CO synthesized from the central one-carbon pool as source for the iron carbonyl in O2-tolerant [NiFe]-hydrogenase

Ingmar Bürstel, Elisabeth Siebert, Stefan Frielingsdorf, Ingo Zebger, Bärbel Friedrich, Oliver Lenz

Hydrogenases are nature’s key catalysts involved in both microbial consumption and production of molecular hydrogen. H2 exhibits a strongly bonded, almost inert electron pair and requires transition metals for activation. Consequently, all hydrogenases are metalloenzymes that contain at least one iron atom in the catalytic center.

For appropriate interaction with H2, the iron moiety demands for a sophisticated coordination environment that cannot be provided just by standard amino acids. This dilemma has been overcome by the introduction of unprecedented chemistry — that is, by ligating the iron with carbon monoxide (CO) and cyanide (or equivalent) groups. These ligands are both unprecedented in microbial metabolism and, in their free form, highly toxic to living organisms. Therefore, the formation of the diatomic ligands relies on dedicated biosynthesis pathways...

Proc. Natl. Acad. Sci. U. S. A. 2016 | DOI: 10.1073/pnas.1614656113
Published online on 5 December 2016.

Design of S-Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl-tRNA Synthetase Variant

Matthias P. Exner, Tilmann Kuenzl, Tuyet Mai T. To, Zhaofei Ouyang, SergejSchwagerus, Michael G. Hoesl, Christian P. R. Hackenberger, Marga C.Lensen, Sven Panke, Nediljko Budisa

The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits a range of biological activities. It is also a small bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function.

Here, we report a simple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploited the exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyl-tRNA synthetase (SacRS) capable of specifically accepting the small, polar amino acid instead of its long and bulky aliphatic natural substrate.

We succeeded in generating a novel and inexpensive strategy for the incorporation of a functionally versatile amino acid. This will help in the conversion of orthogonal translation from a standard technique in academic research to industrial biotechnology.

CHEMBIOCHEM 2016 | DOI: 10.1002/cbic.201600537
Published online on 30 November 2016.

A New Domain of Reactivity for High-Valent Dinuclear [M(µ-O)2M’] Complexes in Oxidation Reactions

Xenia Engelmann, Shenglai Yao, Erik R. Farquhar, Tibor Szilvási, Uwe Kuhlmann, Peter Hildebrandt, Matthias Driess, Kallol Ray

The strikingly different reactivity of a series of homo- and heterodinuclear [(MIII)(µ-O)2(MIII)’]2+ (M=Ni; M’=Fe, Co, Ni and M=M’=Co) complexes with β-diketiminate ligands in electrophilic and nucleophilic oxidation reactions is reported, and can be correlated to the spectroscopic features of the [(MIII)(µ-O)2(MIII)’]2+ core.

In particular, the unprecedented nucleophilic reactivity of the symmetric [NiIII-(µ-O)2NiIII]2+ complex and the decay of the asymmetric [NiIII(µ-O)2CoIII]2+ core through aromatic hydroxylation reactions represent a new domain for high-valent bis(m-oxido)dimetal reactivity.

Angew. Chem. Int. Ed. 2016 | DOI: 10.1002/anie.201607611

Structure of photosystem II and substrate binding at room temperature

Crystal structure of PS II in the dark (S1) state collected at room temperature with femtosecond XFEL pulses.
a, Schematic overview of the reaction centre of PS II showing cofactors involved in light-induced electron transfer and water oxidation.
b, Diffraction pattern of a PS II microcrystal. Insets show enlarged views with the indexing solution.
c, Arrangement of dimers in the unit cells of the crystals. Three dimers (blue, green, yellow) with the view onto the membrane plane from the lumenal side (top) and with the view along the membrane plane (bottom).

Iris D. Young, Holger Dobbek, Athina Zouni and coworkers

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere.

PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC).

Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O–O bond formation and O2 evolution.

A detailed understanding of the O–O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site.

Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II.

Nature 2016, 540, 453-457 | DOI: 10.1038/nature20161
Published online on 21 November 2016.

