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James D. Satterlee - One of the best experts on this subject based on the ideXlab platform.
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Proton NMR assignments and magnetic axes orientations for wild-type yeast iso-1-Ferricytochrome c free in solution and bound to cytochrome c peroxidase.
Biochemistry, 1997Co-Authors: Steven F. Sukits, James E. Erman, James D. SatterleeAbstract:Extensive proton hyperfine-shifted resonance assignments have been made for wild-type yeast iso-1-Ferricytochrome c when it is free in solution and when it is noncovalently complexed to resting state cytochrome c peroxidase. Complete heme proton resonance assignments were made for free iso-1-Ferricytochrome c, while for CcP-complexed iso-1-Ferricytochrome c, 70% of heme proton assignments were made. Additional proton resonance assignments were made for hyperfine-shifted protons of amino acids near the heme. These assignments allowed identification of the most extensive set of complex-induced proton shifts yet reported for CcP/cytochrome c complexes. Several purely dipolar-shifted resonances from heme vicinity amino acid protons were also assigned in both free and complexed iso-1-ferricyt c. Both sets of resonance assignments allowed assessment of the origin of proton complex-induced shifts. Using the assigned dipolar-shifted proton resonances as a basis, the orientations of the principal axis systems of the paramagnetic susceptibility tensors for free and cytochrome c peroxidase-bound iso-1-Ferricytochrome c were elucidated. The results indicated that the iso-1-Ferricytochrome c magnetic axis system orientation shifts significantly upon complex formation. The direction of the complex-induced shifts for heme proton resonances is largely accounted for by the magnetic anisotropy changes. However, analysis of heme complex-induced shifts also reveals local changes in magnetic environment for two heme substituents, presumably through a specific structure change.
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1H NMR Evaluation of Yeast Isoenzyme-1 Ferricytochrome c Equilibrium Exchange Dynamics in Noncovalent Complexes with Two Forms of Yeast Cytochrome c Peroxidase
Journal of the American Chemical Society, 1994Co-Authors: James E. Erman, James D. SatterleeAbstract:Solutions consisting of 2:1 mole excess of yeast isozyme-1 (iso-1) Ferricytochrome c in combination with either resting-state cytochrome c peroxidase (CcP) or cyanide-ligated cytochrome c peroxidase (CcPCN) consist of equimolar concentrations of the noncovalent 1:1 complex (peroxidase/Ferricytochrome c) and Ferricytochrome c free in solution. This work reveals that in these solutions the Ferricytochrome c is in dynamic exchange between the peroxidase bound (b) environment and the free (f) environment. The exchange is in the slow-intermediate regime on the NMR time scale because solutions of these mixtures simultaneously display an iso-1 Ferricytochrome c heme 3-CH 3 resonance from Ferricytochrome c molecules in both free and bound environments
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Studies of protein-protein association between yeast cytochrome c peroxidase and yeast iso-1 Ferricytochrome c by hydrogen-deuterium exchange labeling and proton NMR spectroscopy.
Biochemistry, 1994Co-Authors: James E. Erman, James D. SatterleeAbstract:Hydrogen-deuterium (H-D) exchange labeling and proton NMR have been applied to study the protein-protein association between cytochrome c peroxidase (CcP) and yeast iso-1 Ferricytochrome c. Specifically, the exchange behavior of individual backbone amide protons of yeast iso-1 Ferricytochrome c in both CcP-bound (i.e., complexed) and free (i.e., never in the complex) forms has been investigated and used in an attempt to map the binding site of CcP on yeast iso-1 Ferricytochrome c when the noncovalent complex was formed in very low salt solution. The exchange rates of certain amino acid amide protons were significantly slowed down, by up to 40-fold, in the complex compared to the free form. The protected regions on iso-1 Ferricytochrome c include parts of the 10's helix and the 70's helix surrounding the cytochrome c heme solvent-exposed edge (the so-called "front side" of iso-1 cytochrome c). These regions are very similar to the cytochrome c peroxidase binding interface on iso-1 Ferricytochrome c that has been defined by X-ray crystallographic data. This further supports the direct involvement of the front side of iso-1 cytochrome c in binding with cytochrome c peroxidase. The results from our H-D exchange experiments also indicated that the amide proton exchange rates of Trp59, Asp60, and part of the 90's helix, all of which are located on the opposite side (the "back" side) of Ferricytochrome c from the heme solvent-exposed edge, are also retarded upon complex formation.
