27 results found
Wright JJ, Fedor J, Hirst J, et al., 2020, Using a chimeric respiratory chain and EPR spectroscopy to determine the origin of semiquinone species previously assigned to mitochondrial complex I, BMC Biology, Vol: 18, ISSN: 1741-7007
Background. For decades semiquinone intermediates have been suggested to play an essential role in catalysis by one of the most enigmatic proton-pumping enzymes, respiratory complex I, and different mechanisms have been proposed on their basis. However, the difficulty in investigating complex I semiquinones, due to the many different enzymes embedded in the inner mitochondrial membrane, has resulted in an ambiguous picture and no consensus. Results. In this paper we re-examine the highly debated origin of semiquinone species in mitochondrial membranes using a novel approach. Our combination of a semi-artificial chimeric respiratory chain with pulse EPR spectroscopy (HYSCORE) has enabled us to conclude, unambiguously and for the first time, that the majority of the semiquinones observed in mitochondrial membranes originate from complex III. We also identify a minor contribution from complex II.Conclusions. We are unable to attribute any semiquinone signals unambiguously to complex I and, reconciling our observations with much of the previous literature, conclude that they are likely to have been misattributed to it. We note that, for this earlier work, the tools we have relied on here to deconvolute overlapping EPR signals were not available. Proposals for the mechanism of complex I based on the EPR signals of semiquinone species observed in mitochondrial membranes should thus be treated with caution until future work has succeeded in isolating any complex I semiquinone EPR spectroscopic signatures present.
Bajada MA, Roy S, Warnan J, et al., 2020, A Precious-Metal-Free Hybrid Electrolyzer for Alcohol Oxidation Coupled to CO2-to-Syngas Conversion, ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, ISSN: 1433-7851
Luo H, Papaioannou N, Salvadori E, et al., 2019, Manipulating the Optical Properties of Carbon Dots by Fine-Tuning their Structural Features, CHEMSUSCHEM, Vol: 12, Pages: 4432-4441, ISSN: 1864-5631
Mellor SB, Vinde MH, Nielsen AZ, et al., 2019, Defining optimal electron transfer partners for light-driven cytochrome P450 reactions, Metabolic Engineering, Vol: 55, Pages: 33-43, ISSN: 1096-7176
Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can allevi
Abdiaziz K, Salvadori E, Sokol K, et al., 2019, Protein film electrochemical EPR spectroscopy as a technique to investigate redox reactions in biomolecules, Chemical Communications, Vol: 55, Pages: 8840-8843, ISSN: 1359-7345
Redox reactions and paramagnetic intermediates are ubiquitous in biological chemistry. We report a new method, protein film electrochemical electron paramagnetic resonance spectroscopy (PFE-EPR), that enables the direct and accurate potential control of proteins on the electrode surface for both electrochemical and EPR spectroscopic characterisation of their redox centres.
Cirulli M, Kaur A, Lewis JEM, et al., 2019, Rotaxane-based transition metal complexes: Effect of the mechanical bond on structure and electronic properties, Journal of the American Chemical Society, Vol: 141, Pages: 879-889, ISSN: 0002-7863
Early work by Sauvage revealed that mechanical bonding alters the stability and redox properties of their original catenane metal complexes. However, despite the importance of controlling metal ion properties for a range of applications, these effects have received relatively little attention since. Here we present a series of tri-, tetra-, and pentadentate rotaxane-based ligands and a detailed study of their metal binding behavior and, where possible, compare their redox and electronic properties with their noninterlocked counterparts. The rotaxane ligands form complexes with most of the metal ions investigated, and X-ray diffraction revealed that in some cases the mechanical bond enforces unusual coordination numbers and distorted arrangements as a result of the exclusion of exogenous ligands driven by the sterically crowded binding sites. In contrast, only the noninterlocked equivalent of the pentadentate rotaxane CuII complex could be formed selectively, and this exhibited compromised redox stability compared to its interlocked counterpart. Frozen-solution EPR data demonstrate the formation of an interesting biomimetic state for the tetradentate CuII rotaxane, as well as the formation of stable NiI species and the unusual coexistence of high- and low-spin CoII in the pentadentate framework. Our results demonstrate that readily available mechanically chelating rotaxanes give rise to complexes the noninterlocked equivalent of which are inaccessible, and that the mechanical bond augments the redox behavior of the bound metal ion in a manner analogous to the carefully tuned amino acid framework in metalloproteins.
Martinez-Lumbreras S, Krysztofinska EM, Thapaliya A, et al., 2018, Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality control, BMC Biology, Vol: 16, ISSN: 1741-7007
BackgroundProtein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The C-terminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo.ResultsWe show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain.ConclusionOur results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region.
