Electron paramagnetic resonance studies of molybdenum enzymes

International audienceElectron Paramagnetic Resonance (EPR) is certainly the first and the most widely used spectroscopic technique for studying structure and function of Mo and W enzymes. Although only Mo(v) and W(v) states can be detected, a considerable wealth of data was provided since the seminal EPR works performed on xanthine oxidase and nitrate reductase more than 55 years ago. In this chapter, we give a comprehensive overview of the various applications of EPR on the ubiquitous Mo-enzymes, which exhibit such an extraordinary diversity of substrates and catalyzed reactions. Elucidating the nature of Mo(v) intermediates is a considerable challenge to progress in understanding these processes. The g-tensor analyses are helpful in that aim. But it is essentially thanks to the advances in pulsed EPR methods like ENDOR, ESEEM and HYSCORE, combined with efficient isotopic enrichment strategies, that the measurements of hyperfine couplings of Mo-cofactor with neighbouring magnetic nuclei have brought the most interesting data. Thus, we illustrate how the analysis of hyperfine parameters associated with computational chemistry methods is becoming a powerful way to provide high-resolution structural data on Mo(v) species and enzyme mechanisms. In addition, EPR study of spin–spin couplings between Mo-cofactor and other paramagnetic centres appears as a promising way to gain long-range structural data in these systems

Reductive activation of E. coli respiratory nitrate reductase

International audienceOver the past decades, a number of authors have reported the presence of inactive species in as-prepared samples of members of the Mo/W-bisPGD enzyme family. This greatly complicated the spectroscopic studies of these enzymes, since it is impossible to discriminate between active and inactive species on the basis of the spectroscopic signatures alone. Escherichia coli nitrate reductase A (NarGHI) is a member of the Mo/W-bisPGD family that allows anaerobic respiration using nitrate as terminal electron acceptor. Here, using protein film voltammetry on NarGH films, we show that the enzyme is purified in a functionally heterogeneous form that contains between 20 and 40% of inactive species that activate the first time they are reduced. This activation proceeds in two steps: a non-redox reversible reaction followed by an irreversible reduction. By carefully correlating electrochemical and EPR spectroscopic data, we show that neither the two major Mo(V) signals nor those of the two FeS clusters that are the closest to the Mo center are associated with the two inactive species. We also conclusively exclude the possibility that the major “low-pH” and “high-pH” Mo(V) EPR signatures correspond to species in acid–base equilibrium

HYSCORE Evidence That Endogenous Mena- and Ubisemiquinone Bind at the Same Q Site (Q(D)) of Escherichia coli Nitrate Reductase A.

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The H-bond network surrounding the pyranopterins modulates redox cooperativity in the molybdenum-bisPGD cofactor in arsenite oxidase

International audienceWhile the molybdenum cofactor in the majority of bisPGD enzymes goes through two consecutive 1-electron redox transitions, previous protein-film voltammetric results indicated the possibility of cooperative (n = 2) redox behavior in the bioenergetic enzyme arsenite oxidase (Aio). Combining equilibrium redox titrations, optical and EPR spectroscopies on concentrated samples obtained via heterologous expression, we unambiguously confirm this claim and quantify Aio's redox cooperativity. The stability constant, Ks, of the MoV semi-reduced intermediate is found to be lower than 10− 3. Site-directed mutagenesis of residues in the vicinity of the Mo-cofactor demonstrates that the degree of redox cooperativity is sensitive to H-bonding interactions between the pyranopterin moieties and amino acid residues. Remarkably, in particular replacing the Gln-726 residue by Gly results in stabilization of (low-temperature) EPR-observable MoV with KS = 4. As evidenced by comparison of room temperature optical and low temperature EPR titrations, the degree of stabilization is temperature-dependent. This highlights the importance of room-temperature redox characterizations for correctly interpreting catalytic properties in this group of enzymes.Geochemical and phylogenetic data strongly indicate that molybdenum played an essential biocatalytic roles in early life. Molybdenum's redox versatility and in particular the ability to show cooperative (n = 2) redox behavior provide a rationale for its paramount catalytic importance throughout the evolutionary history of life. Implications of the H-bonding network modulating Molybdenum's redox properties on details of a putative inorganic metabolism at life's origin are discussed

EPR-HYSCORE study of quinone binding in respiratory nitrate reductase: Molecular basis for the adaptation to anaerobiosis-aerobiosis transition

