Conformational and mechanical stability of the isolated large subunit of membrane-bound [NiFe]-hydrogenase from Cupriavidus necator

Comprising at least a bipartite architecture, the large subunit of [NiFe]-hydrogenase harbors the catalytic nickel–iron site while the small subunit houses an array of electron-transferring Fe-S clusters. Recently, some [NiFe]-hydrogenase large subunits have been isolated showing an intact and redox active catalytic cofactor. In this computational study we have investigated one of these metalloproteins, namely the large subunit HoxG of the membrane-bound hydrogenase from Cupriavidus necator (CnMBH), targeting its conformational and mechanical stability using molecular modelling and long all-atom Gaussian accelerated molecular dynamics (GaMD). Our simulations predict that isolated HoxG is stable in aqueous solution and preserves a large portion of its mechanical properties, but loses rigidity in regions around the active site, in contrast to the MBH heterodimer. Inspired by biochemical data showing dimerization of the HoxG protein and IR measurements revealing an increased stability of the [NiFe] cofactor in protein preparations with higher dimer content, corresponding simulations of homodimeric forms were also undertaken. While the monomeric subunit contains several flexible regions, our data predicts a regained rigidity in homodimer models. Furthermore, we computed the electrostatic properties of models obtained by enhanced sampling with GaMD, which displays a significant amount of positive charge at the protein surface, especially in solvent-exposed former dimer interfaces. These data offer novel insights on the way the [NiFe] core is protected from de-assembly and provide hints for enzyme anchoring to surfaces, which is essential information for further investigations on these minimal enzymes

Shedding Light on Proton and Electron Dynamics in [FeFe] Hydrogenases

[FeFe] hydrogenases are highly efficient catalysts for reversible dihydrogen evolution. H2 turnover involves different catalytic intermediates including a recently characterized hydride state of the active site (H-cluster). Applying cryogenic infrared and electron paramagnetic resonance spectroscopy to an [FeFe] model hydrogenase from Chlamydomonas reinhardtii (CrHydA1), we have discovered two new hydride intermediates and spectroscopic evidence for a bridging CO ligand in two reduced H-cluster states. Our study provides novel insights into these key intermediates, their relevance for the catalytic cycle of [FeFe] hydrogenase, and novel strategies for exploring these aspects in detail

