672 research outputs found
Wasserstoffkatalyse in Mikroalgen
Hydrogenasen in GrĂŒnalgen katalysieren die Abgabe von Wasserstoff. Wie lĂ€uft
das auf molekularer Ebene ab? Isotopenmarkierung und Infrarotspektroskopie
helfen, diese Frage zu beantworten
Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis
Metalloenzymes catalyze complex and essential processes, such as
photosynthesis, respiration, and nitrogen fixation. For example, bacteria and
archaea use [NiFe]-hydrogenases to catalyze the uptake and release of
molecular hydrogen (H2). [NiFe]-hydrogenases are redox enzymes composed of a
large subunit that harbors a NiFe(CN)2CO metallo-center and a small subunit
with three ironâsulfur clusters. The large subunit is synthesized with a
C-terminal extension, cleaved off by a specific endopeptidase during
maturation. The exact role of the C-terminal extension has remained elusive;
however, cleavage takes place exclusively after assembly of the
[NiFe]-cofactor and before large and small subunits form the catalytically
active heterodimer. To unravel the functional role of the C-terminal
extension, we used an enzymatic in vitro maturation assay that allows
synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified
components. The maturation process included formation and insertion of the
NiFe(CN)2CO cofactor into the large subunit, endoproteolytic cleavage of the
C-terminal extension, and dimerization with the small subunit. Biochemical and
spectroscopic analysis indicated that the C-terminal extension of the large
subunit is essential for recognition by the maturation machinery. Only upon
completion of cofactor insertion was removal of the C-terminal extension
observed. Our results indicate that endoproteolytic cleavage is a central
checkpoint in the maturation process. Here, cleavage temporally orchestrates
cofactor insertion and protein assembly and ensures that only cofactor-
containing protein can continue along the assembly line toward functional
[NiFe]-hydrogenase
How [FeFe]-hydrogenase facilitates bidirectional proton transfer
Hydrogenases are metalloenzymes that catalyze the conversion of protons and molecular hydrogen, H2. [FeFe]-hydrogenases show particularly high rates of hydrogen turnover and have inspired numerous compounds for biomimetic H2 production. Two decades of research on the active site cofactor of [FeFe]-hydrogenases have put forward multiple models of the catalytic proceedings. In comparison, our understanding of proton transfer is poor. Previously, residues were identified forming a hydrogen-bonding network between active site cofactor and bulk solvent; however, the exact mechanism of catalytic proton transfer remained inconclusive. Here, we employ in situ infrared difference spectroscopy on the [FeFe]-hydrogenase from Chlamydomonas reinhardtii evaluating dynamic changes in the hydrogen-bonding network upon photoreduction. While proton transfer appears to be impaired in the oxidized state (Hox), the presented data support continuous proton transfer in the reduced state (Hred). Our analysis allows for a direct, molecular unique assignment to individual amino acid residues. We found that transient protonation changes of glutamic acid residue E141 and, most notably, arginine R148 facilitate bidirectional proton transfer in [FeFe]-hydrogenases
biodiversity and spectroscopic investigations
Hydrogenases are redox enzymes that catalyze the conversion of protons and molecular hydrogen (H2). Based on the composition of the active site cofactor, the monometallic [Fe]-hydrogenase is distinguished from the bimetallic [NiFe]- or [FeFe]-hydrogenase. The latter has been reported with particularly high turnover activities for both H2 release and H2 oxidation, notably at neutral pH, ambient temperatures, and negligible electric overpotential. Due to these properties, [FeFe]-hydrogenase represents the âgold standardâ in enzymatic hydrogen turnover. Understanding hydrogenase chemistry is crucial for the design of transition metal complexes that serve as potentially sustainable proton reduction or H2 oxidation catalysts, e.g., in electrolytic devices or fuel cells. However, even 20 years after the crystal structures of [FeFe]-hydrogenase have been published, several aspects of biological hydrogen turnover are heatedly discussed. In this perspective, we give an overview on how the diversity of naturally occurring and artificially prepared, semisynthetic [FeFe]-hydrogenases deepens our understanding of hydrogenase chemistry. In parallel, we cover recent results from biophysical techniques that go beyond the scope of conventional X-ray diffraction, EPR, and FTIR spectroscopy. Taking into account both proton transfer and electron transfer as well as the notorious sensitivity of [FeFe]-hydrogenase toward carbon monoxide, the discussion further touches upon the molecular proceedings of biological hydrogen turnover
Infrared Characterization of the Bidirectional Oxygen-Sensitive [NiFe]-Hydrogenase from E. coli
[NiFe]-hydrogenases are gas-processing metalloenzymes that catalyze the conversion of dihydrogen (H2) to protons and electrons in a broad range of microorganisms. Within the framework of green chemistry, the molecular proceedings of biological hydrogen turnover inspired the design of novel catalytic compounds for H2 generation. The bidirectional âO2-sensitiveâ [NiFe]-hydrogenase from Escherichia coli HYD-2 has recently been crystallized; however, a systematic infrared characterization in the presence of natural reactants is not available yet. In this study, we analyze HYD-2 from E. coli by in situ attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR) under quantitative gas control. We provide an experimental assignment of all catalytically relevant redox intermediates alongside the O2- and CO-inhibited cofactor species. Furthermore, the reactivity and mutual competition between H2, O2, and CO was probed in real time, which lays the foundation for a comparison with other enzymes, e.g., âO2-tolerantâ [NiFe]-hydrogenases. Surprisingly, only Ni-B was observed in the presence of O2 with no indications for the âunreadyâ Ni-A state. The presented work proves the capabilities of in situ ATR FTIR spectroscopy as an efficient and powerful technique for the analysis of biological macromolecules and enzymatic small molecule catalysis
Adjuvant radiotherapy improves progression-free survival in intracranial atypical meningioma
BACKGROUND:
Meningiomas are the most common primary tumors of the central nervous system. In patients with WHO grade I meningiomas no adjuvant therapy is recommended after resection. In case of anaplastic meningiomas (WHO grade III), adjuvant fractionated radiotherapy is generally recommended, regardless of the extent of surgical resection. For atypical meningiomas (WHO grade II) optimal postoperative management has not been clearly defined yet.
METHODS:
We conducted a retrospective analysis of patients treated for intracranial atypical meningioma at Charité UniversitÀtsmedizin Berlin from March 1999 to October 2018. Considering the individual circumstances (risk of recurrence, anatomical location, etc.), patients were either advised to follow a wait-and-see approach or to undergo adjuvant radiotherapy. Primary endpoint was progression-free survival (PFS).
RESULTS:
This analysis included 99 patients with atypical meningioma (WHO grade II). Nineteen patients received adjuvant RT after primary tumor resection (intervention group). The remaining 80 patients did not receive any further adjuvant therapy after surgical resection (control group). Median follow-up was 37âmonths. Median PFS after primary resection was significantly longer in the intervention group than in the control group (64âm vs. 37âm, pâ=â0.009, HRâ=â0.204, 95% CIâ=â0.062-0.668). The influence of adjuvant RT was confirmed in multivariable analysis (pâ=â0.041, HRâ=â0.192, 95% CIâ=â0.039-0.932).
CONCLUSIONS:
Our study adds to the evidence that RT can improve PFS in patients with atypical meningioma
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
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
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