39 research outputs found
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 (CNâ) ligands. Extrinsic CO and CNâ, however, inhibit hydrogenases. The mechanism by which CNâ binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CNâ-treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39â
Ă
allowed us to distinguish intrinsic CNâ and CO ligands and to show that extrinsic CNâ binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CNâ 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 (CNâ) 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 CNâ 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 [4Feâ4S]-[FeFe] cofactor, the so-called H-cluster. The catalytic reaction occurs at the diiron site, while the [4Feâ4S] cluster functions as a redox shuttle. In the oxidized resting state (Hox), the iron ions of the diiron site bind one cyanide (CNâ) and carbon monoxide (CO) ligand each and a third carbonyl can be found in the FeâFe bridging position (”CO). 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 (HredÂŽ-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 [4Feâ4S] cluster and an apical CNâ ligand at the diiron site in HredÂŽ-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 [4Feâ4S] 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 [4Feâ4S] cluster is linked to a unique diiron site with three carbon monoxide (CO) and two cyanide (CNâ) 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/CNâ 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 ÎŒH binding seems to exclude Hred and Hsred as catalytic intermediates. Only states with a conservative H-cluster geometry featuring a ÎŒCO 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 Chatelierâs 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 Ăberpotential in der Wasserstoffabgabe-Reaktion
macht die [FeFe]-Hydrogenasen zu einem hervorragenden Beispiel fĂŒr effiziente
Biokatalyse. GegenwÀrtig sind die molekularen Details des Wasserstoffumsatzes
jedoch noch nicht vollstÀndig 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 HredâČ durch protonengekoppelten
Elektronentransport gebildet wird. Ladungskompensation bindet das
ĂŒberschĂŒssige Elektron am [4Fe-4S]-Zentrum und fĂŒhrt zu einer Stabilisierung
der konservativen Konfiguration des [FeFe]-Kofaktors. Die Rolle von HredâČ 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 fĂŒr
Multielektronenprozesse bildet
Die Bindung von Cyanid an [FeFe]âHydrogenasen stabilisiert die alternative Konfiguration des Protonentransferpfads
Hydrogenasen sind H2-umsetzende Metalloenzyme und enthalten katalytische Kofaktoren, deren Eisenionen durch biologisch ungewöhnliche Kohlenmonoxid- (CO) und Cyanid- (CNâ) Liganden koordiniert sind. Externes CO und CNâhemmt Hydrogenasen jedoch. Der molekulare Mechanismus der Bindung von CNâ an [FeFe]-Hydrogenasen ist unbekannt. In dieser Studie prĂ€sentieren wir Kristallstrukturen der mit CNâ behandelten [FeFe]-Hydrogenase CpI aus Clostridium pasteurianum. Auf Grund der hohen Auflösung von 1.39â
Ă
können wir die intrinsischen CO- und CNâ-Liganden voneinander unterscheiden. Wir zeigen, dass externes CNâ die offene Bindestelle des Kofaktors besetzt, an die auch externes CO bindet. Im Gegensatz zu anderen Inhibitoren zeigen die CNâ-behandelten Kristalle KonformationsĂ€nderungen konservierter Reste des Protonentransferpfads, die einen direkten Austausch von Protonen zwischen den AminosĂ€uren E279 und S319 ermöglichen. Diese Konformation wurde als notwendig fĂŒr 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 (CNâ) 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 â complex with the net spin on Fed whereas
Hox-CO shows an apical CNâ at Fed in a [4Fe4S-2Fe(CO)]3 â 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 â complex with an apical superoxide (O2â)
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 â 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 O2â 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