Spektroskopische Untersuchungen an Hydrogenasen

The establishment of sustainable economy may be based on molecular hydrogen as an energy-storage molecule and requires the development of cheap catalysts, which are able to work under various conditions. In nature, this most fundamental reaction, i.e. the reversible reduction of protons, is catalysed by a group of enzymes called hydrogenases. Members of this wide family of enzymes exhibit various properties, such as different activity rates, bias towards a specific direction of the catalytic reaction and a potential tolerance towards oxygen. Vibrational spectroscopy can be used as a tool to clarify molecular mechanism related to the catalytic cycle and reactions with other small molecules (e.g. CO and O 2 ). Such insights could provide a blueprint for the synthesis of molecular catalysts for future applications. In the first part of this thesis, resonance Raman (RR) spectroscopy was applied for the first time on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (HydA1), the smallest catalytic unit of this class of hydrogenase, revealing thereby the first catalytic intermediate in the proton reduction reaction. RR spectroscopy was initially used to characterise the in vitro maturation process of this enzyme on the basis of molecular vibrations related to the Fe-S, Fe-CO or Fe-CN bonds located in its active site, which consists of a [FeFe] centre covalently linked to a [4Fe4S] cluster. A comparison of the spectra of redox-tuned samples of artificially maturated holo-HydA1 revealed a photo-induced intramolecular electron transfer occurring between the two metal centres of the active site. Further experiments with a non-native artificial maturated HydA1 could demonstrate that this electron transfer is coupled with a proton transfer to the secondary amine of the aza-thiolate head group also found in the active site of the native enzyme. Following this hypothesis, the stabilisation of newly discovered H red ’ state, characterised by an oxidised [FeFe] centre and a reduced [4Fe4S] cluster, is related to a charge compensation mechanism. Infrared (IR) spectroscopy is a well-established method to study hydrogenases and can discriminate different redox states of these enzymes with respect to the particular band positions of their CO and CN stretching vibrations. Therefore, this technique was used for control measurements both, at cryogenic and at room temperatures to mimic the conditions of the RR experiments and to validate the procedures for redox tuning, respectively. Within these control experiments, a second light-induced reaction was identified. With help of adequate difference spectra, a set of IR bands could be assigned to a new H sred ’ state, which is probably associated to the final step of the catalytic reaction before a H 2 molecule is released from the active site. This study demonstrates the potential of cryogenic vibrational spectroscopy to probe transient intermediates by kinetically trapping thermodynamically instable states. Contrary to most [FeFe]-hydrogenases, the active site of [NiFe]-hydrogenases does not decompose following an exposure to oxygen. However, so called anaerobic “standard” hydrogenases form the unready Ni u -A state, which requires chemical reduction or very long incubation times with hydrogen to achieve full reactivation. On the other hand, oxygen- tolerant enzymes, such as the membrane-bound hydrogenase (MBH) from Ralstonia eutropha (Re), form only the ready Ni r -B state, which is reactivated after a short lag-time following an exposure to hydrogen. These long and short lag-times are the result of “autocatalytic” intermolecular activation mechanisms proposed for [NiFe] hydrogenases. The second part of the thesis comprises three studies on the ReMBH. Unlike the anaerobic “standard” [NiFe]-hydrogenases, which harbour a cubane-like [4Fe4S] cluster in the proximity of the [NiFe] active site, in oxygen tolerant MBH’s, such as ReMBH, two conserved cysteines in the first coordination sphere of this cofactor lead to the formation of an unusual [4Fe3S]-proximal cluster. This unique cluster is capable to perform two redox transitions, which were previously assumed to prevent the formation of unready states. Two variants of ReMBH were generated, in which one of the two cysteine residues (i.e. cysteine 19 or 120) was replaced by a glycine. A combined RR, IR and electron paramagnetic resonance (EPR) spectroscopic characterisation of redox-tuned samples could show that the cluster of both ReMBH variants (i.e. C19G and C120G) could undergo only a single redox transition, corresponding to one of the respective transitions observed in the wildtype. However, no unready states were detected for both variants after exposure to oxygen. Moreover, in pure electrochemical experiments it was shown