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Toshiko Ichiye - One of the best experts on this subject based on the ideXlab platform.

  • Identifying sequence determinants of reduction potentials of metalloProteins
    JBIC Journal of Biological Inorganic Chemistry, 2013
    Co-Authors: Bradley Scott Perrin, Toshiko Ichiye
    Abstract:

    The reduction potential of an Electron Transfer Protein is one of its most important functional characteristics. Although the type of redox site and the Protein fold are the major determinants of the reduction potential of a redox-active Protein, its amino acid sequence may tune the reduction potential as well. Thus, homologous Proteins can often be divided into different classes, with each class characterized by a biological function and a reduction potential. Site-specific mutagenesis of the sequence determinants of the differences in the reduction potential between classes should change the reduction potential of a Protein in one class to that of the other class. Here, a procedure is presented that combines energetic and bioinformatic analysis of homologous Proteins to identify sequence determinants that are also good candidates for site-specific mutations, using the [4Fe–4S] ferredoxins and the [4Fe–4S] high-potential iron–sulfur Proteins as examples. This procedure is designed to guide site-specific mutations or more computationally expensive studies, such as molecular dynamics simulations. To make the procedure more accessible to the general scientific community, it is being implemented into CHARMMing, a Web-based portal, with a library of density functional theory results for the redox site that are used in the setting up of Poisson–Boltzmann continuum electrostatics calculations for the Protein energetics.

  • Protein Control of Electron Transfer Rates via Polarization: Molecular Dynamics Studies of Rubredoxin
    Biophysical Journal, 2004
    Co-Authors: Elizabeth A. Dolan, Justin T Fischer, Brian W. Beck, Robert B. Yelle, Toshiko Ichiye
    Abstract:

    The Protein matrix of an Electron Transfer Protein creates an electrostatic environment for its redox site, which influences its Electron Transfer properties. Our studies of Fe-S Proteins indicate that the Protein is highly polarized around the redox site. Here, measures of deviations of the environmental electrostatic potential from a simple linear dielectric polarization response to the magnitude of the charge are proposed. In addition, a decomposition of the potential is proposed here to describe the apparent deviations from linearity, in which it is divided into a “permanent” component that is independent of the redox site charge and a dielectric component that linearly responds or polarizes to the charge. The nonlinearity measures and the decomposition were calculated for Clostridium pasteurianum rubredoxin from molecular dynamics simulations. The potential in rubredoxin is greater than expected from linear response theory, which implies it is a better Electron acceptor than a redox site analog in a solvent with a dielectric constant equivalent to that of the Protein. In addition, the potential in rubredoxin is described well by a permanent potential plus a linear response component. This permanent potential allows the Protein matrix to create a favorable driving force with a low activation barrier for accepting Electrons. The results here also suggest that the reduction potential of rubredoxin is determined mainly by the backbone and not the side chains, and that the redox site charge of rubredoxin may help to direct its folding.

  • Computational studies of redox potentials of Electron Transfer Proteins
    Simulation and theory of electrostatic interactions in solution, 1999
    Co-Authors: Toshiko Ichiye
    Abstract:

    The redox potential of an Electron Transfer Protein is an essential property because it determines the driving force in an Electron Transfer reaction. The Protein itself is an important determining factor since the redox potentials of analogs and Proteins with the same metal site or of different Proteins with the same metal site may differ by on the order of 1V. On the other hand, homologous Proteins with the same redox site may have identical redox potentials or may differ by up to 500 mV, although the larger differences generally correspond to significant differences in the backbone fold. Different computational methods for understanding where these differences arise from will be discussed. Particular attention will be placed on how our knowledge of Protein structure function relationships influences the techniques that are used.

Rita Bernhardt - One of the best experts on this subject based on the ideXlab platform.

