Tryptophan regulates Drosophila zinc stores.

Zinc deficiency is commonly attributed to inadequate absorption of the metal. Instead, we show that body zinc stores in Drosophila melanogaster depend on tryptophan consumption. Hence, a dietary amino acid regulates zinc status of the whole insect­a finding consistent with the widespread requirement of zinc as a protein cofactor. Specifically, the tryptophan metabolite kynurenine is released from insect fat bodies and induces the formation of zinc storage granules in Malpighian tubules, where 3-hydroxykynurenine and xanthurenic acid act as endogenous zinc chelators. Kynurenine functions as a peripheral zinc-regulating hormone and is converted into a 3-hydroxykynurenine­zinc­chloride complex, precipitating within the storage granules. Thus, zinc and the kynurenine pathway­well-known modulators of immunity, blood pressure, aging, and neurodegeneration­are physiologically connected.

A Bioinspired Nonheme FeIII-(O22-)-CuII Complex with an St = 1 Ground State.

Cytochrome c oxidase (CcO) is a heme copper oxidase (HCO) that catalyzes the natural reduction of oxygen to water. A profound understanding of some of the elementary steps leading to the intricate 4e-/4H+ reduction of O2 is presently lacking. A total spin St = 1 FeIII-(O22-)-CuII (IP) intermediate is proposed to reduce the overpotentials associated with the reductive O-O bond rupture by allowing electron transfer from a tyrosine moiety without the necessity of any spin-surface crossing. Direct evidence of the involvement of IP in the CcO catalytic cycle is, however, missing. A number of heme copper peroxido complexes have been prepared as synthetic models of IP, but all of them possess the catalytically nonrelevant St = 0 ground state resulting from antiferromagnetic coupling between the S = 1/2 FeIII and CuII centers. In a complete nonheme approach, we now report the spectroscopic characterization and reactivity of the FeIII-(O22-)-CuII intermediates 1 and 2, which differ only by a single -CH3 versus -H substituent on the central amine of the tridentate ligands binding to copper. Complex 1 with an end-on peroxido core and ferromagnetically (St = 1) coupled FeIII and CuII centers performs H-bonding-mediated O-O bond cleavage in the presence of phenol to generate oxoiron(IV) and exchange-coupled copper(II) and PhO• moieties. In contrast, the µ-η2:η1 peroxido complex 2, with a St = 0 ground state, is unreactive toward phenol. Thus, the implications for spin topology contributions to O-O bond cleavage, as proposed for the heme FeIII-(O22-)-CuII intermediate in CcO, can be extended to nonheme chemistry.

Facile and Green Synthesis of Well-Defined Nanocrystal Oxygen Evolution Catalysts by Rational Crystallization Regulation.

The development of catalysts for an economical and efficient oxygen evolution reaction (OER) is critical for clean and sustainable energy storage and conversion. Nickel-iron-based (NiFe) nanostructures are widely investigated as active OER catalysts and especially shape-controlled nanocrystals exhibit optimized surface structure and electronic properties. However, the structural control from amorphous to well-defined crystals is usually time-consuming and requires multiple stages. Here, a universal two-step precipitation-hydrothermal approach is reported to prepare a series of NiFe-based nanocrystals (e.g., hydroxides, sulfides, and molybdates) from amorphous precipitates. Their morphology and evolution of atomic and electronic structure during this process are studied using conclusive microscopy and spectroscopy techniques. The short-term, additive-free, and low-cost method allows for the control of the crystallinity of the materials and facilitates the generation of nanosheets, nanorods, or nano-octahedra with excellent water oxidation activity. The NiFe-based crystalline catalysts exhibit slightly compromised initial activity but more robust long-term stability than their amorphous counterparts during electrochemical operation. This facile, reliable, and universal synthesis method is promising in strategies for fabricating NiFe-based nanostructures as efficient and economically valuable OER electrocatalysts.

Intermetallic Cobalt Indium Nanoparticles as Oxygen Evolution Reaction Precatalyst: A Non-Leaching p-Block Element.