Pseudo-Contact NMR Shifts over the Paramagnetic Metalloprotein CoMMP-12 from First Principles

Ladislav Benda, Jiří Mareš, Enrico Ravera, Giacomo Parigi, Claudio Luchinat, Martin Kaupp, Juha Vaara

Long-range pseudo-contact NMR shifts (PCSs) provide important restraints for the structure refinement of proteins when a paramagnetic metal center is present, either naturally or introduced artificially. Here we show that ab initio quantum-chemical methods and a modern version of the Kurland–McGarvey approach for paramagnetic NMR (pNMR) shifts in the presence of zero-field splitting (ZFS) together provide accurate predictions of all PCSs in a metalloprotein (high-spin cobalt-substituted MMP-12 as a test case). Computations of 314 13C PCSs using g- and ZFS tensors based on multi-reference methods provide a reliable bridge between EPR-parameter- and susceptibility-based pNMR formalisms. Due to the high sensitivity of PCSs to even small structural differences, local structures based either on X-ray diffraction or on various DFT optimizations could be evaluated critically by comparing computed and experimental PCSs. Many DFT functionals provide insufficiently accurate structures. We also found the available 1RMZ PDB X-ray structure to exhibit deficiencies related to binding of a hydroxamate inhibitor. This has led to a newly refined PDB structure for MMP-12 (5LAB) that provides a more accurate coordination arrangement and PCSs.

Angew. Chem. Int. Ed. 2016, 55, 14713-14717 | DOI: 10.1002/anie.201608829

Cyclohexane oxidative dehydrogenation over copper oxide catalysts

Scott L. Nauert, Fabian Schax, Christian Limberg, Justin M. Notestein

Here, we report on structure-reactivity trends for cyclohexane oxidative dehydrogenation (ODH) with silica supported copper oxide catalysts as a function of surface structure. Copper oxide was supported on mesostructured KIT-6 silica at low surface densities <0.2 Cu/nm2 using copper (II) nitrate, ammonium and sodium copper (II) ethylenediaminetetraacetate, and a hexanuclear copper (I) siloxide complex. Copper oxide surface structures were characterized by X-ray absorption spectroscopy as well as ambient and in situ diffuse reflectance UV–visible (DRUV–vis) spectroscopy to determine trends in copper oxide nuclearity. DRUV–vis spectroscopy identifies three copper species based on Cu2+ ligand to metal transfer (LMCT) bands at 238, 266, and >300 nm as well as Cu+ LMCT bands at 235, 296, and 312 nm. Counterintuitively, EXAFS analysis shows that the multinuclear precursor leads to fewer average Cu–Cu interactions than syntheses with mononuclear copper salt precursors. Turnover frequency and selectivity to benzene increase with decreasing copper oxide nuclearity, and thus the multinuclear precursor leads to the highest turnover frequency and benzene production. This work shows the variety of surface species that exist even at extremely low copper surface densities, control of which can improve reactivity of an atypical ODH catalyst up to rates comparable to benchmark vanadia catalysts.

Journal of Catalysis 2016, 341, 180-190 | DOI: 10.1016/j.jcat.2016.07.002

Characterization and Reactivity Studies of a Terminal Copper–Nitrene Species

Teresa Corona, Lídia Ribas, Mireia Rovira, Erik R. Farquhar, Xavi Ribas, Kallol Ray, Anna Company

Kalol Ray and Anna Company

Prof. Kallol Ray and Clara Immerwahr Awardee Dr. Anna Company have published a paper on characterization and reactivity terminal copper–nitrene species. It has resulted from the scientific stay of Ms Teresa Corono in Kallol Ray’s group for a period of four months. Her stay was funded by the Clara Immerwahr Award to Dr. Anna Company.

This paper has been selected as a hot paper in Angewandte Chemie International Edition:

High-valent terminal copper–nitrene species have been postulated as key intermediates in copper-catalyzed aziridination and amination reactions. The high reactivity of these intermediates has prevented their characterization for decades, thereby making the mechanisms ambiguous.

Very recently, the Lewis acid adduct of a copper–nitrene intermediate was trapped at -90°C and shown to be active in various oxidation reactions.

Herein, we describe for the first time the synthesis and spectroscopic characterization of a terminal copper(II)–nitrene radical species that is stable at room temperature in the absence of any Lewis acid.