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Proton NMR study of a non-covalent complex formed between cytochrome c peroxidase-cyanide and tuna Ferricytochrome c
Magnetic Resonance in Chemistry, 1993Co-Authors: James D. Satterlee, James E. ErmanAbstract:A non-covalent complex of cyanide-ligated cytochrome c peroxidase (CcPCN) with tuna Ferricytochrome c, formed in low-concentration KNO3 solutions, was studied by proton NMR spectroscopy. Complex formation affects the Ferricytochrome c spectrum similarly to the spectral changes previously observed for the corresponding complex formed using resting state cytochrome c peroxidase. The CcPCN-tuna Ferricytochrome c complex studied here is also similar to the previously studied CcPCN complexes with both horse and yeast iso-1 Ferricytochromes c. For this complex both proteins are in the low-spin iron(III) form, which make them both paramagnetic and causes severe overlap in the proton hyperfine resonance shift region. Two-dimensional proton NMR spectroscopy has been used to resolve this overlap and make proton resonance assignments. These results complete the set of experiments carried out on the complexes of CcPCN with three species of Ferricytochrome c (hores, yeast and tuna) and reveal that as for the similar complexes of these Ferricytochromes c with the resting-state, high-spin form of the peroxidase, the tuna results closely follow those of the horse Ferricytochrome c complex. These results also extend preliminary work that revealed for the first time that cytochrome c binding was reflected in hyperfine resonance shifts of protons in the peroxidase heme pocket.
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Proton NMR comparison of noncovalent and covalently cross-linked complexes of cytochrome c peroxidase with horse, tuna, and yeast Ferricytochromes c.
Biochemistry, 1992Co-Authors: Susan J. Moench, James E. Erman, Stamatia Chroni, Bih-show Lou, James D. SatterleeAbstract:Proton NMR spectroscopy at 500 and 361 MHz has been used to characterize the noncovalent or electrostatic complexes of yeast cytochrome c peroxidase (CcP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 Ferricytochromes c and the covalently cross-linked complexes of cytochrome c peroxidase with horse and yeast isozyme-1 Ferricytochromes c. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled greater than 90% of complex formed) was found to be 1:1 in all cases. These studies have elucidated significant differences in the proton NMR absorption spectra and the one-dimensional nuclear Overhauser effect difference spectra of the complexes, depending on the specific species of Ferricytochrome c incorporated. In particular, the results indicate that the noncovalent complexes formed between CcP and physiological redox partners (yeast isozyme-1 or yeast isozyme-2 Ferricytochromes c) are distinctly different from the noncovalent complexes formed between CcP and Ferricytochromes c from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochrome c peroxidase with horse Ferricytochrome c, and cytochrome c peroxidase with yeast isozyme-1 Ferricytochrome c further emphasize such cytochrome c-dependent differences, with only the covalently cross-linked complex of physiological redox partners (cytochrome c peroxidase/yeast isozyme-1) displaying NMR spectra characteristic of a heterogeneous mixture of different 1:1 complexes. Finally, one-dimensional nuclear Overhauser effect experiments have proven valuable in selectively and efficiently probing the protein-protein interface in these complexes, including the environment around the cytochrome c heme 3-methyl group and Phe-82.
A G Mauk - One of the best experts on this subject based on the ideXlab platform.
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analysis of the bimolecular reduction of Ferricytochrome c by ferrocytochrome b5 through mutagenesis and molecular modelling
Biochimie, 1994Co-Authors: J G Guillemette, Lindsay D Eltis, P D Barker, Michael Smith, Gary D Brayer, A G MaukAbstract:Site-directed mutagenesis has been used to produce variants of cytochrome c in which selected structural or functional properties of this protein are altered that have been implicated previously in contributing to the rate at which Ferricytochrome c is reduced by ferrocytochrome b5. In total, 18 variants have been studied by kinetics and electrochemical methods to assess the contributions of thermodynamic driving force, surface charge and hydrophobic interactions, and redox-linked structural reorganization of the protein to the rate of electron transfer between these two proteins under conditions where the reaction is bimolecular. While some variants (those at position-38) appear to affect primarily the driving force of the reaction, others appear to influence the rearrangement barrier to electron transfer (those at positions-67 and -52) while the interface between electron donor and acceptor centers is the principal effect of substitutions for a conserved aromatic heme contact residue at the surface of the protein (position-82). Interpretation of these results has been facilitated through the use of energy minimization calculations to refine the hypothetical models previously suggested for the cytochrome c- cytochrome b5 precursor complex on the basis of Brownian dynamics simulations of the bimolecular encounter event.