Merely all transition-metal-based materials reconstruct into similar oxyhydroxides during the electrocatalytic oxygen evolution reaction (OER), severely limiting the options for a tailored OER catalyst design. In such reconstructions, initial constituent p-block elements take a sacrificial role and leach into the electrolyte as oxyanions, thereby losing the ability to tune the catalyst's properties systematically. From a thermodynamic point of view, indium is expected to behave differently and should remain in the solid phase under alkaline OER conditions. However, the structural behavior of transition metal indium phases during the OER remains unexplored. Herein, are synthesized intermetallic cobalt indium (CoIn3 ) nanoparticles and revealed by in situ X-ray absorption spectroscopy and scanning transmission microscopy that they undergo phase segregation to cobalt oxyhydroxide and indium hydroxide. The obtained cobalt oxyhydroxide outperforms a metallic-cobalt-derived one due to more accessible active sites. The observed phase segregation shows that indium behaves distinctively differently from most p-block elements and remains at the electrode surface, where it can form lasting interfaces with the active metal oxo phases.

Development of iron-based single atom materials for general and efficient synthesis of amines.

Heterogeneous catalysts with highly active and at the same time stable isolated metal sites constitute a key factor for the advancement of sustainable and cost-effective chemical synthesis. In particular, the development of more practical, and durable iron-based materials is of central interest for organic synthesis, especially for the preparation of chemical products related to life science applications. Here, we report the preparation of Fe-single atom catalysts (Fe-SACs) entrapped in N-doped mesoporous carbon support with unprecedented potential in the preparation of different kinds of amines. The synthetic protocol of Fe-SACs is based on primary pyrolysis of Fe-nitrogen complexes on SiO2 and subsequent removal of silica resulting in the formation of unique mesoporous N-doped carbon support with the pore size controlled by the size of the original silica nanoparticles. The resulting stable and reusable Fe-SACs allow for the reductive amination of a broad range of aldehydes and ketones with ammonia and amines to produce diverse primary, secondary, and tertiary amines including N-methylated products as well as drugs, agrochemicals, and other biomolecules (amino acid esters and amides) utilizing green hydrogen.

Molecular Dynamics and Structural Studies of Zinc Chloroquine Complexes.

Chloroquine (CQ) is a first-choice drug against malaria and autoimmune diseases. It has been co-administered with zinc against SARS-CoV-2 and soon dismissed because of safety issues. The structural features of Zn-CQ complexes and the effect of CQ on zinc distribution in cells are poorly known. In this study, state-of-the-art computations combined with experiments were leveraged to solve the structural determinants of zinc-CQ interactions in solution and the solid state. NMR, ESI-MS, and X-ray absorption and diffraction methods were combined with ab initio molecular dynamics calculations to address the kinetic lability of this complex. Within the physiological pH range, CQ binds Zn2+ through the quinoline ring nitrogen, forming [Zn(CQH)Clx(H2O)3-x](3+)-x (x = 0, 1, 2, and 3) tetrahedral complexes. The Zn(CQH)Cl3 species is stable at neutral pH and at high chloride concentrations typical of the extracellular medium, but metal coordination is lost at a moderately low pH as in the lysosomal lumen. The pentacoordinate complex [Zn(CQH)(H2O)4]3+ may exist in the absence of chloride. This in vitro/in silico approach can be extended to other metal-targeting drugs and bioinorganic systems.

Spectroscopic Properties of a Biologically Relevant [Fe2 (µ-O)2 ] Diamond Core Motif with a Short Iron-Iron Distance.

Diiron cofactors in enzymes perform diverse challenging transformations. The structures of high valent intermediates (Q in methane monooxygenase and X in ribonucleotide reductase) are debated since Fe-Fe distances of 2.5-3.4 Šwere attributed to "open" or "closed" cores with bridging or terminal oxido groups. We report the crystallographic and spectroscopic characterization of a FeIII 2 (µ-O)2 complex (2) with tetrahedral (4C) centres and short Fe-Fe distance (2.52 Å), persisting in organic solutions. 2 shows a large Fe K-pre-edge intensity, which is caused by the pronounced asymmetry at the TD FeIII centres due to the short Fe-µ-O bonds. A ≈2.5 ŠFe-Fe distance is unlikely for six-coordinate sites in Q or X, but for a Fe2 (µ-O)2 core containing four-coordinate (or by possible extension five-coordinate) iron centres there may be enough flexibility to accommodate a particularly short Fe-Fe separation with intense pre-edge transition. This finding may broaden the scope of models considered for the structure of high-valent diiron intermediates formed upon O2 activation in biology.

