Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H2-driven NAD+-reduction in the presence of O2

Biocatalysts that mediate the H2-dependent reduction of NAD+ to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD+-reducing [NiFe]‑hydrogenase that sustains catalytic activity at high temperatures and in the presence of O2, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD+-reducing [NiFe]‑hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H2-oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H2-mediated NAD+ reduction activity was observed at 80 °C and pH 6.5, and catalytic activity was found to be sustained at low O2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]‑hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD+-reducing [NiFe]‑hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H2-driven cofactor recycling under oxic conditions at elevated temperatures

Stabilizing Monoatomic Two-Coordinate Bismuth(I) and Bismuth(II) Using a Redox Noninnocent Bis(germylene) Ligand

The formation of isolable monatomic BiI complexes and BiII radical species is challenging due to the pronounced reducing nature of metallic bismuth. Here, we report a convenient strategy to tame BiI and BiII atoms by taking advantage of the redox noninnocent character of a new chelating bis(germylene) ligand. The remarkably stable novel BiI cation complex 4, supported by the new bis(iminophosphonamido-germylene)xanthene ligand [(P)GeII(Xant)GeII(P)] 1, [(P)GeII(Xant)GeII(P) = Ph2P(NtBu)2GeII(Xant)GeII(NtBu)2PPh2, Xant = 9,9-dimethyl-xanthene-4,5-diyl], was synthesized by a two-electron reduction of the cationic BiIIII2 precursor complex 3 with cobaltocene (Cp2Co) in a molar ratio of 1:2. Notably, owing to the redox noninnocent character of the germylene moieties, the positive charge of BiI cation 4 migrates to one of the Ge atoms in the bis(germylene) ligand, giving rise to a germylium(germylene) BiI complex as suggested by DFT calculations and X-ray photoelectron spectroscopy (XPS). Likewise, migration of the positive charge of the BiIIII2 cation of 3 results in a bis(germylium)BiIIII2 complex. The delocalization of the positive charge in the ligand engenders a much higher stability of the BiI cation 4 in comparison to an isoelectronic two-coordinate Pb0 analogue (plumbylone; decomposition below −30 °C). Interestingly, 4[BArF] undergoes a reversible single-electron transfer (SET) reaction (oxidation) to afford the isolable BiII radical complex 5 in 5[BArF]2. According to electron paramagnetic resonance (EPR) spectroscopy, the unpaired electron predominantly resides at the BiII atom. Extending the redox reactivity of 4[OTf] employing AgOTf and MeOTf affords BiIII(OTf)2 complex 7 and BiIIIMe complex 8, respectively, demonstrating the high nucleophilic character of BiI cation 4

Bis(silylene)‐Stabilized Monovalent Nitrogen Complexes

The first series of bis(silylene)‐stabilized nitrogen(I) compounds is described. Starting from the 1,2‐bis(N‐heterocyclic silylenyl) 1,2‐dicarba‐closo‐dedocaborane(12) scaffold 1, [1,2‐(LSi)2C2B10H10; L=PhC(NtBu)2], reaction with adamantyl azide (AdN3) affords the terminal N‐μ2‐bridged zwitterionic carborane‐1,2‐bis(silylium) AdN3 adduct 2 with an open‐cage dianionic nido‐C2B10 cluster core. Remarkably, upon one‐electron reduction of 2 with C8K and liberation of N2 and adamantane, the two silylene subunits are regenerated to furnish the isolable bis(silylene)‐stabilized NI complex as an anion of 3 with the nido‐C2B10 cluster cage. On the other hand, one‐electron oxidation of 2 with silver(I) yields the monocationic bis(silylene) NI complex 4 with the closo‐C2B10 cluster core. Moreover, the corresponding neutral NI radical complex 5 results from single‐electron transfer from 3 to 4.DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"TU Berlin, Open-Access-Mittel – 202

Stepwise conversion of the Cys6[4Fe–3S] to a Cys4[4Fe–4S] cluster and its impact on the oxygen tolerance of [NiFe]-hydrogenase