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effects of charged amino acid mutations on the bimolecular kinetics of reduction of yeast iso 1 Ferricytochrome c by bovine ferrocytochrome b5
Biochemistry, 1993Co-Authors: Scott H Northrup, Lindsay D Eltis, P D Barker, J G Guillemette, Kathryn A Thomasson, C M Miller, S C Inglis, A G MaukAbstract:The reduction of wild-type yeast iso-1-Ferricytochrome c (ycytc) and several mutants by trypsin-solubilized bovine liver ferrocytochrome b5 (cytb5) has been studied under conditions in which the electron-transfer reaction is bimolecular. The effect of electrostatic charge modifications and steric changes on the kinetics has been determined by experimental and theoretical observations of the electron-transfer rates of ycytc mutants K79A, K'72A, K79A/K'72A, and R38A (K' is used to signify trimethyllysine (Tml)). A structurally robust Brownian dynamics (BD) method simulating diffusional docking and electron transfer was employed to predict the mutation effect on the rate constants. A realistic model of the electron-transfer event embodied in an intrinsic unimolecular rate constant is used which varies exponentially with donor-acceptor distance. The BD method quantitatively predicts rate constants over a considerable range of ionic strengths. Semiquantitative agreement is obtained in predicting the perturbing influence of the mutations on the rate constants. Both the experimentally observed rate constants and those predicted by BD descend in the following order: native ycytc > K79A > K'72A > K79A/K'72A. Variant R38A was studied at a different ionic strength than this series of mutations, and the theory agreed with experiment in predicting a smaller rate constant for the mutant. In all cases the predicted effect of mutation was in the correct direction, but not as large as that observed. The BD simulations predict that the two proteins dock through essentially a single domain, with a distance of closest approach of the two heme groups in rigid body docking typically around 12 A. Two predominant classes of complexes were calculated, the most frequent involving the quartet of cytb5/ycytc interactions, Glu48-Arg13, Glu56-Lys87, Asp60-Lys86, and heme-Tml72, having an average electrostatic energy of -13.0 kcal/mol. The second most important complexes were of the type previously postulated (Salemme, 1976; Mauk et al., 1986; Rodgers et al., 1988) with interactions Glu44-Lys27, Glu48-Arg13, Asp60-Tml72, and heme-Lys79 and having an energy of -6.4 kcal/mol. The ionic strength dependence of the bimolecular reaction rate was well reproduced using a discontinuous dielectric model, but poorly so for a uniform dielectric model.
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reduction of horse heart Ferricytochrome c by bovine liver ferrocytochrome b5 experimental and theoretical analysis
Biochemistry, 1991Co-Authors: Lindsay D Eltis, R G Herbert, P D Barker, A G Mauk, Scott H NorthrupAbstract:The reduction of horse heart Ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for Ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of Ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.
Scott H Northrup - One of the best experts on this subject based on the ideXlab platform.
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effects of charged amino acid mutations on the bimolecular kinetics of reduction of yeast iso 1 Ferricytochrome c by bovine ferrocytochrome b5
Biochemistry, 1993Co-Authors: Scott H Northrup, Lindsay D Eltis, P D Barker, J G Guillemette, Kathryn A Thomasson, C M Miller, S C Inglis, A G MaukAbstract:The reduction of wild-type yeast iso-1-Ferricytochrome c (ycytc) and several mutants by trypsin-solubilized bovine liver ferrocytochrome b5 (cytb5) has been studied under conditions in which the electron-transfer reaction is bimolecular. The effect of electrostatic charge modifications and steric changes on the kinetics has been determined by experimental and theoretical observations of the electron-transfer rates of ycytc mutants K79A, K'72A, K79A/K'72A, and R38A (K' is used to signify trimethyllysine (Tml)). A structurally robust Brownian dynamics (BD) method simulating diffusional docking and electron transfer was employed to predict the mutation effect on the rate constants. A realistic model of the electron-transfer event embodied in an intrinsic unimolecular rate constant is used which varies exponentially with donor-acceptor distance. The BD method quantitatively predicts rate constants over a considerable range of ionic strengths. Semiquantitative agreement is obtained in predicting the perturbing influence of the mutations on the rate constants. Both the experimentally observed rate constants and those predicted by BD descend in the following order: native ycytc > K79A > K'72A > K79A/K'72A. Variant R38A was studied at a different ionic strength than this series of mutations, and the theory agreed with experiment in predicting a smaller rate constant for the mutant. In all cases the predicted effect of mutation was in the correct direction, but not as large as that observed. The BD simulations predict that the two proteins dock through essentially a single domain, with a distance of closest approach of the two heme groups in rigid body docking typically around 12 A. Two predominant classes of complexes were calculated, the most frequent involving the quartet of cytb5/ycytc interactions, Glu48-Arg13, Glu56-Lys87, Asp60-Lys86, and heme-Tml72, having an average electrostatic energy of -13.0 kcal/mol. The second most important complexes were of the type previously postulated (Salemme, 1976; Mauk et al., 1986; Rodgers et al., 1988) with interactions Glu44-Lys27, Glu48-Arg13, Asp60-Tml72, and heme-Lys79 and having an energy of -6.4 kcal/mol. The ionic strength dependence of the bimolecular reaction rate was well reproduced using a discontinuous dielectric model, but poorly so for a uniform dielectric model.
