X-ray structure of molybdenum-containing carbon monoxide dehydrogenases

· CO-Dehydrogenasen katalysieren die Oxidation von CO mit H2O zu CO2, 2H+ und 2e-. Es sind Mo- und Ni-haltige Formen bekannt, die keinerlei Ähnlichkeit auf der Ebene der Primärstruktur, sowie in der Zusammensetzung ihrer Kofaktoren besitzen. · Mo-haltige CO-Dehydrogenase wird in die Enzym-Familie der Molybdän-Hydroxylasen eingeordnet und teilt neben einer ausgeprägten Homologie auf Primärstruktur-Ebene auch die Zusammensetzung der Kofaktoren mit den meisten Enzymen dieser Familie. Zu den Molybdän-Hydroxylasen gehören eukaryontische und bakterielle Enzyme wie Xanthin-Oxidase/-Dehydrogenase und Aldehyd-Oxidase. · Mo-haltige CO-Dehydrogenase ist als ein Molybdo-Eisen-Schwefel-Flavoprotein bekannt. · Die Kristallstruktur der CO-Dehydrogenase ist sowohl die erste Struktur einer CO-Dehydrogenase, wie auch das erste Molybdo-Eisen-Schwefel-Flavoprotein, dessen Struktur kristallographisch bestimmt wurde. Die Strukturbestimmung geschah unter Verwendung von Pattersonsuch- und MAD-Techniken. Die Charakterisierung der atomar/nahe atomar aufgelösten Strukturen verwendete die Kristalltransformation auf dem free-mounting system zur Optimierung der Diffraktionsqualität der Kristalle. · Die Kristallstruktur der Mo-haltigen CO-Dehydrogenase von Oligotropha carboxidovorans wurde zu einer Auflösung von 1.09 Å für das 277 kDa Protein bestimmt und besteht aus einem Dimer aus Heterotrimeren (LMS)2. Dabei trägt jede Untereinheit eine Art von Kofaktor. Die L-Untereinheit ist das Molybdoprotein und beherbergt das aktive Zentrum. Die M-Untereinheit ist ein Flavoprotein, welches ein nichtkovalent gebundenes FAD-Molekül trägt. Die S-Untereinheit ist das Eisen-Schwefel-Protein und trägt zwei spektroskopisch unterscheidbare [2Fe-2S]-Zentren. · Das aktive Zentrum der CO-Dehydrogenase besteht aus einem neuartigen Cu(I)- und Mo(+VI/+IV)-haltigen zweikernigen Cluster, welcher über einen m-Sulfido-Liganden verbrückt ist. Mo ist dabei verzerrt tetraedrisch von fünf Liganden umgeben, wobei die Dithiolen-Gruppe über einem Scheitelpunkt des Tetraeders sitzt. Neben den beiden Dithiolen-Schwefeln ist Mo von einem Oxo-, einem Hydroxo- und einem Sulfido-Liganden umgeben. Cu(I) ist vom m-Sulfido-Liganden und Sg von Cys 388 verzerrt linear koordiniert. Dieser mit MAD-Methoden entdeckte Cluster ist der erste bekannte Mo- und Cu-haltige Cluster in einem Enzym und widerspricht der Bezeichnung der Molybdopterin-haltigen Enzyme als mononuclear molybdenum enzymes, welche zur Abgrenzung gegen die mehrkernigen Mo-haltigen Nitrogenasen gewählt wurde (Hille 1996). · Kristallographische Studien zum Reaktionsmechanismus dieses Clusters beinhalten die Charakterisierung der oxidierten, der anoxisch CO-, H2- und Dithionit-reduzierten, der CN--inaktivierten und der n-Butylisonitril-(nBIC)-gebundenen Formen des Enzyms bei Auflösungen im atomaren und nahe atomaren Bereich. · Ein auf der Basis dieser Ergebnisse entwickelter Reaktionsmechanismus basiert außerdem auf der Analogie des nBIC-gebunden Zustandes mit einem möglichen Intermediat der CO Oxidation, sowie der bekannten Chemie von Cu(I)-Komplexen. Dabei findet die Bindung und Aktivierung des Substrats CO am substitutionslabilen Cu(I)-Komplex statt, und führt zur Öffnung des Clusters. Diese Öffnung resultiert in einer Destabilisierung des oxidierten Mo(+VI) mit folgender Reduktion des Mo und der Ausbildung einer "spectator-oxo"- Dreifachbindung am Mo. · Der entworfenene Reaktionsmechanismus kann ebenfalls die Aktivierung und Oxidation von H2 am [CuMo]-Cluster erklären. · Aufgrund der hohen Konservierung der direkten Mo-Umgebung erscheint es wahrscheinlich, daß auch andere Mitglieder der Molybdän-Hydroxylase-Familie die Destabilisierung, sowie die Ausbildung eines "spectator oxo"-Liganden zur Stabilisierung des Übergangszustandes nutzen werden. · Neben der Struktur der CO-Dehydrogenase von O. carboxidovorans wurde ebenfalls die Struktur des Enzyms aus Hydrogenophaga pseudoflava bestimmt. Die unter Mo-Mangel im Wachstumsmedium von H. pseudoflava synthetisierte inaktive CO-Dehydrogenase beinhaltet statt des Molybdopterin-Cytosin-Dinukleotid Kofaktors (MCD) nur Cytosin-Diphosphat. Aktive und inaktive Struktur wurden kristallographisch zu Auflösungen von 2.25-2.35 Å bestimmt. Die Mo-enthaltenden Formen der CO-Dehydrogenase-Strukturen aus beiden Organismen sind sich sehr ähnlich.