The membrane-bound [NiFe]-hydrogenase of Cupriavidus necator is a rare example of a truly O2-tolerant hydrogenase. It catalyzes the oxidation of H2 into 2e− and 2H+ in the presence of high O2 concentrations. This characteristic trait is intimately linked to the unique Cys6[4Fe–3S] cluster located in the proximal position to the catalytic center and coordinated by six cysteine residues. Two of these cysteines play an essential role in redox-dependent cluster plasticity, which bestows the cofactor with the capacity to mediate two redox transitions at physiological potentials. Here, we investigated the individual roles of the two additional cysteines by replacing them individually as well as simultaneously with glycine. The crystal structures of the corresponding MBH variants revealed the presence of Cys5[4Fe–4S] or Cys4[4Fe–4S] clusters of different architecture. The protein X-ray crystallography results were correlated with accompanying biochemical, spectroscopic and electrochemical data. The exchanges resulted in a diminished O2 tolerance of all MBH variants, which was attributed to the fact that the modified proximal clusters mediated only one redox transition. The previously proposed O2 protection mechanism that detoxifies O2 to H2O using four protons and four electrons supplied by the cofactor infrastructure, is extended by our results, which suggest efficient shutdown of enzyme function by formation of a hydroxy ligand in the active site that protects the enzyme from O2 binding under electron-deficient conditions

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

Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast

Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl-CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine-tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity

Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli

Hydrogenases are abundant metalloenzymes that catalyze the reversible conversion of molecular H2 into protons and electrons. Important achievements have been made over the past two decades in the understanding of these highly complex enzymes. However, most hydrogenases have low production yields requiring many efforts and high costs for cultivation limiting their investigation. Heterologous production of these hydrogenases in a robust and genetically tractable expression host is an attractive strategy to make these enzymes more accessible. In the present study, we chose the oxygen-tolerant H2-sensing regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha H16 owing to its relatively simple architecture compared to other [NiFe]-hydrogenases as a model to develop a heterologous hydrogenase production system in Escherichia coli. Using screening experiments in 24 deep-well plates with 3 mL working volume, we investigated relevant cultivation parameters, including inducer concentration, expression temperature, and expression time. The RH yield could be increased from 14 mg/L up to >250 mg/L by switching from a batch to an EnPresso B-based fed-batch like cultivation in shake flasks. This yield exceeds the amount of RH purified from the homologous host R. eutropha by several 100-fold. Additionally, we report the successful overproduction of the RH single subunits HoxB and HoxC, suitable for biochemical and spectroscopic investigations. Even though both RH and HoxC proteins were isolated in an inactive, cofactor free apo-form, the proposed strategy may powerfully accelerate bioprocess development and structural studies for both basic research and applied studies. These results are discussed in the context of the regulation mechanisms governing the assembly of large and small hydrogenase subunits.DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"EC/H2020/810856/EU/Twin to Illuminate Metals in Biology and Biocatalysis through Biospectroscopy/TIMB3DFG, 414044773, Open Access Publizieren 2021 - 2022 / Technische Universität Berli

Generally applicable transcriptome-wide analysis of translation using anota2seq

mRNA translation plays an evolutionarily conserved role in homeostasis and when dysregulated contributes to various disorders including metabolic and neurological diseases and cancer. Notwithstanding that optimal and universally applicable methods are critical for understanding the complex role of translational control under physiological and pathological conditions, approaches to analyze translatomes are largely underdeveloped. To address this, we developed the anota2seq algorithm which outperforms current methods for statistical identification of changes in translation. Notably, in contrast to available analytical methods, anota2seq also allows specific identification of an underappreciated mode of gene expression regulation whereby translation acts as a buffering mechanism which maintains protein levels despite fluctuations in corresponding mRNA abundance ('translational buffering'). Thus, the universal anota2seq algorithm allows efficient and hitherto unprecedented interrogation of translatomes which is anticipated to advance knowledge regarding the role of translation in homeostasis and disease

Atomic interferences and the topological phase

International audienc

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