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reduction of horse heart Ferricytochrome c by bovine liver ferrocytochrome b5 experimental and theoretical analysis
Biochemistry, 1991Co-Authors: Lindsay D Eltis, R G Herbert, P D Barker, A G Mauk, Scott H NorthrupAbstract:The reduction of horse heart Ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for Ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of Ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.
Lindsay D Eltis - One of the best experts on this subject based on the ideXlab platform.
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analysis of the bimolecular reduction of Ferricytochrome c by ferrocytochrome b5 through mutagenesis and molecular modelling
Biochimie, 1994Co-Authors: J G Guillemette, Lindsay D Eltis, P D Barker, Michael Smith, Gary D Brayer, A G MaukAbstract:Site-directed mutagenesis has been used to produce variants of cytochrome c in which selected structural or functional properties of this protein are altered that have been implicated previously in contributing to the rate at which Ferricytochrome c is reduced by ferrocytochrome b5. In total, 18 variants have been studied by kinetics and electrochemical methods to assess the contributions of thermodynamic driving force, surface charge and hydrophobic interactions, and redox-linked structural reorganization of the protein to the rate of electron transfer between these two proteins under conditions where the reaction is bimolecular. While some variants (those at position-38) appear to affect primarily the driving force of the reaction, others appear to influence the rearrangement barrier to electron transfer (those at positions-67 and -52) while the interface between electron donor and acceptor centers is the principal effect of substitutions for a conserved aromatic heme contact residue at the surface of the protein (position-82). Interpretation of these results has been facilitated through the use of energy minimization calculations to refine the hypothetical models previously suggested for the cytochrome c- cytochrome b5 precursor complex on the basis of Brownian dynamics simulations of the bimolecular encounter event.
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effects of charged amino acid mutations on the bimolecular kinetics of reduction of yeast iso 1 Ferricytochrome c by bovine ferrocytochrome b5
Biochemistry, 1993Co-Authors: Scott H Northrup, Lindsay D Eltis, P D Barker, J G Guillemette, Kathryn A Thomasson, C M Miller, S C Inglis, A G MaukAbstract:The reduction of wild-type yeast iso-1-Ferricytochrome c (ycytc) and several mutants by trypsin-solubilized bovine liver ferrocytochrome b5 (cytb5) has been studied under conditions in which the electron-transfer reaction is bimolecular. The effect of electrostatic charge modifications and steric changes on the kinetics has been determined by experimental and theoretical observations of the electron-transfer rates of ycytc mutants K79A, K'72A, K79A/K'72A, and R38A (K' is used to signify trimethyllysine (Tml)). A structurally robust Brownian dynamics (BD) method simulating diffusional docking and electron transfer was employed to predict the mutation effect on the rate constants. A realistic model of the electron-transfer event embodied in an intrinsic unimolecular rate constant is used which varies exponentially with donor-acceptor distance. The BD method quantitatively predicts rate constants over a considerable range of ionic strengths. Semiquantitative agreement is obtained in predicting the perturbing influence of the mutations on the rate constants. Both the experimentally observed rate constants and those predicted by BD descend in the following order: native ycytc > K79A > K'72A > K79A/K'72A. Variant R38A was studied at a different ionic strength than this series of mutations, and the theory agreed with experiment in predicting a smaller rate constant for the mutant. In all cases the predicted effect of mutation was in the correct direction, but not as large as that observed. The BD simulations predict that the two proteins dock through essentially a single domain, with a distance of closest approach of the two heme groups in rigid body docking typically around 12 A. Two predominant classes of complexes were calculated, the most frequent involving the quartet of cytb5/ycytc interactions, Glu48-Arg13, Glu56-Lys87, Asp60-Lys86, and heme-Tml72, having an average electrostatic energy of -13.0 kcal/mol. The second most important complexes were of the type previously postulated (Salemme, 1976; Mauk et al., 1986; Rodgers et al., 1988) with interactions Glu44-Lys27, Glu48-Arg13, Asp60-Tml72, and heme-Lys79 and having an energy of -6.4 kcal/mol. The ionic strength dependence of the bimolecular reaction rate was well reproduced using a discontinuous dielectric model, but poorly so for a uniform dielectric model.