· CO-dehydrogenase catalyzes the oxidation of CO with H2O yielding CO2, 2H+ und 2 e-. Mo- und Ni-containing species are known und show no resemblance in their amino acid sequences or cofactor composition. · Mo-containing CO-dehydrogenase has been grouped in the enzyme family of molybdenum hydroxylases und exhibits in addition to a marked sequence homology, also a common cofactor composition with most of these enzymes. The family of molybdenum hydroxylases comprises eu- und prokaryotic enzymes like xanthine oxidases/dehydrogenases und aldehyde oxidase. · Mo-containing CO-dehydrogenases are known as molybdo-iron-sulfur-flavoproteins · The determined crystal structure of the Mo-containing CO-dehydrogenase is the first structure of a CO-dehydrogenase as well as the first structure of a molybdo-iron-sulfur-flavoprotein. The structure has been determined employing Patterson-search techniques und MAD techniques. The characterization of the atomical/near atomical resolved structures has been enabled through crystal transformation on the free-mounting system for the optimization of diffraction quality. · The crystal structure of the Mo-containing CO-dehydrogenase from Oligotropha carboxidovorans has been determined to a resolution of 1.09 Å for the 277 kDa enzyme und is built up by a dimer of heterotrimers (LMS)2. Each subunit carries one type of cofactor. The L subunit is the molybdoprotein und harbours the active site. The M subunit is the flavoprotein, carrying a non-covalently bound FAD molecule. The S subunit is the iron-sulfur protein und carries two spectroscopically distinguishable [2Fe-2S]-Clusters. · The active site of CO-dehydrogenase consists of a novel Cu(I)- und Mo(+VI/+IV)-containing binuclear cluster, bridged by a m-sulfido ligand. The five ligands around Mo form a distorted tetrahedral geometry with the dithiolene sulfurs straddling over one vertex of the tetrahedron. In addition to the two dithiolene sulfurs, Mo is ligated by one oxo-, one hydroxo- und one sulfido-ligand. Cu(I) is coordinated by two ligands in a distorted linear geometry, the m-sulfido ligand und Sg of Cys 388. The cluster has been discovered by MAD methods und is the first Mo- und Cu-containing cluster found in an enzyme. Its existence contradicts a central dogma of molybdopterin containing enzymes, reflected in their name mononuclear molybdenum enzymes in contrast to the polynuclear Mo-containing nitrogenases (Hille 1996). · Crystallographic studies on the reaction mechanism of CO oxidation comprises the characterization of the oxidized, anoxygenically CO-, H2- und dithionite reduced, CN--inactivated und an n-butylisonitril (nBIC) bound state of the enzyme at resolutions in the atomic/near atomic range. · A reaction mechanism developed on the basis of these results is based on the analogy between the nBIC bound state und a possible intermediate of CO oxidation, as well as on the known chemistry of Cu(I) complexes. Binding und activation of the substrate occurs at the substitution-labile Cu(I) complex, leading to the opening of the cluster. This opening results in the destabilization of oxidized Mo(+VI) with the subsequent reduction of Mo und the formation of a "spectator oxo"-triple bond at Mo. · This reaction mechanism can also explain the activation und oxidation of H2 at the [CuMo] cluster. · Because of the remarkable conservation of the direct Mo environment it appears likely, that the destabilization of oxidized Mo together with the formation of a "spectator oxo" triple bond for the stabilization of the reaction intermediate is also used by other members of the molybdenum hydroxylase family. · In cases of Mo-shortage or -depletion in the growth media Hydrogenophaga pseudoflava synthesizes an inactive CO-dehydrogenase containing cytosin diphosphat instead of the molybdopterin-cytosin dinucleotid cofactor, which has been determined by X-ray crystallography together with the active form. The Mo-containing structures of the two carboxidotrophic bacteria are highly similar