  • Structural and Thermodynamic Characterization of the Adrenodoxin-Like Domain of the Electron-Transfer Protein Etp1 from Schizosaccharomyces Pombe.
    Journal of Inorganic Biochemistry, 2011
    Co-Authors: Jürgen Müller, Frank Hannemann, Burkhard Schiffler, Kerstin Maria Ewen, Reinhard Kappl, Udo Heinemann, Rita Bernhardt
    Abstract:

    The Protein Etp1 of Schizosaccharomyces pombe consists of an amino-terminal COX15-like domain and a carboxy-terminal ferredoxin-like domain, Etp1(fd), which is cleaved off after mitochondrial import. The physiological function of Etp1(fd) is supposed to lie in the participation in the assembly of iron-sulfur clusters and the synthesis of heme A. In addition, the Protein was shown to be the first microbial ferredoxin being able to support Electron Transfer in mitochondrial steroid hydroxylating cytochrome P450 systems in vivo and in vitro, replacing thereby the native redox partner, adrenodoxin. Despite a sequence similarity of 39% and the fact that fission yeast is a mesophilic organism, thermodynamic studies revealed that Etp1(fd) has a melting temperature more than 20°C higher than adrenodoxin. The three-dimensional structure of Etp1(fd) has been determined by crystallography. To the best of our knowledge it represents the first three-dimensional structure of a yeast ferredoxin. The structure-based sequence alignment of Etp1(fd) with adrenodoxin yields a rational explanation for their observed mutual exchangeability in the cytochrome P450 system. Analysis of the Electron exchange with the S. pombe redox partner Arh1 revealed differences between Etp1(fd) and adrenodoxin, which might be linked to their different physiological functions in the mitochondria of mammals and yeast.

  • the dipole moment of the Electron carrier adrenodoxin is not critical for redox partner interaction and Electron Transfer
    Journal of Inorganic Biochemistry, 2009
    Co-Authors: Frank Hannemann, Udo Heinemann, Arnaud Guyot, Andy Zollner, Jurgen J Muller, Rita Bernhardt
    Abstract:

    Dipole moments of Proteins arise from helical dipoles, hydrogen bond networks and charged groups at the Protein surface. High Protein dipole moments were suggested to contribute to the electrostatic steering between redox partners in Electron transport chains of respiration, photosynthesis and steroid biosynthesis, although so far experimental evidence for this hypothesis was missing. In order to probe this assumption, we changed the dipole moment of the Electron Transfer Protein adrenodoxin and investigated the influence of this on Protein-Protein interactions and Electron Transfer. In bovine adrenodoxin, the [2Fe-2S] ferredoxin of the adrenal glands, a dipole moment of 803 Debye was calculated for a full-length adrenodoxin model based on the Adx(4-108) and the wild type adrenodoxin crystal structures. Large distances and asymmetric distribution of the charged residues in the molecule mainly determine the observed high value. In order to analyse the influence of the resulting inhomogeneous electric field on the biological function of this Electron carrier the molecular dipole moment was systematically changed. Five recombinant adrenodoxin mutants with successively reduced dipole moment (from 600 to 200 Debye) were analysed for their redox properties, their binding affinities to the redox partner Proteins and for their function during Electron Transfer-dependent steroid hydroxylation. None of the mutants, not even the quadruple mutant K6E/K22Q/K24Q/K98E with a dipole moment reduced by about 70% showed significant changes in the Protein function as compared with the unmodified adrenodoxin demonstrating that neither the formation of the transient complex nor the biological activity of the Electron Transfer chain of the endocrine glands was affected. This is the first experimental evidence that the high dipole moment observed in Electron Transfer Proteins is not involved in electrostatic steering among the Proteins in the redox chain.

  • Functional expression of human mitochondrial CYP11B2 in fission yeast and identification of a new internal Electron Transfer Protein, etp1
    Biochemistry, 2002
    Co-Authors: Matthias Bureik, Burkhard Schiffler, Yasushi Hiraoka, Frank Vogel, Rita Bernhardt
    Abstract:

    Mitochondrial cytochrome P450 enzymes play a crucial role in the steroid biosynthesis in human adrenals, catalyzing regio- and stereospecific hydroxylations. In search of a new model system for the study of these enzymes, we expressed the human CYP11B2 (aldosterone synthase, P450aldo) in fission yeast Schizosaccharomyces pombe. Analysis of the subcellular localization of the P450 enzyme by Western blot analysis, fluorescence microscopy, and Electron microscopy demonstrated that the mitochondrial localization signal of the human Protein is functional in S. pombe. The transformed yeasts show the inducible ability to convert in vivo considerable amounts of 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone, and aldosterone, respectively. Although in mammalian cells, mitochondrial steroid hydroxylases depend for their activity on an Electron transport chain that consists of two Proteins, adrenodoxin and adrenodoxin reductase, no coexpression of these Proteins i...