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reduction of horse heart Ferricytochrome c by bovine liver ferrocytochrome b5 experimental and theoretical analysis
Biochemistry, 1991Co-Authors: Lindsay D Eltis, R G Herbert, P D Barker, A G Mauk, Scott H NorthrupAbstract:The reduction of horse heart Ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for Ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of Ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.
James E. Erman - One of the best experts on this subject based on the ideXlab platform.
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Proton NMR assignments and magnetic axes orientations for wild-type yeast iso-1-Ferricytochrome c free in solution and bound to cytochrome c peroxidase.
Biochemistry, 1997Co-Authors: Steven F. Sukits, James E. Erman, James D. SatterleeAbstract:Extensive proton hyperfine-shifted resonance assignments have been made for wild-type yeast iso-1-Ferricytochrome c when it is free in solution and when it is noncovalently complexed to resting state cytochrome c peroxidase. Complete heme proton resonance assignments were made for free iso-1-Ferricytochrome c, while for CcP-complexed iso-1-Ferricytochrome c, 70% of heme proton assignments were made. Additional proton resonance assignments were made for hyperfine-shifted protons of amino acids near the heme. These assignments allowed identification of the most extensive set of complex-induced proton shifts yet reported for CcP/cytochrome c complexes. Several purely dipolar-shifted resonances from heme vicinity amino acid protons were also assigned in both free and complexed iso-1-ferricyt c. Both sets of resonance assignments allowed assessment of the origin of proton complex-induced shifts. Using the assigned dipolar-shifted proton resonances as a basis, the orientations of the principal axis systems of the paramagnetic susceptibility tensors for free and cytochrome c peroxidase-bound iso-1-Ferricytochrome c were elucidated. The results indicated that the iso-1-Ferricytochrome c magnetic axis system orientation shifts significantly upon complex formation. The direction of the complex-induced shifts for heme proton resonances is largely accounted for by the magnetic anisotropy changes. However, analysis of heme complex-induced shifts also reveals local changes in magnetic environment for two heme substituents, presumably through a specific structure change.
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1H NMR Evaluation of Yeast Isoenzyme-1 Ferricytochrome c Equilibrium Exchange Dynamics in Noncovalent Complexes with Two Forms of Yeast Cytochrome c Peroxidase
Journal of the American Chemical Society, 1994Co-Authors: James E. Erman, James D. SatterleeAbstract:Solutions consisting of 2:1 mole excess of yeast isozyme-1 (iso-1) Ferricytochrome c in combination with either resting-state cytochrome c peroxidase (CcP) or cyanide-ligated cytochrome c peroxidase (CcPCN) consist of equimolar concentrations of the noncovalent 1:1 complex (peroxidase/Ferricytochrome c) and Ferricytochrome c free in solution. This work reveals that in these solutions the Ferricytochrome c is in dynamic exchange between the peroxidase bound (b) environment and the free (f) environment. The exchange is in the slow-intermediate regime on the NMR time scale because solutions of these mixtures simultaneously display an iso-1 Ferricytochrome c heme 3-CH 3 resonance from Ferricytochrome c molecules in both free and bound environments
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Studies of protein-protein association between yeast cytochrome c peroxidase and yeast iso-1 Ferricytochrome c by hydrogen-deuterium exchange labeling and proton NMR spectroscopy.
