Giulia Di Rocco

Enthalpy/entropy compensation phenomena in the reduction thermodynamics of electron transport metalloproteins

Compensation phenomena between the enthalpy and entropy changes of the reduction reaction for all classes of electron transport metalloproteins, namely cytochromes, iron-sulfur, and blue copper proteins, are brought to light. This is the first comprehensive report on such effects for biological redox reactions. Following Grunwald’s approach for the interpretation of H/S compensation for solution reactions, it is concluded that reduction-induced solvent reorganization effects involving the hydration shell of the molecule dominate the reduction thermodynamics in these species, although they have no net effect on the E° values, owing to exact compensation. Thus the reduction potentials of these species are primarily determined by the selective enthalpic stabilization of one of the two oxidation states due to ligand binding interactions and electrostatics at the metal site and by the entropic effects of reduction-induced changes in protein flexibility.

Immobilized cytochrome c bound to cardiolipin exhibits peculiar oxidation state-dependent axial heme ligation and catalytically reduces dioxygen

Mitochondrial 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.

Thermodynamic aspects of the adsorption of cytochrome c and its mutants on kaolinite.

The 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 positions 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

The 1H 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 the high frequency region of the spectrum, which are attributed to the metal binding residues of the mononuclear T1 center. The differences between the patterns of the hyperfine resonances of the free enzyme and its azide derivatives suggest that the alterations in the structural properties of the T3 site induced by the binding of the first azide molecule induce a limited alteration of the spin density distribution over the T1 copper ligands. Overall, these data demonstrate that 1H NMR can be fruitfully applied to characterize the electronic properties of the metal sites of blue oxidases at room temperature.

Computational evidence support the hypothesis of neuroglobin also acting as an electron transfer species

Neuroglobin (Ngb) is a recently identified hexa-coordinated globin, expressed in the nervous system of humans. Its physiological role is still debated: one hypothesis is that Ngb serves as an electron transfer (ET) species, possibly by reducing cytochrome c and preventing it to initiate the apoptotic cascade. Here, we use the perturbed matrix method (PMM), a mixed quantum mechanics/molecular dynamics approach, to investigate the redox thermodynamics of two neuroglobins, namely the human Ngb and GLB-6 from invertebrate Caenorhabditis elegans. In particular, we calculate the reduction potential of the two globins, resulting in an excellent agreement with the experimental values, and we predict the reorganization energies, λ, which have not been determined experimentally yet. The calculated λ values match well those reported for known ET proteins and thereby support a potential involvement in vivo of the two globins in ET processes.

Protein stability and mutations in the axial methionine loop of a minimal cytochrome c

AbstractThe minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.

pH and Solvent H/D Isotope Effects on the Thermodynamics and Kinetics of Electron Transfer for Electrode-Immobilized Native and Urea-Unfolded Stellacyanin

The 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 D(2)O. 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 pK(a) 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 to the role of protein solvation as effector of the thermodynamics and kinetics of ET.

Thermodynamics of the alkaline transition in phytocyanins

The thermodynamics of the alkaline transition which influences the spectral and redox properties of the type 1 copper center in phytocyanins has been determined spectroscopically. The proteins investigated include Rhus vernicifera stellacyanin, cucumber basic protein and its Met89Gln variant, and umecyanin, the stellacyanin from horseradish roots, along with its Gln95Met variant. The changes in reaction enthalpy and entropy within the protein series show partial compensatory behavior. Thus, the reaction free energy change (hence the pKa value) is rather variable. This indicates that species-dependent differences in reaction thermodynamics, although containing an important contribution from changes in the hydrogen-bonding network of water molecules in the hydration sphere of the protein (which feature enthalpy–entropy compensation), are to a large extent protein-based. The data for axial ligand variants are consistent with the hypothesis of a copper-binding His as the deprotonating residue responsible for this transition.

Enhancing Biocatalysis: The Case of Unfolded Cytochrome c Immobilized on Kaolinite

Cytochromes c (cytc) are heme proteins that are involved in several biological processes. The robust protein structure and the covalent attachment of the heme center to the polypeptide matrix allow (at least partial) protein folding and redox function even under hostile physicochemical conditions. The cytc family is among the most thoroughly investigated classes of electron-transfer (ET) proteins owing to its large availability and ease of purification. Moreover, these proteins can be easily engineered by site-directed mutagenesis. In the last decade, several studies have shown that non-native forms of cytc exhibit peroxidase activity (PA) toward several organic substrates including aromatic hydrocarbons, organosulfur compounds, and heterocycles. Given that enzymes are, in general, rather delicate molecules, as they are subjected to inactivation by temperature, pH, and ionic composition of the solution, cytc can be seen as valuable candidates for in vitro and/or industrial oxidative biocatalysis. It is known that cytc shows PA only when the “sixth” axial coordination site of the heme iron is vacant or when it bears a weak ligand.Interestingly, this catalytic property is induced by unfolding agents as a result of the cleavage or weakening of the native axial methionine bond to the iron and to the increase in solvent accessibility of the heme center caused by partial unfolding. 7] Therefore, the PA of cytc is directly related to the availability of the heme iron to undergo H2O2 binding and to the presence of a heme pocket that retains most of its structural features for substrate binding. Moreover, adsorption of cytc on some inorganic surfaces has been found to induce PA. 8–11] This would open the way to the production of a heterogeneous catalyst whose efficiency depends on the surface area; moreover, the use of such a catalyst would facilitate separation and recovery of the reaction products. The possibility of using supported cytc as a heterogeneous catalyst is intriguing because this protein is known to be very versatile and robust and can be easily mutated to work under a wide range of conditions. Therefore, exploitation in bioand environmental catalysis is at hand. We previously investigated the thermodynamic aspects of the immobilization of cytc and some variants on kaolinite (Nak); the variants were obtained by replacing the surface lysine residues in the 72-, 73-, and 79-positions with alanine residues. Cytc is able to strongly and quantitatively adsorb onto the surface of Nak through electrostatic interactions. This bioinorganic interface was found to exhibit appreciable catalytic activity in the oxidation of guaiacol (Gc) to tetraguaiacol (tetraGc) in the presence of H2O2. [6] The aim of this paper is to find conditions under which the catalytic properties are amplified and improved. Protein unfolding is known to increase the PA of cytc, but immobilization on an anionic surface increases conformational stability. 8, 9] For these reasons, we chose to study the effects of the well-known unfolding agent urea, not only on the native protein but also on selected variants. The choice of Lys-to-Ala mutants at the 72-, 73-, and 79-positions was made for the following reasons: 1) These lysine residues are key contributors to the positively charged domain of cytc involved in the binding to Nak. Therefore, these residues may influence the adsorption geometry and, consequently, may affect the PA of immobilized cytc. 2) These lysine residues are placed near the crevice exposing the heme to the solvent and, therefore, could control access of a substrate to the heme center. 3) These lysine residues, and in particular Lys79, are located near the foldon, which is first involved in urea-induced unfolding. The PA was spectrophotometrically determined by following the oxidation of Gc to tetraGc due to H2O2 (Supporting Information S3):

Electrostatic effects on the thermodynamics of protonation of reduced plastocyanin

The 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.