Volbeda A, Mouesca JM, Darnault C, et al., 2018, X- ray structural, functional and computational studies of the O2-sensitive E. coli hydrogenase-1 C19G variant reveal an unusual [ 4Fe-4S] cluster+, CHEMICAL COMMUNICATIONS, Vol: 54, Pages: 7175-7178, ISSN: 1359-7345
Roessler MM, Salvadori E, 2018, Principles and applications of EPR spectroscopy in the chemical sciences, CHEMICAL SOCIETY REVIEWS, Vol: 47, Pages: 2534-2553, ISSN: 0306-0012
Le Breton N, Wright JJ, Jones AJY, et al., 2017, Using hyperfine electron paramagnetic resonance spectroscopy to define the proton-coupled electron transfer reaction at Fe-S cluster N2 in respiratory complex I, Journal of the American Chemical Society, Vol: 139, Pages: 16319-16326, ISSN: 1520-5126
Energy-transducing respiratory complex I (NADH:ubiquinone oxidoreductase) is one of the largest and most complicated enzymes in mammalian cells. Here, we used hyperfine electron paramagnetic resonance (EPR) spectroscopic methods, combined with site-directed mutagenesis, to determine the mechanism of a single proton-coupled electron transfer reaction at one of eight iron-sulfur clusters in complex I, [4Fe-4S] cluster N2. N2 is the terminal cluster of the enzyme's intramolecular electron-transfer chain and the electron donor to ubiquinone. Because of its position and pH-dependent reduction potential, N2 has long been considered a candidate for the elusive "energy-coupling" site in complex I at which energy generated by the redox reaction is used to initiate proton translocation. Here, we used hyperfine sublevel correlation (HYSCORE) spectroscopy, including relaxation-filtered hyperfine and single-matched resonance transfer (SMART) HYSCORE, to detect two weakly coupled exchangeable protons near N2. We assign the larger coupling with A(1H) = [-3.0, -3.0, 8.7] MHz to the exchangeable proton of a conserved histidine and conclude that the histidine is hydrogen-bonded to N2, tuning its reduction potential. The histidine protonation state responds to the cluster oxidation state, but the two are not coupled sufficiently strongly to catalyze a stoichiometric and efficient energy transduction reaction. We thus exclude cluster N2, despite its proton-coupled electron transfer chemistry, as the energy-coupling site in complex I. Our work demonstrates the capability of pulse EPR methods for providing detailed information on the properties of individual protons in even the most challenging of energy-converting enzymes.
Adamson H, Robinson M, Wright JJ, et al., 2017, Retuning the catalytic bias and overpotential of a [NiFe]-Hydrogenase via a single amino acid exchange at the electron entry/exit site, Journal of the American Chemical Society, Vol: 139, Pages: 10677-10686, ISSN: 1520-5126
The redox chemistry of the electron entry/exit site in Escherichia coli hydrogenase-1 is shown to play a vital role in tuning biocatalysis. Inspired by nature, we generate a HyaA-R193L variant to disrupt a proposed Arg–His cation−π interaction in the secondary coordination sphere of the outermost, “distal”, iron–sulfur cluster. This rewires the enzyme, enhancing the relative rate of H2 production and the thermodynamic efficiency of H2 oxidation catalysis. On the basis of Fourier transformed alternating current voltammetry measurements, we relate these changes in catalysis to a shift in the distal [Fe4S4]2+/1+ redox potential, a previously experimentally inaccessible parameter. Thus, metalloenzyme chemistry is shown to be tuned by the second coordination sphere of an electron transfer site distant from the catalytic center.
Wright JJ, Salvadori E, Bridges HR, et al., 2016, Small-volume potentiometric titrations: EPR investigations of Fe-S cluster N2 in mitochondrial complex I, Journal of Inorganic Biochemistry, Vol: 162, Pages: 201-206, ISSN: 0162-0134
EPR-based potentiometric titrations are a well-established method for determining the reduction potentials of cofactors in large and complex proteins with at least one EPR-active state. However, such titrations require large amounts of protein. Here, we report a new method that requires an order of magnitude less protein than previously described methods, and that provides EPR samples suitable for measurements at both X- and Q-band microwave frequencies. We demonstrate our method by determining the reduction potential of the terminal [4Fe-4S] cluster (N2) in the intramolecular electron-transfer relay in mammalian respiratory complex I. The value determined by our method, Em7 = − 158 mV, is precise, reproducible, and consistent with previously reported values. Our small-volume potentiometric titration method will facilitate detailed investigations of EPR-active centres in non-abundant and refractory proteins that can only be prepared in small quantities.