Biochemistry, 1994Co-Authors: James E. Erman, James D. SatterleeAbstract:Hydrogen-deuterium (H-D) exchange labeling and proton NMR have been applied to study the protein-protein association between cytochrome c peroxidase (CcP) and yeast iso-1 Ferricytochrome c. Specifically, the exchange behavior of individual backbone amide protons of yeast iso-1 Ferricytochrome c in both CcP-bound (i.e., complexed) and free (i.e., never in the complex) forms has been investigated and used in an attempt to map the binding site of CcP on yeast iso-1 Ferricytochrome c when the noncovalent complex was formed in very low salt solution. The exchange rates of certain amino acid amide protons were significantly slowed down, by up to 40-fold, in the complex compared to the free form. The protected regions on iso-1 Ferricytochrome c include parts of the 10's helix and the 70's helix surrounding the cytochrome c heme solvent-exposed edge (the so-called "front side" of iso-1 cytochrome c). These regions are very similar to the cytochrome c peroxidase binding interface on iso-1 Ferricytochrome c that has been defined by X-ray crystallographic data. This further supports the direct involvement of the front side of iso-1 cytochrome c in binding with cytochrome c peroxidase. The results from our H-D exchange experiments also indicated that the amide proton exchange rates of Trp59, Asp60, and part of the 90's helix, all of which are located on the opposite side (the "back" side) of Ferricytochrome c from the heme solvent-exposed edge, are also retarded upon complex formation.
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Proton NMR study of a non-covalent complex formed between cytochrome c peroxidase-cyanide and tuna Ferricytochrome c
Magnetic Resonance in Chemistry, 1993Co-Authors: James D. Satterlee, James E. ErmanAbstract:A non-covalent complex of cyanide-ligated cytochrome c peroxidase (CcPCN) with tuna Ferricytochrome c, formed in low-concentration KNO3 solutions, was studied by proton NMR spectroscopy. Complex formation affects the Ferricytochrome c spectrum similarly to the spectral changes previously observed for the corresponding complex formed using resting state cytochrome c peroxidase. The CcPCN-tuna Ferricytochrome c complex studied here is also similar to the previously studied CcPCN complexes with both horse and yeast iso-1 Ferricytochromes c. For this complex both proteins are in the low-spin iron(III) form, which make them both paramagnetic and causes severe overlap in the proton hyperfine resonance shift region. Two-dimensional proton NMR spectroscopy has been used to resolve this overlap and make proton resonance assignments. These results complete the set of experiments carried out on the complexes of CcPCN with three species of Ferricytochrome c (hores, yeast and tuna) and reveal that as for the similar complexes of these Ferricytochromes c with the resting-state, high-spin form of the peroxidase, the tuna results closely follow those of the horse Ferricytochrome c complex. These results also extend preliminary work that revealed for the first time that cytochrome c binding was reflected in hyperfine resonance shifts of protons in the peroxidase heme pocket.
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Proton NMR comparison of noncovalent and covalently cross-linked complexes of cytochrome c peroxidase with horse, tuna, and yeast Ferricytochromes c.
Biochemistry, 1992Co-Authors: Susan J. Moench, James E. Erman, Stamatia Chroni, Bih-show Lou, James D. SatterleeAbstract:Proton NMR spectroscopy at 500 and 361 MHz has been used to characterize the noncovalent or electrostatic complexes of yeast cytochrome c peroxidase (CcP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 Ferricytochromes c and the covalently cross-linked complexes of cytochrome c peroxidase with horse and yeast isozyme-1 Ferricytochromes c. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled greater than 90% of complex formed) was found to be 1:1 in all cases. These studies have elucidated significant differences in the proton NMR absorption spectra and the one-dimensional nuclear Overhauser effect difference spectra of the complexes, depending on the specific species of Ferricytochrome c incorporated. In particular, the results indicate that the noncovalent complexes formed between CcP and physiological redox partners (yeast isozyme-1 or yeast isozyme-2 Ferricytochromes c) are distinctly different from the noncovalent complexes formed between CcP and Ferricytochromes c from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochrome c peroxidase with horse Ferricytochrome c, and cytochrome c peroxidase with yeast isozyme-1 Ferricytochrome c further emphasize such cytochrome c-dependent differences, with only the covalently cross-linked complex of physiological redox partners (cytochrome c peroxidase/yeast isozyme-1) displaying NMR spectra characteristic of a heterogeneous mixture of different 1:1 complexes. Finally, one-dimensional nuclear Overhauser effect experiments have proven valuable in selectively and efficiently probing the protein-protein interface in these complexes, including the environment around the cytochrome c heme 3-methyl group and Phe-82.