Dynamic water bridging and proton transfer at a surface carboxylate cluster of photosystem II

Proton-transfer proteins are often exposed to the bulk clusters of carboxylate groups that might bind protons transiently. This raises important questions as to how the carboxylate groups of a protonated cluster interact with each other and with water, and how charged protein groups and hydrogen-bonded waters could have an impact on proton transfers at the cluster. We address these questions by combining classical mechanical and quantum mechanical computations with the analysis of cyanobacterial photosystem II crystal structures from Thermosynechococcus elongatus. The model system we use consists of an interface between PsbO and PsbU, which are two extrinsic proteins of photosystem II. We find that a protonated carboxylate pair of PsbO is part of a dynamic network of protein–water hydrogen bonds which extends across the protein interface. Hydrogen-bonded waters and a conserved lysine sidechain largely shape the energetics of proton transfer at the carboxylate cluster

Protein dynamics in the reductive activation of a B12-containing enzyme

B12-dependent proteins are involved in methyl transfer reactions ranging from the biosynthesis of methionine in humans to the formation of acetyl-CoA in anaerobic bacteria. During their catalytic cycle, they undergo large conformational changes to interact with various proteins. Recently, the crystal structure of the B12-containing corrinoid iron–sulfur protein (CoFeSP) in complex with its reductive activator (RACo) was determined, providing a first glimpse of how energy is transduced in the ATP-dependent reductive activation of corrinoid-containing methyltransferases. The thermodynamically uphill electron transfer from RACo to CoFeSP is accompanied by large movements of the cofactor-binding domains of CoFeSP. To refine the structure-based mechanism, we analyzed the conformational change of the B12-binding domain of CoFeSP by pulsed electron–electron double resonance and Förster resonance energy transfer spectroscopy. We show that the site-specific labels on the flexible B12-binding domain and the small subunit of CoFeSP move within 11 Å in the RACo:CoFeSP complex, consistent with the recent crystal structures. By analyzing the transient kinetics of formation and dissociation of the RACo:CoFeSP complex, we determined values of 0.75 μM–1 s–1 and 0.33 s–1 for rate constants kon and koff, respectively. Our results indicate that the large movement observed in crystals also occurs in solution and that neither the formation of the protein encounter complex nor the large movement of the B12-binding domain is rate-limiting for the ATP-dependent reductive activation of CoFeSP by RACo

Axial Ligation and Redox Changes at the Cobalt Ion in Cobalamin Bound to Corrinoid Iron-Sulfur Protein (CoFeSP) or in Solution Characterized by XAS and DFT

A cobalamin (Cbl) cofactor in corrinoid iron-sulfur protein (CoFeSP) is the primary methyl group donor and acceptor in biological carbon oxide conversion along the reductive acetyl-CoA pathway. Changes of the axial coordination of the cobalt ion within the corrin macrocycle upon redox transitions in aqua-, methyl-, and cyano-Cbl bound to CoFeSP or in solution were studied using X-ray absorption spectroscopy (XAS) at the Co K-edge in combination with density functional theory (DFT) calculations, supported by metal content and cobalt redox level quantification with further spectroscopic methods. Calculation of the highly variable pre-edge X-ray absorption features due to core-to-valence (ctv) electronic transitions, XANES shape analysis, and cobalt-ligand bond lengths determination from EXAFS has yielded models for the molecular and electronic structures of the cobalt sites. This suggested the absence of a ligand at cobalt in CoFeSP in α-position where the dimethylbenzimidazole (dmb) base of the cofactor is bound in Cbl in solution. As main species, (dmb)CoIII(OH2), (dmb)CoII(OH2), and (dmb)CoIII(CH3) sites for solution Cbl and CoIII(OH2), CoII(OH2), and CoIII(CH3) sites in CoFeSP-Cbl were identified. Our data support binding of a serine residue from the reductive-activator protein (RACo) of CoFeSP to the cobalt ion in the CoFeSP-RACo protein complex that stabilizes Co(II). The absence of an α-ligand at cobalt not only tunes the redox potential of the cobalamin cofactor into the physiological range, but is also important for CoFeSP reactivation

