Marco Sola

Redox properties of heme peroxidases

Peroxidases are heme enzymes found in bacteria, fungi, plants and animals, which exploit the reduction of hydrogen peroxide to catalyze a number of oxidative reactions, involving a wide variety of organic and inorganic substrates. The catalytic cycle of heme peroxidases is based on three consecutive redox steps, involving two high-valent intermediates (Compound I and Compound II), which perform the oxidation of the substrates. Therefore, the thermodynamics and the kinetics of the catalytic cycle are influenced by the reduction potentials of three redox couples, namely Compound I/Fe3+, Compound I/Compound II and Compound II/Fe3+. In particular, the oxidative power of heme peroxidases is controlled by the (high) reduction potential of the latter two couples. Moreover, the rapid H2O2-mediated two-electron oxidation of peroxidases to Compound I requires a stable ferric state in physiological conditions, which depends on the reduction potential of the Fe3+/Fe2+ couple. The understanding of the molecular determinants of the reduction potentials of the above redox couples is crucial for the comprehension of the molecular determinants of the catalytic properties of heme peroxidases. This review provides an overview of the data available on the redox properties of Fe3+/Fe2+, Compound I/Fe3+, Compound I/Compound II and Compound II/Fe3+ couples in native and mutated heme peroxidases. The influence of the electron donor properties of the axial histidine and of the polarity of the heme environment is analyzed and the correlation between the redox properties of the heme group with the catalytic activity of this important class of metallo-enzymes is discussed.

The Reversible Opening of Water Channels in Cytochrome c Modulates the Heme Iron Reduction Potential

Dynamic protein-solvent interactions are fundamental for life processes, but their investigation is still experimentally very demanding. Molecular dynamics simulations up to hundreds of nanoseconds can bring to light unexpected events even for extensively studied biomolecules. This paper reports a combined computational/experimental approach that reveals the reversible opening of two distinct fluctuating cavities in Saccharomyces cerevisiae iso-1-cytochrome c. Both channels allow water access to the heme center. By means of a mixed quantum mechanics/molecular dynamics (QM/MD) theoretical approach, the perturbed matrix method (PMM), that allows to reach long simulation times, changes in the reduction potential of the heme Fe(3+)/Fe(2+) couple induced by the opening of each cavity are calculated. Shifts of the reduction potential upon changes in the hydration of the heme propionates are observed. These variations are relatively small but significant and could therefore represent a tool developed by cytochrome c for the solvent driven, fine-tuning of its redox functionality.

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.

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c.

Abstract The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50  °C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E°′ observed with increasing ionic strength [ΔE°′IS =(E°′)I=0.1 M–(E°′)I=0 M=–0.035 V at 25  °C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factorized out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pKa for a residue deprotonation is the key event of this conformational change.

Medium and Temperature Effects on the Redox Chemistry of Cytochrome c

Cytochromes c (cytc) are ubiquitous heme-containing metalloproteins that shuttle electrons in a variety of electron-transport chains, most often central to the production of the chemical energy necessary for cell life. The reduction potential (E°′) of the Fe3+/2+ couple is central to the physiological role of these species in that it influences the thermodynamic and kinetic features of electron-exchange reactions with redox partners. In the last two decades, voltammetric techniques exploiting the heterogeneous electron exchange between cytc and solid electrodes have proved to be particularly valuable for the determination of E°′ values for these species and for characterizing the mechanistic and kinetic aspects of the redox process for the various cytc conformers under a variety of solution conditions. The understanding of how, and to what extent, different molecular factors control the E°′ value in these species has been the subject of much debate. First coordination sphere effects on the heme iron and the interactions of the heme group with the surrounding polypeptide chain and the solvent are the main factors affecting E°′ in cytc. These interactions are sensitive to medium effects such as the pH and the nature and ionic composition of the solvent. E°′ is also strongly affected by the temperature. This article summarizes the authors’ work on the effects on the selective stabilization of the two redox states of class I cytochromes c exerted by acid-base equilibria, general ionic strength effects, specific anion binding, the presence of non-aqueous solvents, and the temperature. The temperature dependence of E°′ allows the determination of the enthalpy and entropy changes that accompany protein reduction. These parameters have proved to be informative with regard to the interplay between first coordination sphere effects and electrostatics at the heme−protein interface, including solvent dipoles, which mainly affect the reduction enthalpy, and solvent reorganization effects and differences in protein dynamics between the two oxidation states, which control the reduction entropy instead.

