617 research outputs found
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
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
Developmental dynamics of two bipotent thymic epithelial progenitor types
T cell development in the thymus is essential for cellular immunity and depends on the organotypic thymic epithelial microenvironment. In comparison with other organs, the size and cellular composition of the thymus are unusually dynamic, as exemplified by rapid growth and high T cell output during early stages of development, followed by a gradual loss of functional thymic epithelial cells and diminished naive T cell production with age. Single-cell RNA sequencing (scRNA-seq) has uncovered an unexpected heterogeneity of cell types in the thymic epithelium of young and aged adult mice; however, the identities and developmental dynamics of putative pre- and postnatal epithelial progenitors have remained unresolved. Here we combine scRNA-seq and a new CRISPRâCas9-based cellular barcoding system in mice to determine qualitative and quantitative changes in the thymic epithelium over time. This dual approach enabled us to identify two principal progenitor populations: an early bipotent progenitor type biased towards cortical epithelium and a postnatal bipotent progenitor population biased towards medullary epithelium. We further demonstrate that continuous autocrine provision of Fgf7 leads to sustained expansion of thymic microenvironments without exhausting the epithelial progenitor pools, suggesting a strategy to modulate the extent of thymopoietic activity
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
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
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
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
Protonation/reduction dynamics at the [4Feâ4S] cluster of the hydrogen-forming cofactor in [FeFe]-hydrogenases
The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen
conversion catalysts in nature. Their active-site cofactor (H-cluster)
comprises a [4Feâ4S] cluster linked to a unique diiron site that binds three
carbon monoxide (CO) and two cyanide (CNâ) ligands. Understanding microbial
hydrogen conversion requires elucidation of the interplay of proton and
electron transfer events at the H-cluster. We performed real-time spectroscopy
on [FeFe]-hydrogenase protein films under controlled variation of atmospheric
gas composition, sample pH, and reductant concentration. Attenuated total
reflection Fourier-transform infrared spectroscopy was used to monitor shifts
of the CO/CNâ vibrational bands in response to redox and protonation changes.
Three different [FeFe]-hydrogenases and several protein and cofactor variants
were compared, including element and isotopic exchange studies. A protonated
equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially
accumulated at acidic pH and under reducing conditions. We show that the one-
electron reduced state HredâČ represents an intrinsically protonated species.
Interestingly, the formation of HoxH and HredâČ was independent of the
established proton pathway to the diiron site. Quantum chemical calculations
of the respective CO/CNâ infrared band patterns favored a cysteine ligand of
the [4Feâ4S] cluster as the protonation site in HoxH and HredâČ. We propose
that proton-coupled electron transfer facilitates reduction of the [4Feâ4S]
cluster and prevents premature formation of a hydride at the catalytic diiron
site. Our findings imply that protonation events both at the [4Feâ4S] cluster
and at the diiron site of the H-cluster are important in the hydrogen
conversion reaction of [FeFe]-hydrogenases
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
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Understanding the Mind or Predicting Signal-Dependent Action? Performance of Children With and Without Autism on Analogues of the False-Belief Task
To evaluate the claim that correct performance on unexpected transfer false-belief tasks specifically involves mental-state understanding, two experiments were carried out with children with autism, intellectual disabilities, and typical development. In both experiments, children were given a standard unexpected transfer false-belief task and a mental-state-free, mechanical analogue task in which participants had to predict the destination of a train based on true or false signal information. In both experiments, performance on the mechanical task was found to correlate with that on the false-belief task for all groups of children. Logistic regression showed that performance on the mechanical analogue significantly predicted performance on the false-belief task even after accounting for the effects of verbal mental age. The findings are discussed in relation to possible common mechanisms underlying correct performance on the two tasks
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