Protein crystallization and initial neutron diffraction studies of the photosystem II subunit PsbO

The PsbO protein of photosystem II stabilizes the active-site manganese cluster and is thought to act as a proton antenna. To enable neutron diffraction studies, crystals of the β-barrel core of PsbO were grown in capillaries. The crystals were optimized by screening additives in a counter-diffusion setup in which the protein and reservoir solutions were separated by a 1% agarose plug. Crystals were cross-linked with glutaraldehyde. Initial neutron diffraction data were collected from a 0.25 mm3 crystal at room temperature using the MaNDi single-crystal diffractometer at the Spallation Neutron Source, Oak Ridge National Laboratory

Structural insights into the light-driven auto-assembly process of the water- oxidizing Mn4CaO5-cluster in photosystem II

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light- driven splitting of water at a protein-bound Mn4CaO5-cluster, the water- oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre- organized ligand shell for kinetically competent and error-free photo-assembly of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of Mn(III) ions, as an assembly intermediate

Assignment of Individual Metal Redox States in a Metalloprotein by Crystallographic Refinement at Multiple X-ray Wavelengths

A method is presented to derive anomalous scattering contributions for individual atoms within a protein crystal by collecting several sets of diffraction data at energies spread along an X-ray absorption edge of the element in question. The method has been applied to a [2Fe:2S] ferredoxin model system with localized charges in the reduced state of the iron−sulfur cluster. The analysis shows that upon reduction the electron resides at the iron atom closer to the protein surface. The technique should be sufficiently sensitive for more complex clusters with noninteger redox states and is generally applicable given that crystals are available

a FD-FT THz-EPR study

A combined X-band and frequency-domain Fourier-transform THz electron paramagnetic resonance (FD-FT THz-EPR) approach has been employed to determine heme Fe(III) S = 5/2 zero-field splitting (ZFS) parameters of frozen metHb and metMb solutions, both with fluoro and aquo ligands. Frequency-domain EPR measurements have been carried out by an improved synchrotron-based FD-FT THz- EPR spectrometer. ZFS has been determined by field dependence of spin transitions within the mS = ±1/2 manifold, for all four protein systems, and by zero-field spin transitions between mS = ±1/2 and mS = ±3/2 levels, for metHb and metMb flouro-states. FD-FT THz-EPR data were simulated with a novel numerical routine based on Easyspin, which allows now for direct comparison of EPR spectra in field and frequency domain. We found purely axial ZFSs of D = 5.0(1) cm−1 (flouro-metMb), D = 9.2(4) cm−1 (aquo-metMb), D = 5.1(1) cm−1 (flouro-metHB) and D = 10.4(2) cm−1 (aquo-metHb)

A Morphing [4Fe-3S-nO]-Cluster within a Carbon Monoxide Dehydrogenase Scaffold

Ni,Fe-containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of CO2 to CO. Several anaerobic microorganisms encode multiple CODHs in their genome, of which some, despite being annotated as CODHs, lack a cysteine of the canonical binding motif for the active site Ni,Fe-cluster. Here, we report on the structure and reactivity of such a deviant enzyme, termed CooS-VCh. Its structure reveals the typical CODH scaffold, but contains an iron-sulfur-oxo hybrid-cluster. Although closely related to true CODHs, CooS-VCh catalyzes neither CO oxidation, nor CO2 reduction. The active site of CooS-VCh undergoes a redox-dependent restructuring between a reduced [4Fe-3S]-cluster and an oxidized [4Fe-2S-S*-2O-2(H2O)]-cluster. Hydroxylamine, a slow-turnover substrate of CooS-VCh, oxidizes the hybrid-cluster in two structurally distinct steps. Overall, minor changes in CODHs are sufficient to accommodate a Fe/S/O-cluster in place of the Ni,Fe-heterocubane-cluster of CODHs

Bacterial Chaperone Domain Insertions Convert Human FKBP12 into an Excellent Protein-Folding Catalyst—A Structural and Functional Analysis

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain.Fonds der Chemischen Industrie and the Deutsche ForschungsgemeinschaftPeer Reviewe