Exploring the Biosynthetic Potential of TsrM, a B12 -dependent Radical SAM Methyltransferase Catalyzing Non-radical Reactions.

B12 -dependent radical SAM enzymes are an emerging enzyme family with approximately 200,000 proteins. These enzymes have been shown to catalyze chemically challenging reactions such as methyl transfer to sp2- and sp3-hybridized carbon atoms. However, to date we have little information regarding their complex mechanisms and their biosynthetic potential. Here we show, using X-ray absorption spectroscopy, mutagenesis and synthetic probes that the vitamin B12 -dependent radical SAM enzyme TsrM catalyzes not only C- but also N-methyl transfer reactions further expanding its synthetic versatility. We also demonstrate that TsrM has the unique ability to directly transfer a methyl group to the benzyl core of tryptophan, including the least reactive position C4. Collectively, our study supports that TsrM catalyzes non-radical reactions and establishes the usefulness of radical SAM enzymes for novel biosynthetic schemes including serial alkylation reactions at particularly inert C-H bonds.

Trapping an Oxidized and Protonated Intermediate of the [FeFe]-Hydrogenase Cofactor under Mildly Reducing Conditions.

The H-cluster is the catalytic cofactor of [FeFe]-hydrogenase, a metalloenzyme that catalyzes the formation of dihydrogen (H2). The catalytic diiron site of the H-cluster carries two cyanide and three carbon monoxide ligands, making it an excellent target for IR spectroscopy. In previous work, we identified an oxidized and protonated H-cluster species, whose IR signature differs from that of the oxidized resting state (Hox) by a small but distinct shift to higher frequencies. This "blue shift" was explained by a protonation at the [4Fe-4S] subcomplex of the H-cluster. The novel species, denoted HoxH, was preferentially accumulated at low pH and in the presence of the exogenous reductant sodium dithionite (NaDT). When HoxH was reacted with H2, the hydride state (Hhyd) was formed, a key intermediate of [FeFe]-hydrogenase turnover. A recent publication revisited our protocol for the accumulation of HoxH in wild-type [FeFe]-hydrogenase, concluding that inhibition by NaDT decay products rather than cofactor protonation causes the spectroscopic "blue shift". Here, we demonstrate that HoxH formation does not require the presence of NaDT (or its decay products), but accumulates also with the milder reductants tris(2-carboxyethyl)phosphine, dithiothreitol, or ascorbic acid, in particular at low pH. Our data consistently suggest that HoxH is accumulated when deprotonation of the H-cluster is impaired, thereby preventing the regain of the oxidized resting state Hox in the catalytic cycle.

Water Oxidation by Pentapyridyl Base Metal Complexes? A Case Study.

The design of molecular water oxidation catalysts (WOCs) requires a rational approach that considers the intermediate steps of the catalytic cycle, including water binding, deprotonation, storage of oxidizing equivalents, O-O bond formation, and O2 release. We investigated several of these properties for a series of base metal complexes (M = Mn, Fe, Co, Ni) bearing two variants of a pentapyridyl ligand framework, of which some were reported previously to be active WOCs. We found that only [Fe(Py5OMe)Cl]+ (Py5OMe = pyridine-2,6-diylbis[di-(pyridin-2-yl)methoxymethane]) showed an appreciable catalytic activity with a turnover number (TON) = 130 in light-driven experiments using the [Ru(bpy)3]2+/S2O82- system at pH 8.0, but that activity is demonstrated to arise from the rapid degradation in the buffered solution leading to the formation of catalytically active amorphous iron oxide/hydroxide (FeOOH), which subsequently lost the catalytic activity by forming more extensive and structured FeOOH species. The detailed analysis of the redox and water-binding properties employing electrochemistry, X-ray absorption spectroscopy (XAS), UV-vis spectroscopy, and density-functional theory (DFT) showed that all complexes were able to undergo the MIII/MII oxidation, but none was able to yield a detectable amount of a MIV state in our potential window (up to +2 V vs SHE). This inability was traced to (i) the preference for binding Cl- or acetonitrile instead of water-derived species in the apical position, which excludes redox leveling via proton coupled electron transfer, and (ii) the lack of sigma donor ligands that would stabilize oxidation states beyond MIII. On that basis, design features for next-generation molecular WOCs are suggested.