Effects of solvent on the redox properties of cytochrome c: cyclic voltammetry and 1H NMR experiments in mixed water-dimethylsulfoxide solutions

Abstract Bovine heart cytochrome c (cyt c) was studied through cyclic voltammetry, 1H NMR and circular dichroism measurements in mixed water-dimethylsulfoxide (DMSO) solutions containing up to 50% DMSO by volume, under different conditions of temperature and pH. The effect of DMSO on the reduction potential of native cyt c was found to be determined mainly the he decrease in dielectric constant of the medium. No appreciable specific DMSO-protein interactions were detected. Instead, DMSO affects to some extent the conformation of alkaline cyt c and notably, stabilizes both redox states of this form to the detriment of the native form. In particular, DMSO lowers the pKa of the native to alkaline transition for oxidized cyt c and increases the electrochemical reversibility of the voltammetric wave of the alkaline form. DMSO-induced changes in the reduction entropy for native and alkaline cyt c were also determined and interpreted tentatively in terms of solvation properties of the heme and structural features of the protein environment.

Redox thermodynamics, acid-base equilibria and salt-induced effects for the cucumber basic protein. General implications for blue-copper proteins

Abstract The reduction potential of the basic blue-copper protein from cucumber peels (CBP) was determined through voltammetric techniques in different conditions of temperature, pH and ionic composition of the medium. The most notable properties of CBP include a positive entropy change upon reduction, a low-pH protonation and detachment of a metal-binding histidine in the reduced protein, and specific binding interactions with a number of anions present in common laboratory buffers, which influence to some extent the redox thermodynamics. The enthalpy and entropy changes accompanying reduction of the Cu(II) center were compared with those for other blue-copper proteins and correlated with spectroscopic data, structural properties and theoretical calculations. This allows some general considerations to be offered regarding the determinants of the reduction potential in this protein class. It emerges, in line with previous studies of the electronic structure of blue-copper sites, that the enthalpic contribution to the reduction potential is mainly modulated by the metal-binding interactions in the trigonal N2S ligand set, and particularly by the Cu-cysteinate bond, while the entropy term is mainly affected by solvation properties and possibly by the weak axial bond to copper. The role of solvent exposure of the metal site in the active-site protonations in reduced blue-copper proteins is discussed. Finally, it is shown that the Nernst-Debye-Huckel model qualitatively accounts for the ionic strength dependence of the reduction potential.

Solvent-based deuterium isotope effects on the redox thermodynamics of cytochrome c.

The reduction thermodynamics of cytochrome c (cytc), determined electrochemically, are found to be sensitive to solvent H/D isotope effects. Reduction of cytochrome c is enthalpically more favored in D2O with respect to H2O, but is disfavored on entropic grounds. This is consistent with a reduction-induced strengthening of the H-bonding network within the hydration sphere of the protein. No significant changes in E°′ occur, since the above variations are compensative. As a main result, this work shows that the oxidation-state-dependent differences in protein solvation, including electrostatics and solvent reorganization effects, play an important role in determining the individual enthalpy and entropy changes of the reduction process. It is conceivable that this is a common thermodynamic feature of all electron transport metalloproteins. The isotope effects turn out to be sensitive to buffer anions which specifically bind to cytc. Evidence is gained that the solvation thermodynamics of both redox forms of cytc are sensibly affected by strongly hydrated anions.

Understanding the Mechanism of Short-Range Electron Transfer Using an Immobilized Cupredoxin

The hydrophobic patch of azurin (AZ) from Pseudomonas aeruginosa is an important recognition surface for electron transfer (ET) reactions. The influence of changing the size of this region, by mutating the C-terminal copper-binding loop, on the ET reactivity of AZ adsorbed on gold electrodes modified with alkanethiol self-assembled monolayers (SAMs) has been studied. The distance-dependence of ET kinetics measured by cyclic voltammetry using SAMs of variable chain length, demonstrates that the activation barrier for short-range ET is dominated by the dynamics of molecular rearrangements accompanying ET at the AZ-SAM interface. These include internal electric field-dependent low-amplitude protein motions and the reorganization of interfacial water molecules, but not protein reorientation. Interfacial molecular dynamics also control the kinetics of short-range ET for electrostatically and covalently immobilized cytochrome c. This mechanism therefore may be utilized for short-distance ET irrespective of the type of metal center, the surface electrostatic potential, and the nature of the protein-SAM interaction.

Computational approaches to structural and functional analysis of plastocyanin and other blue copper proteins

Computational techniques are becoming increasingly important in structural and functional biology, in particular as tools to aid the interpretation of experimental results and the design of new systems. This review reports on recent studies employing a variety of computational approaches to unravel the microscopic details of the structure-function relationships in plastocyanin and other proteins belonging to the blue copper superfamily. Aspects covered include protein recognition, electron transfer and protein-solvent interaction properties of the blue copper protein family. The relevance of integrating diverse computational approaches to address the analysis of a complex protein system, such as a cupredoxin metalloprotein, is emphasized.