Angew. Chem. Int. Ed. 2016 | DOI: 10.1002/anie.201607238

Chemical looping as reactor concept for the oxidative coupling of methane over a Na2WO4/Mn/SiO2 catalyst

Vinzenz Fleischer, Patrick Littlewood, Samira Parishan, Reinhard Schomäcker

In this work we present chemical looping and simulated chemical looping as a new reactor concept for the oxidative coupling of methane over Na2WO4/Mn/SiO2. As a consequences of an alternating feed of oxygen and methane to the catalyst bed side reactions are avoided and the selectivity of the coupling reaction is greatly increased.

By variation of methane pulse contact time and temperature a maximum yield of 0.25 is obtained. Although this does not exceed the often discussed yield limitation of OCM, it is achieved from a substantially lower amount of converted methane. A time on stream experiment were carried out at 775 and 800 °C for 150 min and showed stable performance and C2 yield.

Chemical Engineering Journal 2016, 306, 646-654 | DOI: 10.1016/j.cej.2016.07.094
Published online on 28 July 2016.

Vibrational spectroscopy reveals the initial steps of biological hydrogen evolution

S. Katz, J. Noth, M. Horch,H. S. Shafaat, T. Happe, P. Hildebrandt, I. Zebger

[FeFe] hydrogenases are biocatalytic model systems for the exploitation and investigation of catalytichydrogen evolution. Here, we used vibrational spectroscopic techniques to characterize, in detail, redoxtransformations of the [FeFe] and [4Fe4S] sub-sites of the catalytic centre (H-cluster) in a monomeric[FeFe] hydrogenase.

Through the application of low-temperature resonance Raman spectroscopy, wediscovered a novel metastable intermediate that is characterized by an oxidized [FeIFeII] centre anda reduced [4Fe4S]1+ cluster.

Based on this unusual configuration, this species is assigned to the first,deprotonated H-cluster intermediate of the [FeFe] hydrogenase catalytic cycle. Providing insights intothe sequence of initial reaction steps, the identification of this species represents a key finding towardsthe mechanistic understanding of biological hydrogen evolution.

Chemical Science 2016, 7, 6746–6752 | DOI: 10.1039/c6sc01098a

Silica material variation for the MnxOy-Na2WO4/SiO2

M. Yildiz, Y. Aksu, U. Simon, T. Otremba, K. Kailasam, C. Göbel, F. Girgsdies, O. Görke, F. Rosowski, A. Thomas, R. Schomäcker, S. Arndt

The oxidative coupling of methane (OCM) is one of the best methods for the direct conversion of methane. Among the known OCM catalysts, MnxOy-Na2WO4/SiO2 is a promising candidate for an industrial application, showing a high methane conversion and C2 selectivity, with a good stability during long-term catalytic activity tests.

In the present study, some results have been already published and discussed briefly in our previous short communication. However, we herein investigated comprehensively the influence of various silica support materials on the performance of the MnxOy-Na2WO4/SiO2 system in the OCM by means of ex situ and in situ XRD, BET, SEM and TEM characterization methods and showed new results to reveal possible support effects on the catalyst.

The catalytic performance of most MnxOy-Na2WO4/SiO2 catalysts supported by different silica support materials did not differ substantially. However, the performance of the SBA-15 supported catalyst was outstanding and the methane conversion was nearly twofold higher in comparison to the other silica supported catalysts at similar C2 selectivity as shown before in the communication. The reason of this substantial increase in performance could be the ordered mesoporous structure of the SBA-15 support material, homogeneous dispersion of active components and high number of active sites responsible for the OCM.

Applied Catalysis A: General 2016, 525, 168-179 | DOI: 10.1016/j.apcata.2016.06.034
Published online on 1 July 2016.

Oxidative coupling of methane on the Na2WO4-MnxOy catalyst: COK-12 as an inexpensive alternative to SBA-15

M.G. Colmenares, U. Simon, M. Yildiz, S. Arndt, R. Schomaecker, A. Thomas, F. Rosowski, A. Gurlo, O. Goerke

Recent investigations have revealed that SBA-15 supported Na2WO4-MnxOy catalyst exhibits an enhanced performance compared to other silica supports for the oxidative coupling of methane. We report on the use of the inexpensive and upscalable SBA-15 analog denoted as COK-12 as a support material for this catalytic system.

COK-12 is synthesized at room temperature and almost neutral pH using an inexpensive silica source. A performance matching that of the SBA-15 supported catalyst was achieved. We observe a decrease in activity of the catalyst as a result of pelletizing by pressing.