Bimetallic Mn, Fe, Co, and Ni Sites in a Four-Helix Bundle Protein: Metal Binding, Structure, and Peroxide Activation.

Bimetallic active sites in enzymes catalyze small-molecule conversions that are among the top 10 challenges in chemistry. As different metal cofactors are typically incorporated in varying protein scaffolds, it is demanding to disentangle the individual contributions of the metal and the protein matrix to the activity. Here, we compared the structure, properties, and hydrogen peroxide reactivity of four homobimetallic cofactors (Mn(II)2, Fe(II)2, Co(II)2, Ni(II)2) that were reconstituted into a four-helix bundle protein. Reconstituted proteins were studied in solution and in crystals. All metals bind with high affinity and yield similar cofactor structures. Cofactor variants react with H2O2 but differ in their turnover rates, accumulated oxidation states, and trapped peroxide-bound intermediates. Varying the metal composition thus creates opportunities to tune the reactivity of the bimetallic cofactor and to study and functionalize reactive species.

Operando tracking of oxidation-state changes by coupling electrochemistry with time-resolved X-ray absorption spectroscopy demonstrated for water oxidation by a cobalt-based catalyst film.

Transition metal oxides are promising electrocatalysts for water oxidation, i.e., the oxygen evolution reaction (OER), which is critical in electrochemical production of non-fossil fuels. The involvement of oxidation state changes of the metal in OER electrocatalysis is increasingly recognized in the literature. Tracing these oxidation states under operation conditions could provide relevant information for performance optimization and development of durable catalysts, but further methodical developments are needed. Here, we propose a strategy to use single-energy X-ray absorption spectroscopy for monitoring metal oxidation-state changes during OER operation with millisecond time resolution. The procedure to obtain time-resolved oxidation state values, using two calibration curves, is explained in detail. We demonstrate the significance of this approach as well as possible sources of data misinterpretation. We conclude that the combination of X-ray absorption spectroscopy with electrochemical techniques allows us to investigate the kinetics of redox transitions and to distinguish the catalytic current from the redox current. Tracking of the oxidation state changes of Co ions in electrodeposited oxide films during cyclic voltammetry in neutral pH electrolyte serves as a proof of principle.

A Pseudotetrahedral Terminal Oxoiron(IV) Complex: Mechanistic Promiscuity in C-H Bond Oxidation Reactions.

S=2 oxoiron(IV) species act as reactive intermediates in the catalytic cycle of nonheme iron oxygenases. The few available synthetic S=2 FeIV =O complexes known to date are often limited to trigonal bipyramidal and very rarely to octahedral geometries. Herein we describe the generation and characterization of an S=2 pseudotetrahedral FeIV =O complex 2 supported by the sterically demanding 1,4,7-tri-tert-butyl-1,4,7-triazacyclononane ligand. Complex 2 is a very potent oxidant in hydrogen atom abstraction (HAA) reactions with large non-classical deuterium kinetic isotope effects, suggesting hydrogen tunneling contributions. For sterically encumbered substrates, direct HAA is impeded and an alternative oxidative asynchronous proton-coupled electron transfer mechanism prevails, which is unique within the nonheme oxoiron community. The high reactivity and the similar spectroscopic parameters make 2 one of the best electronic and functional models for a biological oxoiron(IV) intermediate of taurine dioxygenase (TauD-J).

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins.