that both variants exhibit some catalytic activity under aerobic condition. Using surface enhanced infrared absorption (SEIRA) spectroscopy, structural data was obtained to compliment the electrochemical measurements. This spectro- electrochemical approach could show no qualitative difference in terms of redox states composition of the active site, between the two variants and the wildtype after an exposure to oxygen under catalytic conditions. These results suggest that the additional redox transition of the modified proximal cluster of the wildtype plays mainly a regulating role in the kinetic of the oxygen-detoxification mechanism and that the formation of the unready states in ReMBH is prevented by other modifications, presumably near the active site. A study on another variant of ReMBH, namely the D117S variant, with a modified active site environment demonstrates the role of proton transfer as a charge compensation mechanism, which occurs during the activation process of ReMBH’s. Moreover, this study could provide the first spectroscopic evidence for an “autocatalytic” reactivation mechanism in these enzymes. In a third study, a redox titration of the artificially cross-linked heterotrimer of ReMBH as well as IR and EPR spectroscopic characterisation of membrane samples enriched with wildtype and variants of ReMBH could elucidate the role of the native configuration as a super-complex of heterotrimers in de- and reactivation processes. Thereby, it was shown that full reoxidation of the proximal cluster as well as the prevention of the formation of some inactive states of the active site could occur only in the native environment, presumably due to an intact electron relay of ReMBH. These studies contributed to an identification of structural and redox arrangements in ReMBH, which tightly regulate the chemistry occurring at the active site, thereby enabling the oxidation of molecular hydrogen in the presence of oxygen.Die Etablierung einer nachhaltigen Wirtschaft die auf molekularem Wasserstoff als Energiespeichermolekül basiert, erfordert die Entwicklung kostengünstiger Katalysatoren, die unter den verschiedensten Bedingungen arbeiten können. In der Natur wird die Elementarreaktion, die reversible Reduktion von Protonen, durch eine Gruppe von Enzymen katalysiert, die als Hydrogenasen bezeichnet werden. Mitglieder dieser großen Familie von Enzymen weisen verschiedene Eigenschaften auf, wie unterschiedliche Aktivitätsraten, Neigung zu einer spezifischen Richtung der katalytischen Reaktion sowie die etwaige Fähigkeit diese in Gegenwart von Sauerstoff ausführen zu können. Die Schwingungsspektroskopie kann verwendet werden, um den molekularen Mechanismus im Zusammenhang mit dem katalytischen Zyklus sowie den Reaktionen mit anderen kleinen Molekülen (z. B. CO und O 2 ) aufzuklären. Solche Erkenntnisse könnten ein Modell für die Synthese molekularer Katalysatoren zukünftiger Anwendungen liefern. Im ersten Teil dieser Arbeit wurden erstmals Resonanz Raman (RR) spektroskopischen Messungen an der [FeFe] Hydrogenase von Chlamydomonas reinhardtii (HydA1) durchgeführt, die die kleinste katalytische Einheit dieser Klasse von Hydrogenasen aufweist. Dabei konnte das erste katalytische Intermediat der Protonen-Reduktionsreaktion nachgewiesen werden. Die RR-Spektroskopie wurde zunächst verwendet, um den in vitro Reifungsprozess dieses Enzyms auf der Grundlage molekularer Schwingungen zu charakterisieren, die mit den Fe-S-, Fe-CO- oder Fe-CN-Bindungen in seinem aktiven Zentrum zusammenhängen Dieses aktive Zentrum besteht aus einem [FeFe] Zentrum, welches kovalent an einen [4Fe4S] Cluster gebunden ist. Ein Vergleich der Spektren von festeingestellten Redox-Zuständen in den Proben von artifiziell maturiertem Holo-HydA1 deutete auf einen fotoinduzierten intramolekularen Elektronentransfer zwischen den beiden Metallzentren des aktiven Zentrums hin. Weitere Versuche mit einem nicht-nativen artifiziell maturierten HydA1 zeigten, dass dieser Elektronentransfer mit einem Protonentransfer zum sekundären Amin der Aza-Thiolat-Kopfgruppe gekoppelt ist, die auch im aktiven Zentrum des nativen Enzyms gefunden wird. Gemäß dieser Hypothese steht die Stabilisierung des neu entdeckten H red ‘ Zustands, der durch ein oxidiertes [FeFe] Zentrum und einen reduzierten [4Fe4S] Cluster charakterisiert ist, mit einem Ladungsausgleichsmechanismus im Zusammenhang. Die Infrarot (IR) Spektroskopie ist eine etablierte Methode zur Untersuchung von Hydrogenasen und kann verschiedene Redox-Zustände dieser Enzyme hinsichtlich der jeweiligen Bandposition ihrer CO- und CN-Streckschwingungen unterscheiden. Daher wurde diese Technik für Kontrollmessungen sowohl bei kryogenen Temperaturen zur Nachstellung der Bedingungen für die RR-Experimente, als auch bei Raumtemperatur zur Validierung des Verfahrens zur Redox-Zustandseinstellung verwendet. Im Rahmen dieser Kontrollexperimente wurde eine zweite lichtinduzierte Reaktion identifiziert. Mit Hilfe geeigneter Differenzspektren konnte eine Reihe von IR-Banden einem neuen H sred ‘-Zustand zugeordnet werden, der wahrscheinlich mit dem letzten Schritt der katalytischen Reaktion zusammenhängt, bevor ein H 2 -Molekül aus dem aktiven Zentrum freigesetzt wird. Diese Studie demonstriert das Potenzial kryogener Schwingungsspektroskopie zur Untersuchung transienter Intermediate durch ein kinetisches „Einfangen“ thermodynamisch instabiler Zustände. Im Unterschied zu den meisten [FeFe] Hydrogenasen zersetzt sich das aktive Zentrum von [NiFe] Hydrogenasen nach Sauerstoffeinwirkung nicht. Solche anaeroben "Standard" Hydrogenasen bilden jedoch einen sogenannten „unready“ Ni u -A-Zustand, der Umsetzung mit chemischen Reduktionsmitteln oder sehr lange Inkubationszeiten mit Wasserstoff benötigt, um vollständig reaktiviert zu werden. Auf der anderen Seite bilden sauerstofftolerante Enzyme wie die membrangebundene Hydrogenase (MBH) von Ralstonia eutropha (Re) nur den „ready“ Ni r -B-Zustand aus, der nach einer kurzen Verzögerungszeit nach Wasserstoffeinwirkung wieder aktiviert wird. Diese langen und kurzen Verzögerungszeiten sind das Ergebnis der für solche Enzyme vorgeschlagenen "autokatalytischen" intermolekularen Aktivierungsmechanismen. Der zweite Teil der Arbeit umfasst drei Studien an der ReMBH. Im Gegensatz zu den anaeroben "Standard"-[NiFe]-Hydrogenasen, die einen Cuban-ähnlichen [4Fe4S]-Cluster in der Nähe des aktiven NiFe-Zentrums beherbergen, befinden sich in sauerstofftoleranten MBHs, wie ReMBH, zwei konservierte Cysteine in der ersten Koordinationssphäre von diesem Kofaktor und er bildet einen ungewöhnlichen proximalen [4Fe3S] Cluster. Dieses einzigartige Cluster ist in der Lage, zwei Redox-Übergänge durchzuführen, von denen angenommen wurde, dass sie die Bildung von „unready“ Zuständen verhindern. Es wurden zwei Varianten von ReMBH präpariert, bei denen einer der beiden Cysteinreste (d. h. Cystein 19 oder 120) durch ein Glycin ersetzt wurde. Eine kombinierte RR-, IR- und elektronen-paramagnetische-Resonanz- (EPR) spektroskopische Charakterisierung von auf bestimmten Redox-Zustände eingestellten Proben konnte zeigen, dass der Cluster beider ReMBH-Varianten (d.h. C19G und C120G) nur einen einzelnen Redox-Übergang durchlaufen kann, der einen der jeweils im Wildtyp beobachteten Übergänge entspricht. Es wurden jedoch für beide Varianten nach Einwirkung von Sauerstoff keine „unready“ Zustände nachgewiesen. In reinen elektrochemischen Experimenten konnte zudem gezeigt werden, dass beide Varianten unter aeroben Bedingungen katalytisch aktiv sind. Unter Verwendung der Oberflächeverstäkten-Infrarot-Absorptionsspektroskopie (SEIRA) wurden Strukturdaten erhalten, um die rein elektrochemischen Messungen zu ergänzen. Dieser spektro-elektrochemische Ansatz konnte keinen qualitativen Unterschied hinsichtlich der Zusammensetzung des aktiven Zentrums nach einer Sauerstoffexposition unter katalytischen Bedingungen zwischen den beiden Varianten und dem Wildtyp zeigen. Diese Ergebnisse deuten darauf hin, dass der zusätzliche Redox-Übergang des modifizierten proximalen Clusters des Wildtyps hauptsächlich eine regulierende kinetische Rolle im „Entgiftungsmechanismus“ des Sauerstoffs spielt und dass die Bildung der „unready“ Zustände in der ReMBH durch andere Modifikationen, vermutlich in der Nähe des aktiven Zentrums, verhindert wird. Eine Studie zu einer anderen Variante von ReMBH, der D117S Variante, mit einer modifizierten Umgebung des aktiven Zentrums zeigt die Rolle des Protonentransfers als Ladungskompensationsmechanismus auf, der während des Aktivierungsprozesses von der ReMBH auftritt. Darüber hinaus konnte diese Studie den ersten spektroskopischen Nachweis für den sogenannten „autokatalytischen“ Reaktivierungsmechanismus in solchen Enzymen liefern. In einer dritten Studie, konnte eine Redox-Titration des künstlich vernetzten Heterotrimers der ReMBH, sowie die IR- und EPR-spektroskopische Charakterisierung von mit Wildtyp und Varianten von mit ReMBH angereicherten Membranproben, die Rolle der nativen Konfiguration als Superkomplex von Heterotrimeren in den De- und Reaktivierungsprozessen erklären. Dabei konnte gezeigt werden, dass eine vollständige Reoxidation des proximalen Clusters sowie die Verhinderung der Bildung einiger inaktiver Zustände des aktiven Zentrums nur in der natürlichen Umgebung auftreten können, vermutlich aufgrund eines intakten Elektronentransferkette in der ReMBH. Diese Studien trugen dazu bei, strukturelle Anordnungen in der ReMBH zu identifizieren, die die am aktiven Zentrum stattfindende Chemie streng regulieren und so die Oxidation von molekularem Wasserstoff in der Gegenwart von Sauerstoff ermöglichen

Structural Determinants of the Catalytic Nia-L Intermediate of [NiFe]-Hydrogenase

[NiFe]-hydrogenases catalyze the reversible cleavage of H2 into two protons and two electrons at the inorganic heterobimetallic NiFe center of the enzyme. Their catalytic cycle involves at least four intermediates, some of which are still under debate. While the core reaction, including H2/H- binding, takes place at the inorganic cofactor, a major challenge lies in identifying those amino acid residues that contribute to the reactivity and how they stabilize (short-lived) intermediate states. Using cryogenic infrared and electron paramagnetic resonance spectroscopy on the regulatory [NiFe]-hydrogenase from Cupriavidus necator, a model enzyme for the analysis of catalytic intermediates, we deciphered the structural basis of the hitherto elusive Nia-L intermediates. We unveiled the protonation states of a proton-accepting glutamate and a Ni-bound cysteine residue in the Nia-L1, Nia-L2, and the hydride-binding Nia-C intermediates, as well as previously unknown conformational changes of amino acid residues in proximity of the bimetallic active site. As such, this study unravels the complexity of the Nia-L intermediate and reveals the importance of the protein scaffold in fine-tuning proton and electron dynamics in [NiFe]-hydrogenase

X-ray Crystallography and Vibrational Spectroscopy Reveal the Key Determinants of Biocatalytic Dihydrogen Cycling by [NiFe] Hydrogenases

[NiFe] hydrogenases are complex model enzymes for the reversible cleavage of dihydrogen (H2). However, structural determinants of efficient H2 binding to their [NiFe] active site are not properly understood. Here, we present crystallographic and vibrational-spectroscopic insights into the unexplored structure of the H2-binding [NiFe] intermediate. Using an F420-reducing [NiFe]-hydrogenase from Methanosarcina barkeri as a model enzyme, we show that the protein backbone provides a strained chelating scaffold that tunes the [NiFe] active site for efficient H2 binding and conversion. The protein matrix also directs H2 diffusion to the [NiFe] site via two gas channels and allows the distribution of electrons between functional protomers through a subunit-bridging FeS cluster. Our findings emphasize the relevance of an atypical Ni coordination, thereby providing a blueprint for the design of bio-inspired H2-conversion catalysts

Local electric field changes during thephotoconversion of the bathy phytochrome Agp2

Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause–effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective nitrile stretching modes in the various states of photoconversion (vibrational Stark effect). Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural model for the Pfr state of F192pCNF, a molecular mechanical–quantum mechanical approach was employed to calculate the electric field at the nitrile group and the respective stretching frequency, in excellent agreement with the experiment. These calculations serve as a reference for determining the electric field changes in the photoinduced states of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the theoretical approach is not applicable. However, in both variants, the largest changes of the nitrile stretching modes occur in the last step of the photoconversion, supporting the view that the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"DFG, 221545957, SFB 1078: Proteinfunktion durch Protonierungsdynami

