1,721,018 research outputs found
Thermodynamic Aspects of the Adsorption of Cytochrome c and Its Mutants on Kaolinite
No full textThe adsorption of native, wild-type and engineered cytochrome c on sodium-exchanged kaolinite was investigated by spectroscopic means. The variants of yeast cytochrome c were obtained replacing surface lysines in position 72, 73 and 79 with alanine residues. All proteins are strongly adsorbed onto kaolinite. In particular, the presence of the lysine residue in position 73 remarkably favors adsorption. A detailed characterization of the thermodynamic aspects of the adsorption process has been performed. Most notably, adsorbed cytochrome c maintains its moderate peroxidase activity against guaiacol. This investigation is prodromal to the exploitation of the catalytic activity of engineered cytochrome c immobilized on a polydisperse system
1H NMR of native and azide-inhibited laccase from Rhus vernicifera
No full textThe H NMR spectra of the fully oxidized Rhus vernicifera laccase and of its 1:1 and 2:1 azide adducts are reported for the first time.These spectra, which are the first so far reported for a multi copper oxidase, contain a number of broad hyperfine-shifted resonances in thehigh frequency region of the spectrum, which are attributed to the metal binding residues of the mononuclear T1 center. The differencesbetween the patterns of the hyperfine resonances of the free enzyme and its azide derivatives suggest that the alterations in the structuralproperties of the T3 site induced by the binding of the first azide molecule induce a limited alteration of the spin density distribution over1 the T1 copper ligands. Overall, these data demonstrate that H NMR can be fruitfully applied to characterize the electronic properties ofthe metal sites of blue oxidases at room temperature
Role of Met80 and Tyr67 in the Low-pH Conformational Equilibria ofCytochrome c
No full textThe low-pH conformational equilibria of ferricyeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, andM80A/Y67A variants were studied from pH 7 to 2 at low ionicstrength through electronic absorption, magnetic circulardichroism, and resonance Raman spectroscopies. For wild-typeycc, the protein structure, axial heme ligands, and spin state ofthe iron atom convert from the native folded His/Met low-spin(LS) form to a molten globule His/H2O high-spin (HS) formand a totally unfolded bis-aquo HS state, in a single cooperativetransition with an apparent pKa of ∼3.0. An analogouscooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pKa of ∼4.2, and by an equilibrium between a His/OH−-ligated LS and a His/H2O-ligated HS conformer, with a pKa of∼5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increasedstability of the molten globule state that is formed at higher pH values than the other species. These data show that removal ofthe axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pHconformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule andrenders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the threedimensionalstructure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues71−85 and 40−57
pH and Solvent H/D Isotope Effects on the Thermodynamics and Kinetics of Electron Transfer for Electrode-Immobilized Native and Urea-Unfolded Stellacyanin
No full textThe thermodynamics of Cu(II) to Cu(I) reduction and the kinetics of the electron transfer (ET) process for Rhus vernicifera stellacyanin (STC) immobilized on a decane-1-thiol coated gold
electrode have been measured through cyclic voltammetry at varying pH and temperature, in the presence of urea and in D2O. Immobilized STC undergoes a limited conformational change that
mainly results in an enhanced exposure of one or both copper binding histidines to solvent which slightly stabilizes the cupric state and increases histidine basicity. The large immobilization-induced increase in the pKa for the acid transition (from 4.5 to 6.3) makes this electrode-SAM-protein construct an attractive candidate as a biomolecular ET switch operating near neutral pH in molecular electronics. Such a potential interest is increased by the robustness of this interface against chemical unfolding as it undergoes only moderate changes in the reduction thermodynamics and in the ET rate in the presence of up to 8 M urea. The sensitivity of these parameters to solvent H/D isotope effects testifies the role of protein solvation as effector of the thermodynamics and kinetics of ET
Electrostatic effects on the thermodynamics of protonation of reduced plastocyanin
No full textThe L12E, L12K, Q88E, and Q88K variants of spinach plastocyanin have been electrochemically investigated. The effects of insertion of net charges near the metal site on the thermodynamics of protonation and detachment from the copper(I) ion of the His87 ligand have been evaluated. The mutation-induced changes in transition enthalpy cannot be explained by electrostatic considerations. The existence of enthalpy/entropy (H/S) compensation within the protein series indicates that solvent-reorganization effects control the differences in transition thermodynamics. Once these compensating contributions are factorized out, the resulting modest differences in transition enthalpies turn out to be those that can be expected on purely electrostatic grounds. Therefore, this work shows that the acid transition in cupredoxins involves a reorganization of the H-bonding network within the hydration sphere of the molecule in the proximity of the metal center that dominates the observed transition thermodynamics and masks the differences that are due to protein-based effects
Free energy of transition for the individual alkaline conformers of yeast iso-1-cytochrome c