A heterobimetallic Mn/Fe cofactor is present in the R2 subunit of class Ic ribonucleotide reductases (R2c) and in R2-like ligand-binding oxidases (R2lox). Although the protein-derived metal ligands are the same in both groups of proteins, the connectivity of the two metal ions and the chemistry each cofactor performs are different: in R2c, a one-electron oxidant, the Mn/Fe dimer is linked by two oxygen bridges (µ-oxo/µ-hydroxo), whereas in R2lox, a two-electron oxidant, it is linked by a single oxygen bridge (µ-hydroxo) and a fatty acid ligand. Here, we identified a second coordination sphere residue that directs the divergent reactivity of the protein scaffold. We found that the residue that directly precedes the N-terminal carboxylate metal ligand is conserved as a glycine within the R2lox group but not in R2c. Substitution of the glycine with leucine converted the resting-state R2lox cofactor to an R2c-like cofactor, a µ-oxo/µ-hydroxo-bridged MnIII/FeIII dimer. This species has recently been observed as an intermediate of the oxygen activation reaction in WT R2lox, indicating that it is physiologically relevant. Cofactor maturation in R2c and R2lox therefore follows the same pathway, with structural and functional divergence of the two cofactor forms following oxygen activation. We also show that the leucine-substituted variant no longer functions as a two-electron oxidant. Our results reveal that the residue preceding the N-terminal metal ligand directs the cofactor's reactivity toward one- or two-electron redox chemistry, presumably by setting the protonation state of the bridging oxygens and thereby perturbing the redox potential of the Mn ion.

Stoichiometric Formation of an Oxoiron(IV) Complex by a Soluble Methane Monooxygenase Type Activation of O2 at an Iron(II)-Cyclam Center.

In soluble methane monooxygenase enzymes (sMMO), dioxygen (O2) is activated at a diiron(II) center to form an oxodiiron(IV) intermediate Q that performs the challenging oxidation of methane to methanol. An analogous mechanism of O2 activation at mono- or dinuclear iron centers is rare in the synthetic chemistry. Herein, we report a mononuclear non-heme iron(II)-cyclam complex, 1-trans, that activates O2 to form the corresponding iron(IV)-oxo complex, 2-trans, via a mechanism reminiscent of the O2 activation process in sMMO. The conversion of 1-trans to 2-trans proceeds via the intermediate formation of an iron(III)-superoxide species 3, which could be trapped and spectroscopically characterized at -50 °C. Surprisingly, 3 is a stronger oxygen atom transfer (OAT) agent than 2-trans; 3 performs OAT to 1-trans or PPh3 to yield 2-trans quantitatively. Furthermore, 2-trans oxidizes the aromatic C-H bonds of 2,6-di-tert-butylphenol, which, together with the strong OAT ability of 3, represents new domains of oxoiron(IV) and superoxoiron(III) reactivities.

[FeFe]-hydrogenase maturation: H-cluster assembly intermediates tracked by electron paramagnetic resonance, infrared, and X-ray absorption spectroscopy.

[FeFe]-hydrogenase enzymes employ a unique organometallic cofactor for efficient and reversible hydrogen conversion. This so-called H-cluster consists of a [4Fe-4S] cubane cysteine linked to a diiron complex coordinated by carbon monoxide and cyanide ligands and an azadithiolate ligand (adt = NH(CH2S)2)·[FeFe]-hydrogenase apo-protein binding only the [4Fe-4S] sub-complex can be fully activated in vitro by the addition of a synthetic diiron site precursor complex ([2Fe]adt). Elucidation of the mechanism of cofactor assembly will aid in the design of improved hydrogen processing synthetic catalysts. We combined electron paramagnetic resonance, Fourier-transform infrared, and X-ray absorption spectroscopy to characterize intermediates of H-cluster assembly as initiated by mixing of the apo-protein (HydA1) from the green alga Chlamydomonas reinhardtii with [2Fe]adt. The three methods consistently show rapid formation of a complete H-cluster in the oxidized, CO-inhibited state (Hox-CO) already within seconds after the mixing. Moreover, FTIR spectroscopy support a model in which Hox-CO formation is preceded by a short-lived Hred'-CO-like intermediate. Accumulation of Hox-CO was followed by CO release resulting in the slower conversion to the catalytically active state (Hox) as well as formation of reduced states of the H-cluster.

Temperature Dependence of Structural Dynamics at the Catalytic Cofactor of [FeFe]-hydrogenase.