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Demethylmenaquinol is a substrate of Escherichia coli nitrate reductase A (NarGHI) and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site

International audienceQuinones are essential building blocks of respiration, a universal process dedicated to efficient harvesting of environmental energy and its conversion into a transmembrane chemiosmotic potential. Quinones differentiate mostly by their midpoint redox potential. As such, γ-proteobacteria such as Escherichia coli are characterized by the presence of demethylmenaquinone (DMK) with an intermediate redox potential between low-potential (menaquinone) and high-potential (ubiquinone) quinones. In this study, we show that demethylmenaquinol (DMKH2) is a good substrate for nitrate reductase A (NarGHI) in nitrate respiration in E. coli. Kinetic studies performed with quinol analogs on NarGHI show that removal of the methyl group on the naphthoquinol ring impacts modestly the catalytic constant but not the KM. EPR-monitored redox titrations of NarGHI-enriched membrane vesicles reveal that endogeneous demethylmenasemiquinone (DMSK) intermediates are stabilized in the enzyme. The measured midpoint potential of the DMK/DMKH2 redox couple in NarGHI (E′m,7.5 (DMK/DMKH2) ~− 70 mV) is significantly lower than that previously measured for unbound species. High resolution pulsed EPR experiments demonstrate that DMSK are formed within the NarGHI quinol oxidation site. Overall, our results provide the first characterization of a protein-bound DMSK and allows for comparison for distinct use of three quinones at a single Q-site in NarGHI

Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective 2 H and 15 N Labeling, HYSCORE Spectroscopy, and DFT Modeling

International audienceIn vivo specific isotope labeling at the residue or substituent level is used to probe menasemiquinone (MSK) binding to the quinol oxidation site of respiratory nitrate reductase A (NarGHI) from E. coli. 15N selective labeling of His15Nδ or Lys15Nζ in combination with hyperfine sublevel correlation (HYSCORE) spectroscopy unambiguously identified His15Nδ as the direct hydrogen-bond donor to the radical. In contrast, an essentially anisotropic coupling to Lys15Nζ consistent with a through-space magnetic interaction was resolved. This suggests that MSK does not form a hydrogen bond with the side chain of the nearby Lys86 residue. In addition, selective 2H labeling of the menaquinone methyl ring substituent allows unambiguous characterization of the 2H—and hence of the 1H—methyl isotropic hyperfine coupling by 2H HYSCORE. DFT calculations show that a simple molecular model consisting of an imidazole Nδ atom in a hydrogen-bond interaction with a MSK radical anion satisfactorily accounts for the available spectroscopic data. These results support our previously proposed one-sided binding model for MSK to NarGHI through a single short hydrogen bond to the Nδ of His66, one of the distal heme axial ligands. This work establishes the basis for future investigations aimed at determining the functional relevance of this peculiar binding mode

Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

International audienceA major gap of knowledge in metalloproteins is the identity of the prefolded state of the protein before cofactor insertion. This holds for molybdoenzymes serving multiple purposes for life, especially in energy harvesting. This large group of prokaryotic enzymes allows for coordination of molybdenum or tungsten cofactors (Mo/W-bisPGD) and Fe/S clusters. Here we report the structural data on a cofactor-less enzyme, the nitrate reductase respiratory complex and characterize the conformational changes accompanying Mo/W-bisPGD and Fe/S cofactors insertion. Identified conformational changes are shown to be essential for recognition of the dedicated chaperone involved in cofactors insertion. A solvent-exposed salt bridge is shown to play a key role in enzyme folding after cofactors insertion. Furthermore, this salt bridge is shown to be strictly conserved within this prokaryotic molybdoenzyme family as deduced from a phylogenetic analysis issued from 3D structure-guided multiple sequence alignment. A biochemical analysis with a distantly-related member of the family, respiratory complex I, confirmed the critical importance of the salt bridge for folding. Overall, our results point to a conserved cofactors insertion mechanism within the Mo/W-bisPGD family

Structural evidence for a reaction intermediate mimic in the active site of a sulfite dehydrogenase†

By combining X-ray crystallography, electron paramagnetic resonance techniques and density functional theory-based modelling, we provide evidence for a direct coordination of the product analogue, phosphate, to the molybdenum active site of a sulfite dehydrogenase.This interaction is mimicking the still experimentally uncharacterized reaction intermediate proposed to arise during the catalytic cycle of this class of enzymes. This work opens new perspectives for further deciphering the reaction mechanism of this nearly ubiquitous class of oxidoreductase