No full textDirect protein electrochemistry was used to obtain the thermodynamic parameters of transition from the native (state III) to the alkaline (state IV) conformer for untrimethylated Saccharomyces cerevisiae iso-1-cytochrome c expressed in E. coli and its single and multiple lysine-depleted variants. In these variants, one or more of the lysine residues involved in axial Met substitution (Lys72, Lys73, and Lys79) was mutated to alanine. The aim of this work is to determine the thermodynamic affinity of each of the substituting lysines for the heme iron and evaluate the interplay of enthalpic and entropic factors. The equilibrium constants for the deprotonation reaction of Lys72, 73, and 79 were computed for the minimized MD average structures of the wild-type and mutated proteins, applying a modified Tanford-Kirkwood calculation. Solvent accessibility calculations for the substituting lysines in all variants were also performed. The transition enthalpy and entropy values within the protein series show a compensatory behavior, typical of a process involving extensive solvent reorganization effects. The experimental and theoretical data indicate that Lys72 most readily deprotonates and replaces M80 as the axial heme iron ligand, whereas Lys73 and Lys79 show comparably higher pK(a) values and larger transition free energies. A good correlation is found within the series between the lowest calculated Lys pK(a) value and the corresponding experimental pK(a) value, which can be interpreted as indicative of the deprotonating lysine itself acting as the triggering group for the conformational transition. The triple Lys to Ala mutant, in which no lysine residues are available for heme iron binding, features transition thermodynamics consistent with a hydroxide ion replacing the axial methionine ligand
Immobilized cytochrome c bound to cardiolipin exhibits peculiar oxidation state-dependent axial heme ligation and catalytically reduces dioxygen
No full textMitochondrial cytochrome c (cytc) plays an important role in programmed cell death upon binding to cardiolipin (CL), a negatively charged phospholipid of the inner mitochondrial membrane (IMM). Although this binding has been thoroughly investigated in solution, little is known on the nature and reactivity of the adduct (cytc–CL) immobilized at IMM. In this work, we have studied electrochemically cytc–CL immobilized on a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol. This construct would reproduce the motional restriction and the nonpolar environment experienced by cytc–CL at IMM. Surface-enhanced resonance Raman (SERR) studies allowed the axial heme iron ligands to be identified, which were found to be oxidation state dependent and differ from those of cytc–CL in solution. In particular, immobilized cytc–CL experiences an equilibrium between a low-spin (LS) 6c His/His and a high-spin (HS) 5c His/− coordination states. The former prevails in the oxidized and the latter in the reduced form. Axial coordination of the ferric heme thus differs from the (LS) 6c His/Lys and (LS) 6c His/OH– states observed in solution. Moreover, a relevant finding is that the immobilized ferrous cytc–CL is able to catalytically reduce dioxygen, likely to superoxide ion. These findings indicate that restriction of motional freedom due to interaction with the membrane is an additional factor playing in the mechanism of cytc unfolding and cytc-mediated peroxidation functional to the apoptosis cascade
Heterogeneous Electron Transfer of a Two-Centered Heme Protein: Redox and Electrocatalytic Properties of Surface-Immobilized Cytochrome c4
No full textThe recombinant di-heme cytochrome c4 from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature an hydroxide ion axially bound to the heme iron at the N- and C-terminal domain, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups facilitates analysis of the heterogeneous and intra-heme electron transfer for these two-centered systems in which the high- and low-potential heme are swept over in the bilobal protein framework. The voltammetric behavior of these species, which experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode, unequivocally shows that intra-heme electron transfer is activated only in the immobilized proteins, as proposed previously for the homologous species from Pseudomonas stutzeri. T-dependent kinetic measurements show that for both proteins the C-lobe faces the HOOC-terminated SAM-coated electrode at a distance of slightly more than 7 Å. The reduction thermodynamics for the native and mutated heme (measured for the first time for a di-heme cytochrome c) in the diffusing regime reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. Protein-electrode orientation and efficient intra-heme ET enable the His,OH--ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes an unprecedented two-centered heme-base biocatalytic interface to be exploited for “third-generation” amperometric biosensing
Axial ligation and polypeptide matrix effects on the reduction potential of heme proteins probed on their cyanide adducts
No full textThe enthalpic and entropic changes accompanying the reduction reaction of the six-coordinate cyanide adducts of cytochrome c, microperoxidase-11 and a few plant peroxidases were measured electrochemically. Once the compensating changes in reduction enthalpy and entropy due to solvent reorganization effects are factorized out, it is found that cyanide binding stabilizes enthalpically the ferriheme following the order: cyochrome c > peroxidase > microperoxidase-11. The effect is inversely correlated to the solvent accessibility of the heme. Comparison of the reduction thermodynamics for the cyanide adducts of cytochrome c and plant peroxidases with those for microperoxidase-11 and myoglobin, respectively, yielded an estimate of the consequences of protein encapsulation and of the anionic character of the proximal histidine on the reduction potential of the heme-cyanide group. Insertion of the heme-CN group into the folded peptide chain of cyt c induces an enthalpy-based decrease in E-o' of approximately 100 mV, consistent with the lower net charge of the oxidized as compared to the reduced iron center, whereas a full imidazolate character of the proximal histidine stabilizes enthalpically the ferriheme by approximately 400 mV. The latter value should be best considered as an upper limit since it also includes some solvation effects arising from the nature of the protein systems being compared
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