.

– Lucia Banci, Centro Risonanze Magnetiche, University of Florence; NMR analysis of the biosynthetic intermediates.

Serena DeBeer, Max-Planck-Institute für chemische Energiekonversion, X-ray absorption spectroscopic analysis of the biosynthetic precursors.

Holger Dobbek, Humboldt Universität Berlin and Lorenz Adrian, The Helmholtz Centre for Environmental Research Physiology, protein analysis of methanogenic archaea.

Lolland Lill, Marburg University; heterologous production of Hcg proteins in yeast and circular dichroism spectroscopy.

– Gary Sawers, University Halle-Wittenberg; Johann Heider, Marburg University and Constanze Pinske, University Halle-Wittenberg, crystallization of hydrogenases and other iron-sulfur proteins.

An in vitro system for the study of [NiFe]-hydrogenase maturation

Basem Soboh
Freie Universität Berlin

Our research focuses on the in vitro biosynthesis of complex Fe-S cofactors. We currently employ hydrogenases that make or oxidize hydrogen gas. [NiFe]-hydrogenase has a complicated catalytic active site that contains nickel and iron bound to the protein in addition to unique non-protein ligands.

The biosynthesis of hydrogenases is a multi-step process that requires the coordinated activity of several accessory proteins. Our interest lies in determining how this complex cofactor is assembled and incorporated into these enzymes.

Our strategy concept of an in vitro system is based on isolation of an active maturation machinery, followed by “watching” the stepwise synthesis and assembly of cofactor over time. This concept involves a broad range of methodologies ranging from microbiology, molecular biology and protein biochemistry to biophysical methods including X-ray crystallography, FT-IR, EPR, Raman and EXAFS spectroscopy.

Recently we could demonstrate the in vitro synthesis of active [NiFe]-hydrogenase using only purified components. This finding allows us to exert much greater control over the hydrogenase maturation machinery. It provides the possibility to study each of maturation steps individually at molecular level. This study includes determination of the temporal sequence of events and analysis of the compositions of functional complexes at each maturation step along the path of cofactor biosynthesis. A more thorough spectroscopic and structural investigation of an all-pure-component-based in vitro system is required to conclusively trace the intermediates of the assembly process of [NiFe]-hydrogenase.


AG DeBeer/Neese: EXAFS analysis of intermediates of [NiFe]-hydrogenases

AG Shima: Anaerobic crystallization of functional protein complexes and intermediates

AG Pinske: Generation of chromosomal mutants and variants

AG Einsle: Crystallization, FeMo-co biogenesis and catalysis

AG Schünemann: Mössbauer spectroscopy for the study f Fe-containing maturation proteins

AG Lill: 55Fe-labeling to identify the source, route and incorporation of iron into hydrogenase

AG Pierik: EPR spectroscopy of maturation proteins and intermediates

AG Adrian: Identification of protein/cofactor modifications and analysis of the compositions of functional protein complexes by Mass-spectrometry.

AG Schaffrath: characterization of electron transfer reactions

Simulating the Coupling of Catalysis and Electron Transfer along Chains of FeS-Clusters in Molybdo- and Tungsto-Pterin Enzymes

Matthias Ullmann
Universität Bayreuth

Iron-sulfur (FeS) clusters play a role in enzyme catalysis and electron transfer. In many enzymes, iron sulfur clusters align to enable electron transfer over large distances and often connect two catalytic centers of enzymes. Examples are molybdo- and tungsto-pterin enzymes. While the catalytic action of some of these enzymes has been investigated in detail, its connection to the electron transfer reactions has not been studied to the same level. The goal of this project is to lie the foundations for simulating the coupling of the chemical reactions and the electron transfer between the catalytic sites. For this purpose, we plan to extend our method to simulate the electron transfer dynamics using a master equation to enzyme catalysis. The enzyme is modeled as a system of sites (redox active site, protonatable sites, the catalytic centers). In each microstates, the protonation, redox state, and conformation of all sites are exactly defined. The energy of each microstate is obtained as a sum of site energies and the interactions between the sites. The site energies and the interactions are calculated from electrostatic calculations using the Poisson-Boltzmann equation. Rate constant for going from one microstate to the other are obtained from Marcus theory for charge transfer reactions and form QM/MM calculation for more complex chemical reactions in the active site. From these simulations, we will be able to deduce enzymatic mechanism of even very complex enzymes with several active sites. This method has so far only been applied to simple electron transfer reaction. In this project, we plan to applied it to molybdo- and tungsto-pterin enzymes that connect to active sites by a chain FeS clusters. In order to be able to do such calculations, we need to obtain reliable “model redox potentials”, i.e., redox potential data the FeS clusters would have in aqueous solutions. These values will be calculated on the basis of experimental redox potential of small proteins and synthetic model compounds of the active sites of enzymes. The simulation method will be applied to analyze the coupling of the chemical catalysis in Xanthine Dehydrogenase to the electron transfer to NAD+ via a chain of [2Fe2S] clusters and a FAD. This simulation will be a first test case our method. Moreover, we want to apply the method to study the mechanism of BamBC, an enzyme involved in the degradation of aromatic compounds. In the second funding period, we envision that the developed methodology can be applied to even more complex enzymes such as the whole Bam B-I complex. Also the study interplay of Nitrate Reductase and Formate Dehydrogenase in the bioenergetics of E. coli is an potential goal for the second funding period of the priority program.


Oliver Einsle:  Structure of Various Small FeS Proteins

Thorsten Friedrich:  Redox Potential Determination

Matthias Boll: Structural and Functional Studies of the Bam Complex

Franc Meyer:  Model Compounds of FeS Complexes

Carola Schulzke:  Model Compounds of Molybdo- and Tungstopterin Complexes

Silke Leimkühler:  Functional Studies on Molybdo- and Tungstopterin Enzymes