Modulation of Bacillus pasteurii cytochrome c553 reduction potential by structural and solution parameters

Direct cyclic voltammetry and H-1 NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c(553) isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c(553) at a pyrolitic graphite edge microelectrode. The temperature dependence of the reduction potential, measured using a non-isothermal electrochemical cell: revealed a discontinuity at 308 K. The thermodynamic parameters determined in the low-temperature range (275-308 K; Delta S degrees'= -162.7 +/- 1.2 J mol(-1) K-1, Delta H degrees' = -53.0 +/- 0.5 kJ mol(-1), Delta G degrees' = -4.5 +/- 0.1 kJ mol(-1), E degrees' = +47.0 +/- 0.6 mV) indicate the presence of large enthalpic and entropic effects, leading, respectively, to stabilization and destabilization of the reduced form of cytochrome c(553). Both effects are more accentuated in the high-temperature range (308-323 K; Delta S degrees'= -294.1 +/- 8.4 J mol(-1) K-1. Delta H degrees' = -93.4+\-3.1 kJ mol(-1), Delta G degrees'= -5.8 +/- 0.6 kJ mol(-1), E degrees' = +60.3 +/- 5.8 mV), with the net result being a slight increase of the standard reduction potential. These thermodynamic parameters are interpreted using the compensation theory of hydration of biopolymers as indicating the extrusion, upon reduction, of water molecules from the hydration sphere of the cytochrome. The low-T and high-ir conformers differ by the number of water molecules in the solvation sphere: in the high-T conformer, the number of water molecules extruded upon reduction increases, as compared to the low-T conformer. The ionic strength dependence of the reduction potential at 298 K, treated within the frame of extended Debye-Huckel theory, yields values of E-(I=0)degrees',= -25.4 +/- 1.4 mV, z(red)=-11.3, and z(ox)= -10.3. The pH dependence of the reduction potential at 298 K shows a plateau in the pH range 7-10 and an increase at more acidic pH, allowing the calculation of pK(O) = 5.5 and pK(R) = 5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c(553). H-1 NMR spectra of the oxidized paramagnetic cytochrome c(553) indicate the presence of a His-Met axial coordination of the low-spin (S=1/2) heme iron, which is maintained in the temperature interval 288-340 K at pH 7 and in the pH range 4.8-10.0 at 298 K. The temperature dependence of the hyperfine-shifted signals shows both Curie-type and anti-Curie-type behavior, with marked deviations from linearity, interpreted as indicating the presence of a fast equilibrium between the low-T and high-T conformers, having slightly different heme electronic structures resulting from the T-induced conformational change. Increasing the NaCl concentration in the range 0-0.2 M causes a slight change of the H-1 NMR chemical shifts of the hyperfine-shifted signals, with no influence on their linewidth. The calculated lower limit value of the apparent affinity constant for specific ion binding is estimated as 5,2 +/- 1.1 M-1. The pH dependence of the isotropically shifted H-1 NMR signals of the oxidized cytochrome displays at least one ionization step with pK(O)=5.7. The thermodynamic and spectroscopic data indicate a large solvent-derived entropic effect as the main cause for the observed low reduction potential of B. pasteurii cytochrome c(553).