Catalysis Communications 2016, 85, 75-78 | DOI: 10.1016/j.catcom.2016.06.025
Published online on 27 June 2016.

Leader Peptide-Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease-Targeted Libraries

Emmanuel Reyna-Gonzlez, Bianca Schmid, Daniel Petras, Roderich D. Süssmuth, Elke Dittmann

Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases.

Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins.

The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure–activity relationships for respective target proteases.

Angew. Chem. Int. Ed. 2016, 55, 9398 –9401 | DOI: 10.1002/anie.201604345

Biochemical Dissection of the Natural Diversification of Microcystin Provides Lessons for Synthetic Biology of NRPS

Sabine Meyer, Jan-Christoph Kehr, Andi Mainz, Daniel Dehm, Daniel Petras, Roderich D. Süssmuth, Elke Dittmann

The cyanobacterial hepatotoxin microcystin is assembled at a non-ribosomal peptide synthetase (NRPS) complex. The enormous structural diversity of this peptide, which is also found in closely related strains, is the result of frequent recombination events and point mutations.

Here, we have compared the in vitro activation profiles of related monospecific and multispecific modules that either strictly incorporate leucine or arginine or incorporate chemically diverse amino acids in parallel into microcystin. By analyzing di- and tri-domain proteins we have dissected the role of adenylation and condensation domains for substrate specificity.

We have further analyzed the role of subdomains and provide evidence for an extended gatekeeping function for the condensation domains of multispecific modules. By reproducing natural point mutations, we could convert a monospecific module into a multispecific module. Our findings may inspire novel synthetic biology approaches and demonstrate how recombination platforms of NRPSs have developed in nature.

Cell Chemical Biology, 23, 462-471 | DOI: 10.1016/j.chembiol.2016.03.011

Krypton Derivatization of an O2-Tolerant Membrane-Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport

Jacqueline Kalms, Andrea Schmidt, Stefan Frielingsdorf, Peter van der Linden, David von Stetten, Oliver Lenz, Philippe Carpentier, Patrick Scheerer

[NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies.

Here we describe the crystal structure of the O2-tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2-tolerant and O2-sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance.

Angew. Chem. Int. Ed. 2016, 55, 5586–5590 | DOI: 10.1002/anie.201508976

When the inhibitor tells more than the substrate: the cyanide-bound state of a carbon monoxide dehydrogenase

Alexandre Ciaccafava, Daria Tombolelli, Lilith Domnik, Jochen Fesseler, Jae-Hun Jeoung, Holger Dobbek, Maria Andrea Mroginski, Ingo Zebger, Peter Hildebrandt

Carbon monoxide dehydrogenase (CODH) is a key enzyme for reversible CO interconversion. To elucidate structural and mechanistic details of CO binding at the CODH active site (C-cluster), cyanide is frequently used as an iso-electronic substitute and inhibitor. However, previous studies revealed conflicting results on the structure of the cyanide-bound complex and the mechanism of cyanide-inhibition. To address this issue in this work, we have employed IR spectroscopy, crystallography, site directed mutagenesis, and theoretical methods to analyse the cyanide complex of the CODH from Carboxydothermus hydrogenoformans (CODHIICh).

IR spectroscopy demonstrates that a single cyanide binds to the Ni ion. Whereas the inhibitor could be partially removed at elevated temperature, irreversible degradation of the C-cluster occurred in the presence of an excess of cyanide on the long-minute time scale, eventually leading to the formation of [Fe(CN)6]4− and [Ni(CN)4]2− complexes. Theoretical calculations based on a new high-resolution structure of the cyanide-bound CODHIICh indicated that cyanide binding to the Ni ion occurs upon dissociation of the hydroxyl ligand from the Fe1 subsite of the C-cluster.

The hydroxyl group is presumably protonated by Lys563 which, unlike to His93, does not form a hydrogen bond with the cyanide ligand. A stable deprotonated ε-amino group of Lys563 in the cyanide complex is consistent with the nearly unchanged CN stretching in the Lys563Ala variant of CODHIICh. These findings support the view that the proton channel connecting the solution phase with the active site displays a strict directionality, controlled by the oxidation state of the C-cluster.­

 Chemical Science | DOI: 10.1039/C5SC04554A