Alexander Dikiy

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

Abstract Direct cyclic voltammetry and 1H NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c553 isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c553 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;ΔS°′=–162.7±1.2 J mol–1 K–1, ΔH°′=–53.0±0.5 kJ mol–1, ΔG°′=–4.5±0.1 kJ mol–1, E°′=+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 c553. Both effects are more accentuated in the high-temperature range (308–323 K;ΔS°′=–294.1±8.4 J mol–1 K–1, ΔH°′=–93.4±3.1 kJ mol–1, ΔG°′=–5.8±0.6 kJ mol–1, E°′=+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-T 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-Hückel theory, yields values of E°′(I=0)=–25.4±1.4 mV, zred=–11.3, and zox=–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 pKO=5.5 and pKR=5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c553. 1H NMR spectra of the oxidized paramagnetic cytochrome c553 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 1H 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 1H NMR signals of the oxidized cytochrome displays at least one ionization step with pKO=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 c553.

NMR Solution Structure, Backbone Mobility, and Homology Modeling of c-Type Cytochromes from Gram-Positive Bacteria

The solution structure of oxidized cytochrome c553 (71 amino acid residues) from the Gram‐positive bacterium Bacillus pasteurii is here reported and compared with the available crystal structure. The solution structure is obtained from 1609 meaningful NOE data (22.7 per residue), 76 dihedral angles, and 59 pseudocontact shifts. The root mean square deviations from the average structure are 0.25±0.07 and 0.59±0.13 Å for the backbone and all heavy atoms, respectively, and the quality assessment of the structure is satisfactory. The solution structure closely reproduces the fold observed in the crystal structure. The backbone mobility was then investigated through amide 15N relaxation rate and 15N–1H NOE measurements. The protein is rigid in both the sub‐nanosecond and millisecond time scales, probably due to the relatively large heme:number of amino acids ratio. Modeling of eight c‐type cytochromes from other Gram‐positive bacteria with a high sequence identity (>30 %) to the present cytochrome c553 was performed. Analysis of consensus features accounts for the relatively low reduction potential as being due to extensive heme hydration and indicates residues 34–35, 44–46, 69–72, and 75 as a conserved hydrophobic patch for the interaction with a protein partner. At variance with mitochondrial c‐type cytochrome, this protein does not experience pH‐dependent coordination equilibria. The reasons for this difference are analyzed.

Isolation and physico-chemical characterization of a cytochrome c from the methylotrophic yeast Hansenula polymorpha.

Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential.