Cyanide Binding to [FeFe]-Hydrogenase Stabilizes the Alternative Configuration of the Proton Transfer Pathway

Hydrogenases are H2 converting enzymes that harbor catalytic cofactors in which iron (Fe) ions are coordinated by biologically unusual carbon monoxide (CO) and cyanide (CN−) ligands. Extrinsic CO and CN−, however, inhibit hydrogenases. The mechanism by which CN− binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CN−-treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39 Å allowed us to distinguish intrinsic CN− and CO ligands and to show that extrinsic CN− binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CN− treated crystals show conformational changes of conserved residues within the proton transfer pathway which could allow a direct proton transfer between E279 and S319. This configuration has been proposed to be vital for efficient proton transfer, but has never been observed structurally

The Geometry of the Catalytic Active Site in [FeFe]-hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

[FeFe]-hydrogenases are efficient metalloenzymes that catalyze the oxidation and evolution of molecular hydrogen, H2. They serve as a blueprint for the design of synthetic H2-forming catalysts. [FeFe]-hydrogenases harbor a six-iron cofactor that comprises a [4Fe-4S] cluster and a unique diiron site with cyanide, carbonyl, and hydride ligands. To address the ligand dynamics in catalytic turnover and upon carbon monoxide (CO) inhibition, we replaced the native aminodithiolate group of the diiron site by synthetic dithiolates, inserted into wild-type and amino acid variants of the [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. The reactivity with H2 and CO was characterized using in situ and transient infrared spectroscopy, protein crystallography, quantum chemical calculations, and kinetic simulations. All cofactor variants adopted characteristic populations of reduced species in the presence of H2 and showed significant changes in CO inhibition and reactivation kinetics. Differences were attributed to varying interactions between polar ligands and the dithiolate head group and/or the environment of the cofactor (i.e., amino acid residues and water molecules). The presented results show how catalytically relevant intermediates are stabilized by inner-sphere hydrogen bonding suggesting that the role of the aminodithiolate group must not be restricted to proton transfer. These concepts may inspire the design of improved enzymes and biomimetic H2-forming catalysts

Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN−) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of 13CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN− at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective 13CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle

Spectroscopical Investigations on the Redox Chemistry of [FeFe]-Hydrogenases in the Presence of Carbon Monoxide

[FeFe]-hydrogenases efficiently catalyzes hydrogen conversion at a unique [4Fe–4S]-[FeFe] cofactor, the so-called H-cluster. The catalytic reaction occurs at the diiron site, while the [4Fe–4S] cluster functions as a redox shuttle. In the oxidized resting state (Hox), the iron ions of the diiron site bind one cyanide (CN−) and carbon monoxide (CO) ligand each and a third carbonyl can be found in the Fe–Fe bridging position (µCO). In the presence of exogenous CO, A fourth CO ligand binds at the diiron site to form the oxidized, CO-inhibited H-cluster (Hox-CO). We investigated the reduced, CO-inhibited H-cluster (Hred´-CO) in this work. The stretching vibrations of the diatomic ligands were monitored by attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR). Density functional theory (DFT) at the TPSSh/TZVP level was employed to analyze the cofactor geometry, as well as the redox and protonation state of the H-cluster. Selective 13CO isotope editing, spectro-electrochemistry, and correlation analysis of IR data identified a one-electron reduced, protonated [4Fe–4S] cluster and an apical CN− ligand at the diiron site in Hred´-CO. The reduced, CO-inhibited H-cluster forms independently of the sequence of CO binding and cofactor reduction, which implies that the ligand rearrangement at the diiron site upon CO inhibition is independent of the redox and protonation state of the [4Fe–4S] cluster. The relation of coordination dynamics to cofactor redox and protonation changes in hydrogen conversion catalysis and inhibition is discussed

Bridging hydride at reduced H-cluster species in [FeFe]-hydrogenases revealed by infrared spectroscopy, isotope editing, and quantum chemistry

[FeFe]-Hydrogenases contain a H2-converting cofactor (H-cluster) in which a canonical [4Fe–4S] cluster is linked to a unique diiron site with three carbon monoxide (CO) and two cyanide (CN–) ligands (e.g., in the oxidized state, Hox). There has been much debate whether reduction and hydrogen binding may result in alternative rotamer structures of the diiron site in a single (Hred) or double (Hsred) reduced H-cluster species. We employed infrared spectro-electrochemistry and site-selective isotope editing to monitor the CO/CN– stretching vibrations in [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. Density functional theory calculations yielded vibrational modes of the diatomic ligands for conceivable H-cluster structures. Correlation analysis of experimental and computational IR spectra has facilitated an assignment of Hred and Hsred to structures with a bridging hydride at the diiron site. Pronounced ligand rotation during μH binding seems to exclude Hred and Hsred as catalytic intermediates. Only states with a conservative H-cluster geometry featuring a μCO ligand are likely involved in rapid H2 turnover

Accumulating the hydride state in the catalytic cycle of [FeFe]-hydrogenases

H2 turnover at the [FeFe]-hydrogenase cofactor (H-cluster) is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydrido-species which again is double-oxidized to release another proton. Three of the four presumed catalytic intermediates (Hox, Hred/Hred and Hsred) were characterized, using various spectroscopic techniques. However, in catalytically active enzyme, the state containing the hydrido-species, which is eponymous for the proposed heterolytic mechanism, has yet only been speculated about. We use different strategies to trap and spectroscopically characterize this transient hydride state (Hhyd) for three wild-type [FeFe]-hydrogenases. Applying a novel set-up for real-time attenuated total- reflection Fourier-transform infrared spectroscopy, we monitor compositional changes in the state-specific infrared signatures of [FeFe]-hydrogenases, varying buffer pH and gas composition. We selectively enrich the equilibrium concentration of Hhyd, applying Le Chatelier’s principle by simultaneously increasing substrate and product concentrations (H2/H+). Site-directed manipulation, targeting either the proton-transfer pathway or the adt ligand, significantly enhances Hhyd accumulation independent of pH

Protonengekoppelte Reduktion des katalytischen [4Fe-4S]-Zentrums in [FeFe]-Hydrogenasen

In der Natur katalysieren [FeFe]-Hydrogenasen die Abgabe und Aufnahme von molekularem Wasserstoff (H2) an einem einzigartigen Eisen-Schwefel-Kofaktor. Das geringe elektrochemische Überpotential in der Wasserstoffabgabe-Reaktion macht die [FeFe]-Hydrogenasen zu einem hervorragenden Beispiel für effiziente Biokatalyse. Gegenwärtig sind die molekularen Details des Wasserstoffumsatzes jedoch noch nicht vollständig verstanden. Daher adressieren wir in dieser Untersuchung die initiale Reduktion des katalytischen Zentrums der [FeFe]-Hydrogenasen mittels Infrarotspektroskopie und Elektrochemie und zeigen, dass der reduzierte Zustand Hred′ durch protonengekoppelten Elektronentransport gebildet wird. Ladungskompensation bindet das überschüssige Elektron am [4Fe-4S]-Zentrum und führt zu einer Stabilisierung der konservativen Konfiguration des [FeFe]-Kofaktors. Die Rolle von Hred′ beim Wasserstoffumsatz und mögliche Auswirkungen auf den katalytischen Mechanismus werden diskutiert. Es liegt nahe, dass die Regulation elektronischer Eigenschaften in der Umgebung von metallischen Kofaktoren die Grundlage für Multielektronenprozesse bildet

Die Bindung von Cyanid an [FeFe]‐Hydrogenasen stabilisiert die alternative Konfiguration des Protonentransferpfads

Hydrogenasen sind H2-umsetzende Metalloenzyme und enthalten katalytische Kofaktoren, deren Eisenionen durch biologisch ungewöhnliche Kohlenmonoxid- (CO) und Cyanid- (CN−) Liganden koordiniert sind. Externes CO und CN−hemmt Hydrogenasen jedoch. Der molekulare Mechanismus der Bindung von CN− an [FeFe]-Hydrogenasen ist unbekannt. In dieser Studie präsentieren wir Kristallstrukturen der mit CN− behandelten [FeFe]-Hydrogenase CpI aus Clostridium pasteurianum. Auf Grund der hohen Auflösung von 1.39 Å können wir die intrinsischen CO- und CN−-Liganden voneinander unterscheiden. Wir zeigen, dass externes CN− die offene Bindestelle des Kofaktors besetzt, an die auch externes CO bindet. Im Gegensatz zu anderen Inhibitoren zeigen die CN−-behandelten Kristalle Konformationsänderungen konservierter Reste des Protonentransferpfads, die einen direkten Austausch von Protonen zwischen den Aminosäuren E279 und S319 ermöglichen. Diese Konformation wurde als notwendig für einen effizienten Protonentransfer vorgeschlagen, doch wurde sie bisher nicht strukturell nachgewiesen

Hydrogen and oxygen trapping at the H-cluster of [FeFe]-hydrogenase revealed by site-selective spectroscopy and QM/MM calculations

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN−) ligands. Hydrogen (H2) and oxygen (O2) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO- inhibited (Hox-CO) species in wildtype enzyme. 57Fe labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fed in a [4Fe4S-2Fe]3 − complex with the net spin on Fed whereas Hox-CO shows an apical CN− at Fed in a [4Fe4S-2Fe(CO)]3 − complex with net spin sharing among Fep and Fed (proximal or distal iron ions in [2Fe]). At ambient O2 pressure, a novel H-cluster species (Hox-O2) accumulated in C169A, assigned to a [4Fe4S-2Fe(O2)]3 − complex with an apical superoxide (O2−) carrying the net spin bound at Fed. H2 exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)]3 − complex with the net spin on the reduced cubane, an apical hydride at Fed, and a proton at a cysteine ligand. Hox-O2 and Hhyd are stabilized by impaired O2– protonation or proton release after H2 cleavage due to interruption of the proton path towards and out of the active site

Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site

Elucidating the distribution of intermediates at the active site of redox metalloenzymes is vital to understanding their highly efficient catalysis. Here we demonstrate that it is possible to generate, and detect, the key catalytic redox states of an [FeFe]-hydrogenase in a protein crystal. Individual crystals of the prototypical [FeFe]-hydrogenase I from Clostridium pasteurianum (CpI) are maintained under electrochemical control, allowing for precise tuning of the redox potential, while the crystal is simultanaously probed via Fourier Transform Infrared (FTIR) microspectroscopy. The high signal/noise spectra reveal potential-dependent variation in the distribution of redox states at the active site (H-cluster) according to state-specific vibrational bands from the endogeneous CO and CN- ligands. CpI crystals are shown to populate the same H-cluster states as those detected in solution, including the oxidised species Hox, the reduced species Hred/HredH+, the super-reduced HsredH+ and the hydride species Hhyd. The high sensitivity and precise redox control offered by this approach also facilitates the detection and characterisation of low abundance species that only accumulate within a narrow window of conditions, revealing new redox intermediates