[FeFe]-hydrogenases are nature's blueprint for efficient hydrogen turnover. Understanding their enzymatic mechanism may improve technological H2 fuel generation. The active-site cofactor (H-cluster) consists of a [4Fe-4S] cluster ([4Fe]H), cysteine-linked to a diiron site ([2Fe]H) carrying an azadithiolate (adt) group, terminal cyanide and carbon monoxide ligands, and a bridging carbon monoxide (µCO) in the oxidized protein (Hox). Recently, the debate on the structure of reduced H-cluster states was intensified by the assignment of new species under cryogenic conditions. We investigated temperature effects (4-280 K) in infrared (IR) and X-ray absorption spectroscopy (XAS) data of [FeFe]-hydrogenases using fit analyses and quantum-chemical calculations. IR data from our laboratory and literature sources were evaluated. At ambient temperatures, reduced H-cluster states with a bridging hydride (µH-, in Hred and Hsred) or with an additional proton at [4Fe]H (Hred') or at the distal iron of [2Fe]H (Hhyd) prevail. At cryogenic temperatures, these species are largely replaced by states that hold a µCO, lack [4Fe]H protonation, and bind an additional proton at the adt nitrogen (HredH+ and HsredH+). XAS revealed the atomic coordinate dispersion (i.e., the Debye-Waller parameter, 2σ2) of the iron-ligand bonds and Fe-Fe distances in the oxidized and reduced H-cluster. 2σ2 showed a temperature dependence typical for the so-called protein-glass transition, with small changes below ∼200 K and a pronounced increase above this "breakpoint". This behavior is attributed to the freezing-out of larger-scale anharmonic motions of amino acid side chains and water species. We propose that protonation at [4Fe]H as well as ligand rearrangement and µH- binding at [2Fe]H are impaired because of restricted molecular mobility at cryogenic temperatures so that protonation can be biased toward adt. We conclude that a H-cluster with a µCO, selective [4Fe]H or [2Fe]H protonation, and catalytic proton transfer via adt facilitates efficient H2 conversion in [FeFe]-hydrogenase.

Anion Binding and Oxidative Modification at the Molybdenum Cofactor of Formate Dehydrogenase from Rhodobacter capsulatus Studied by X-ray Absorption Spectroscopy.

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo═S) and oxo (Mo═O) bonds, were observed in the presence of azide (N3-) or cyanate (OCN-). Azide provided best protection against O2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.

Effective intermediate-spin iron in O2-transporting heme proteins.

Proteins carrying an iron-porphyrin (heme) cofactor are essential for biological O2 management. The nature of Fe-O2 bonding in hemoproteins is debated for decades. We used energy-sampling and rapid-scan X-ray Kß emission and K-edge absorption spectroscopy as well as quantum chemistry to determine molecular and electronic structures of unligated (deoxy), CO-inhibited (carboxy), and O2-bound (oxy) hemes in myoglobin (MB) and hemoglobin (HB) solutions and in porphyrin compounds at 20-260 K. Similar metrical and spectral features revealed analogous heme sites in MB and HB and the absence of low-spin (LS) to high-spin (HS) conversion. Amplitudes of Kß main-line emission spectra were directly related to the formal unpaired Fe(d) spin count, indicating HS Fe(II) in deoxy and LS Fe(II) in carboxy. For oxy, two unpaired Fe(d) spins and, thus by definition, an intermediate-spin iron center, were revealed by our static and kinetic X-ray data, as supported by (time-dependent) density functional theory and complete-active-space self-consistent-field calculations. The emerging Fe-O2 bonding situation includes in essence a ferrous iron center, minor superoxide character of the noninnocent ligand, significant double-bond properties of the interaction, and three-center electron delocalization as in ozone. It resolves the apparently contradictory classical models of Pauling, Weiss, and McClure/Goddard into a unifying view of O2 bonding, tuned toward reversible oxygen transport.

Identification of YdhV as the First Molybdoenzyme Binding a Bis-Mo-MPT Cofactor in Escherichia coli.

The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco)-containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homology with a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. The cofactor was identified to be of a bis-Mo-MPT type with no guanine nucleotides present, which represents a form of Moco that has not been found previously in any molybdoenzyme. Our studies showed that YdhV has a preference for bis-Mo-MPT over bis-W-MPT to be inserted into the enzyme. In-depth characterization of YdhV by X-ray absorption and electron paramagnetic resonance spectroscopies revealed that the bis-Mo-MPT cofactor in YdhV is redox active. The bis-Mo-MPT and bis-W-MPT cofactors include metal centers that bind the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors.