Lodovica Loschi

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.

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.

Redox properties of the basic blue protein (plantacyanin) from spinach

Abstract The reduction potential of spinach plantacyanin was determined through direct electrochemistry as a function of temperature and pH. This species shows a higher reduction potential than the homologous cucumber basic protein (CBP) ( E 0 =+345 mV vs. +304 mV for CBP, in 0.1 M NaCl, pH 7, T =25°C), which turns out to be primarily the result of a more negative reduction enthalpy. Like CBP, spinach plantacyanin undergoes a positive entropy change upon reduction, at variance with most cupredoxins. Both species show a low-pH increase in E 0 , indicative of protonation and detachment from the Cu(I) center of a histidine ligand. However, the p K a value for the spinach protein is sensibly higher (5.7 vs. about 3.5 for CBP). It is concluded that the copper site differs to some extent in the two species, although the main coordination features are likely conserved. The differences likely involve solvation properties, and, possibly, protein sequence in the metal domain.

1H NMR of oxidized blue copper proteins

Abstract The hyperfine-shifted 1 H NMR resonances arising from the Cu(II) ligands of cucumber stellacyanin, plantacyanin (CBP) and plastocyanin, horseradish umecyanin, and of spinach plastocyanin and its L12G mutant are reported. It is shown that these broad paramagnetic signals are diagnostic of structural, electronic and solvation properties of the metal site and can be exploited to probe for differences and/or analogies among type I copper centers.