Salvatore Cannistraro - One of the best experts on this subject based on the ideXlab platform.

  • Optical investigation of the Electron Transfer Protein azurin–gold nanoparticle system
    Biophysical Chemistry, 2009
    Co-Authors: Ines Delfino, Salvatore Cannistraro
    Abstract:

    The hybrid system obtained by conjugating the Protein azurin, which is a very stable and well-described Protein showing a unique interplay among its Electron Transfer and optical properties, with 20-nm sized gold nanoparticles has been investigated. Binding of azurin molecules to gold nanoparticle surface results in the red shift of the nanoparticle resonance plasmon band and in the quenching of the azurin single tryptophan fluorescence signal. These findings together with the estimate of the hydrodynamic radius of the composite, obtained by means of Dynamic Light Scattering, are consistent with the formation of a monolayer of Protein molecules, with preserved natural folding, on nanoparticle surface. The fluorescence quenching of azurin bound molecules is explained by an energy Transfer from Protein to metal surface and it is discussed in terms of the involvement of the Az Electron Transfer route in the interaction of the Protein with the nanoparticle.

  • optical investigation of the Electron Transfer Protein azurin gold nanoparticle system
    Biophysical Chemistry, 2009
    Co-Authors: Ines Delfino, Salvatore Cannistraro
    Abstract:

    The hybrid system obtained by conjugating the Protein azurin, which is a very stable and well-described Protein showing a unique interplay among its Electron Transfer and optical properties, with 20-nm sized gold nanoparticles has been investigated. Binding of azurin molecules to gold nanoparticle surface results in the red shift of the nanoparticle resonance plasmon band and in the quenching of the azurin single tryptophan fluorescence signal. These findings together with the estimate of the hydrodynamic radius of the composite, obtained by means of Dynamic Light Scattering, are consistent with the formation of a monolayer of Protein molecules, with preserved natural folding, on nanoparticle surface. The fluorescence quenching of azurin bound molecules is explained by an energy Transfer from Protein to metal surface and it is discussed in terms of the involvement of the Az Electron Transfer route in the interaction of the Protein with the nanoparticle.

Ines Delfino - One of the best experts on this subject based on the ideXlab platform.

  • Optical investigation of the Electron Transfer Protein azurin–gold nanoparticle system
    Biophysical Chemistry, 2009
    Co-Authors: Ines Delfino, Salvatore Cannistraro
    Abstract:

    The hybrid system obtained by conjugating the Protein azurin, which is a very stable and well-described Protein showing a unique interplay among its Electron Transfer and optical properties, with 20-nm sized gold nanoparticles has been investigated. Binding of azurin molecules to gold nanoparticle surface results in the red shift of the nanoparticle resonance plasmon band and in the quenching of the azurin single tryptophan fluorescence signal. These findings together with the estimate of the hydrodynamic radius of the composite, obtained by means of Dynamic Light Scattering, are consistent with the formation of a monolayer of Protein molecules, with preserved natural folding, on nanoparticle surface. The fluorescence quenching of azurin bound molecules is explained by an energy Transfer from Protein to metal surface and it is discussed in terms of the involvement of the Az Electron Transfer route in the interaction of the Protein with the nanoparticle.

  • optical investigation of the Electron Transfer Protein azurin gold nanoparticle system
    Biophysical Chemistry, 2009
    Co-Authors: Ines Delfino, Salvatore Cannistraro
    Abstract:

    The hybrid system obtained by conjugating the Protein azurin, which is a very stable and well-described Protein showing a unique interplay among its Electron Transfer and optical properties, with 20-nm sized gold nanoparticles has been investigated. Binding of azurin molecules to gold nanoparticle surface results in the red shift of the nanoparticle resonance plasmon band and in the quenching of the azurin single tryptophan fluorescence signal. These findings together with the estimate of the hydrodynamic radius of the composite, obtained by means of Dynamic Light Scattering, are consistent with the formation of a monolayer of Protein molecules, with preserved natural folding, on nanoparticle surface. The fluorescence quenching of azurin bound molecules is explained by an energy Transfer from Protein to metal surface and it is discussed in terms of the involvement of the Az Electron Transfer route in the interaction of the Protein with the nanoparticle.