Hirst J, Roessler MM, 2016, Energy conversion, redox catalysis and generation of reactive oxygen species by respiratory complex I, BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, Vol: 1857, Pages: 872-883, ISSN: 0005-2728
Armstrong FA, Evans RM, Hexter SV, et al., 2016, Guiding Principles of Hydrogenase Catalysis Instigated and Clarified by Protein Film Electrochemistry, ACCOUNTS OF CHEMICAL RESEARCH, Vol: 49, Pages: 884-892, ISSN: 0001-4842
Flanagan LA, Wright JJ, Roessler MM, et al., 2016, Re-engineering a NiFe hydrogenase to increase the H-2 production bias while maintaining native levels of O-2 tolerance, CHEMICAL COMMUNICATIONS, Vol: 52, Pages: 9133-9136, ISSN: 1359-7345
Murphy BJ, Hidalgo R, Roessler MM, et al., 2015, Discovery of Dark pH-Dependent H+ Migration in a [NiFe]-Hydrogenase and Its Mechanistic Relevance: Mobilizing the Hydrido Ligand of the Ni-C Intermediate, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 137, Pages: 8484-8489, ISSN: 0002-7863
Roessler MM, Evans RM, Davies RA, et al., 2013, Erratum: EPR spectroscopic studies of the Fe-S clusters in the O <inf>2</inf>-Tolerant [NiFe]-Hydrogenase Hyd-1 from escherichia coli and characterization of the unique [4Fe-3S] Cluster by HYSCORE (Journal of the American Chemical Society (2012) 134 (15581-15594) DOI: 10.1021/ja307117y), Journal of the American Chemical Society, Vol: 135, ISSN: 0002-7863
Evans RM, Parkin A, Roessler MM, et al., 2013, Principles of Sustained Enzymatic Hydrogen Oxidation in the Presence of Oxygen - The Crucial Influence of High Potential Fe-S Clusters in the Electron Relay of [NiFe]-Hydrogenases, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 135, Pages: 2694-2707, ISSN: 0002-7863
Roessler MM, Evans RM, Davies RA, et al., 2012, EPR Spectroscopic Studies of the Fe-S Clusters in the O-2-Tolerant [NiFe]-Hydrogenase Hyd-1 from Escherichia coli and Characterization of the Unique [4Fe-3S] Cluster by HYSCORE, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 134, Pages: 15581-15594, ISSN: 0002-7863
Volbeda A, Amara P, Darnault C, et al., 2012, X-ray crystallographic and computational studies of the O-2-tolerant [NiFe]-hydrogenase 1 from Escherichia coli, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 109, Pages: 5305-5310, ISSN: 0027-8424
Parkin A, Bowman L, Roessler MM, et al., 2012, How Salmonella oxidises H-2 under aerobic conditions, FEBS LETTERS, Vol: 586, Pages: 536-544, ISSN: 0014-5793
Lukey MJ, Roessler MM, Parkin A, et al., 2011, Oxygen-Tolerant [NiFe]-Hydrogenases: The Individual and Collective Importance of Supernumerary Cysteines at the Proximal Fe-S Cluster, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 133, Pages: 16881-16892, ISSN: 0002-7863
Lee C-Y, Stevenson GP, Parkin A, et al., 2011, Theoretical and experimental investigation of surface-confined two-center metalloproteins by large-amplitude Fourier transformed ac voltammetry, JOURNAL OF ELECTROANALYTICAL CHEMISTRY, Vol: 656, Pages: 293-303, ISSN: 1572-6657
Lukey MJ, Parkin A, Roessler MM, et al., 2010, How Escherichia coli is equipped to oxidize hydrogen under different redox conditions., Journal of Biological Chemistry, Vol: 285, Pages: 20421.5-20421, ISSN: 0021-9258
Lukey MJ, Parkin A, Roessler MM, et al., 2010, Erratum: How Escherichia coli is equipped to oxidize hydrogen under different redox conditions (The Journal Of Biological Chemistry (2010) 285, (3928-3938) DOI: 10.1074/jbc.A109.067751), Journal of Biological Chemistry, Vol: 285, ISSN: 0021-9258
Lukey MJ, Parkin A, Roessler MM, et al., 2010, How Escherichia coli Is Equipped to Oxidize Hydrogen under Different Redox Conditions, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 285, Pages: 3928-3938
Roessler MM, King MS, Robinson AJ, et al., 2010, Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron-electron resonance, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 107, Pages: 1930-1935, ISSN: 0027-8424
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