Potential Distribution across Model Membranes

Membrane models assembled on electrodes are widely used tools to study potential-dependent molecular processes at or in membranes. However, the relationship between the electrode potential and the potential across the membrane is not known. Here we studied lipid bilayers immobilized on mixed self-assembled monolayers (SAM) on Au electrodes. The mixed SAM was composed of thiol derivatives of different chain lengths such that between the islands of the short one, mercapto­benzonitrile (MBN), and the tethered lipid bilayer an aqueous compartment was formed. The nitrile function of MBN, which served as a reporter group for the vibrational Stark effect (VSE), was probed by surface-enhanced infrared absorption spectroscopy to determine the local electric field as a function of the electrode potential for pure MBN, mixed SAM, and the bilayer system. In parallel, we calculated electric fields at the VSE probe by molecular dynamics (MD) simulations for different charge densities on the metal, thereby mimicking electrode potential changes. The agreement with the experiments was very good for the calculations of the pure MBN SAM and only slightly worse for the mixed SAM. The comparison with the experiments also guided the design of the bilayer system in the MD setups, which were selected to calculate the electrode potential dependence of the transmembrane potential, a quantity that is not directly accessible by the experiments. The results agree very well with estimates in previous studies and thus demonstrate that the present combined experimental–theoretical approach is a promising tool for describing potential-dependent processes at biomimetic interfaces

Unusual structures and unknown roles of FeS clusters in metalloenzymes seen from a resonance Raman spectroscopic perspective

Funding Information: This work was financially supported by: Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionaliza??o (POCI) and by national funds through FCT - Funda?a?o para a Cie?ncia e a Tecnologia; by the European Union's Horizon 2020 research and innovation programme, TIMB3, GA No 810856. ST acknowledges PTDC/BTM-SAL/29507/2017 and CMS acknowledges PTDC/BIA-BFS/31026/2017 project and CB acknowledges 2020.05017.BD fellowship granted by FCT. GC, IZ and PH thank the Einstein Foundation Berlin (grant number EVF-2016-277) for funding. This work was also supported through the cluster of excellence ?UniSysCat? funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany?s Excellence Strategy-EXC2008/1-390540038 and the SPP 1927 ?Iron sulfur for Life? - ZE 510/2-2 (IZ). Funding Information: This work was financially supported by: Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT - Fundação para a Ciência e a Tecnologia; by the European Union’s Horizon 2020 research and innovation programme, TIMB 3 , GA No 810856. ST acknowledges PTDC/BTM-SAL/29507/2017 and CMS acknowledges PTDC/BIA-BFS/31026/2017 project and CB acknowledges 2020.05017.BD fellowship granted by FCT. GC, IZ and PH thank the Einstein Foundation Berlin (grant number EVF-2016-277) for funding. This work was also supported through the cluster of excellence “UniSysCat“ funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germanýs Excellence Strategy-EXC2008/1-390540038 and the SPP 1927 ”Iron sulfur for Life” - ZE 510/2-2 (IZ). Publisher Copyright: © 2021 The AuthorsThe universe of known biological FeS clusters is constantly enlarging. Besides the conventional, well described [2Fe–2S], [3Fe–4S] and cubane [4Fe–4S] clusters, novel, unprecedented structures are emerging. They include unusually coordinated clusters, with additional sulfur atoms, e.g., [4Fe–5S], [5Fe–5S], [4Fe–4S]-5S-[4Fe–4S], [8Fe–7S], [8Fe–9S] and [8Fe–8S–C] and heteronuclear clusters, e.g., [Ni–4Fe–4S], [2Ni–4Fe–4S], [4Fe–4S]-[2Ru], [Me–7Fe–9S–C–(homocitrate)] that undertake versatile physiological roles in the activation of small molecules (H2, CO2, CO and N2) and in the sulfuration of different compounds (e.g., t-RNAs, biotin and lipoic acid) in biology. A few structures are characterized by highly distorted geometries, e.g., the non-cubane [4Fe–4S] center and the hydrogenase-related [4Fe–3S] cluster, and contain atypical ligations or vacant coordination sites, which confer them novel functions far from the common electron transfer. Herein, we single out clusters found in i) hydrogenases that ensure sustainable hydrogen cycling, promising a clean fuel production in the future, ii) radical-SAM enzymes that can inspire applied catalysis due to an intrinsic flexibility of the radical chemistry, and iii) standard [4Fe–4S] cluster with still unknown function in DNA repair enzymes, which offer a possibility to interfere with DNA repair in pathogens or improve it in humans. Focusing on the abovementioned enzymes, we demonstrate the unique power of resonance Raman spectroscopy to unveil remarkable features in FeS centers, which has contributed to our understanding of unusual structures and disentangling of unknown functions.publishersversionpublishe