Ivano Bertini - One of the best experts on this subject based on the ideXlab platform.

  • Cytochrome c and SDS: a molten globule Protein with altered axial ligation.
    Journal of Molecular Biology, 2004
    Co-Authors: Ivano Bertini, Paola Turano, Paul R Vasos, Arnaud Bondon, Soizic Chevance, Gérard Simonneaux
    Abstract:

    Saccharomices cerevisiae (yeast iso-1) cytochrome c has been investigated in the presence of 100 mM SDS in order to simulate the interaction of cytochrome c with membrane. Under these circumstances, a high spin species with detached methionine axial ligand is observed through NMR, in analogy to findings on the horse heart Protein. However, at variance with the latter system, for the yeast Protein also a low spin species is detected, which appears to be present with a concentration of about 40% with respect to that of the high spin species. The R(1), R(2), [1H]-15N NOE of backbone amides which are not affected by paramagnetism are homogeneous and allow a simultaneous analysis of the data for the two species. The result is that the rotational correlation time is larger than in water and larger than expected on the basis of viscosity of the SDS-containing solution. This finding suggests interactions of cytochrome c with SDS. Furthermore, it appears that there is subnanosecond backbone mobility, which also accounts for the decreased intensity of NOE cross-peaks and may be associated with equilibria between helical and random coil structure. The dynamic behavior appears to be a common feature of the high spin and low spin species and is consistent with the presence of a molten globule state. The molten globule nature of the Protein could account for the presence of the different axial coordination of the heme iron. Such findings are meaningful with respect to the physiology of cytochrome c as Electron Transfer Protein and as promoter of apoptosis.

  • solution structure of oxidized cytochrome c6 from the green alga monoraphidium braunii
    Biochemistry, 1998
    Co-Authors: Lucia Banci, Dionysios Koulougliotis, José A. Navarro, Ivano Bertini, Miguel A. De La Rosa, Olaf Walter
    Abstract:

    Cytochrome c6 from Monoraphidium braunii, an 89-amino acid Electron Transfer Protein, has been investigated by NMR in solution, in its oxidized form, at pH 7 and 300 K. By using a combination of COSY, TOCSY, and NOESY experiments, 84% of the proton resonances have been assigned. A total of 1668 experimental NOE constraints, 1109 of which were meaningful, together with 288 pseudocontact shifts, have been used to determine the structure in solution. This is represented as a family of 40 structures which have been energy minimized. The rmsd values with respect to the mean structure are 0.57 ± 0.08 and 0.94 ± 0.09 A for the backbone and heavy atoms, respectively. The structure has been found to be very similar to that of the reduced form, except for a rearrangement in propionate 7, a feature which has been observed in all c-type cytochromes investigated so far. Such a feature could be relevant for the efficiency of the Electron Transfer pathway with either the oxidizing or the reducing partners. Other differe...

  • nmr characterization and solution structure determination of the oxidized cytochrome c7 from desulfuromonas acetoxidans
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Lucia Banci, Pornthep Sompornpisut, Mireille Bruschi, Ivano Bertini, Paola Turano
    Abstract:

    The solution structure of the three-heme Electron Transfer Protein cytochrome c7 from Desulfuromonas acetoxidans is reported. The determination of the structure is obtained through NMR spectroscopy on the fully oxidized, paramagnetic form. The richness of structural motifs and the presence of three prosthetic groups in a Protein of 68 residues is discussed in comparison with the four-heme cytochromes c3 already characterized through x-ray crystallography. In particular, the orientation of the three hemes present in cytochrome c7 is similar to that of three out of four hemes of cytochromes c3. The reduction potentials of the individual hemes, which have been obtained through the sequence-specific assignment of the heme resonances, are discussed with respect to the properties of the Protein matrix. This information is relevant for any attempt to understand the Electron Transfer pathway.