Hydrogen ion titration of horse heart ferricytochrome c.

Continuous hydrogen ion titration curves of deionized solutions of horse heart ferricytochrome c have been obtained at 25 degrees C. at a constant ionic strength of 0.10 from pH 3.0 to 11.0. Titration of the oxidized protein in KCl required 28.4 equiv over that pH range, and a small hysteresis between the forward and reverse limbs was displayed. The Linderstrom-Lang approximation, which takes into account electrostatic interactions between charged groups on the protein surface, was used in a computer simulation program to analyze the forward and reverse limbs of the titration curve separately. The results indicated 1 alpha-, 12 beta- and gamma-, and 1 heme propionic carboxylic, 1 imidazole, 1 phenolic, and 18 epsilon-amino residues appear to titrate normally. Variations in the electrostatic interaction factor omega suggest conformational changes in the protein at the extremes of pH, although the relationship of the variations in omega to the magnitude of the conformational changes does not appear to be strictly quantitative for cytochrome c. These results show the acid-base behavior of cytochrome c to be complex in nature, and suggest that the Lindenstrom-Lang model may not be adequate for cytochrome c.     

Reduction of horse heart ferricytochrome c by glutathione: evidence for two different conformational forms of ferricytochrome c in neutral and slightly alkaline solutions.

The reduction of horse heart ferricytochrome c in neutral and slightly alkaline solutions follows biphasic kinetics. The obtained results are consistent with the existence of two conformational forms of ferricytochrome c, cyt c and cyt c¹. The conversion of cyt c to cyt c¹ occurs by deprotonation (pK～7), followed by a slow conformational change in the protein structure.     

Kinetics and mechanism of the reduction of horse heart ferricytochrome c by glutathione.

A detailed investigation of the reduction of cytochrome c by glutathione has shown that the reaction proceeds through several steps. A rapid combination of the reducing agent with the cytochrome leads to the formation of a glutathione-cytochrome intermediate in which the glutathione most likely interacts with the edge of the heme moiety. The electron transfer takes place in a subsequent slower step. Since cytochrome c(III) exists in two conformational forms at neutral pH [Kujundzic, N., & Everse, J. (1978) Biochem. Biophys. Res. Commun. 82, 1211], the reduction of cytochrome c by glutathione may be represented by cyt c(III) + GS- reversible K1 cyt c(III) ... GS- reversible k1 products cyt c*(III) + GS- reversible K2 cyt c*(III) ... GS- reversible k2 products At 25 degrees C, pH 7.5, and an ionic strength of 1.0 (NaCl), k1 = 1.2 X 10(-3) S-1, k2 = 2.0 X 10(-3) S-1, k1 = 2.9 X 10(3) M-1, and K2 = 5.3 X 10(3) M-1. The reaction is catalyzed by trisulfides, and second-order rate constants of 4.55 X 10(3) and 7.14 X 10(3) M-1 S-1 were obtained for methyl trisulfide and cysteine trisulfide, respectively.     

Reduction of ferricytochrome c by tyrosyltyrosylphenylalanine

Cytochrome c (cyt c) was reduced by a tyrosine-containing peptide, tyrosyltyrosylphenylalanine (TyrTyrPhe), at pH 6.0–8.0, while tyrosinol or tyrosyltyrosine (TyrTyr) could not reduce cyt c effectively under the same condition. Cyt c was reduced at high peptide concentration, whereas the reaction did not occur effectively at low concentration. The reaction rate varied with time owing to a decrease in the TyrTyrPhe concentration and the production of tyrosine derivatives during the reaction. The initial rate constants were 2.4×10^–4 and 8.1×10^–4 s^–1 at pH 7.0 and 8.0, respectively, for the reaction with 1.0 mM TyrTyrPhe in 10 mM phosphate buffer at 15°C. The reciprocal initial rate constant (1/k_int) increased linearly against the reciprocal peptide concentration and against the linear proton concentration, whereas logk_int decreased linearly against the root of the ionic strength. These results show that deprotonated (TyrTyrPhe)^–, presumably deprotonated at a tyrosine site, reduces cyt c by formation of an electrostatic complex. No significant difference in the reaction rate was observed between the reaction under nitrogen and oxygen atmospheres. From the matrix-assisted laser desorption ionization time-of-flight mass spectra of the reaction products, formation of a quinone and other tyrosine derivatives of the peptide was supported. These products should have been produced from a tyrosyl radical. We interpret the results that a cyt c_ox/(TyrTyrPhe)^–cyt c_red/(TyrTyrPhe) equilibrium is formed, which is usually shifted to the left. This equilibrium may shift to the right by reaction of the produced tyrosyl radical with the tyrosine sites of unreacted TyrTyrPhe peptides.     

Evolutionary change of the heme c electronic structure: ferricytochrome c-551 from Pseudomonas aeruginosa and horse heart ferricytochrome c.

Individual assignments of the ¹H n.m.r. lines of heme c in reduced and oxidized cytochrome c-551 from $\text{Pseudomonas aeruginosa}$ were obtained by nuclear Overhauser enhancement and saturation transfer experiments. Comparison with the corresponding data on horse heart cytochrome c showed that the locations of high spin density on the heme c periphery as well as the in-plane principal axes x and y of the electronic g-tensor are rotated by approximately 90° in ferricytochrome c-551 relative to horse ferricytochrome c. High spin density in ferricytochrome c-551 is thus localized on the pyrrole ring III. While this pyrrole ring is well shielded in the interior of mammalian-type cytochromes c, it is more easily accessible in cytochrome c-551. It is suggested that this evolutionary change of the heme c electronic structure would be compatible with the hypothesis that the electron transfer in both species is via solvent exposed peripheral ring carbon atoms.     

A denaturation-induced proton-uptake study of horse ferricytochrome c.

The observation that 6 M-urea denatures horse ferricytochrome c in the pH range 4-6, but not horse ferrocytochrome c, has been exploited to determine the denaturation-induced proton uptake of ferricytochrome c. This is related to the pKa values of ionizable groups buried within the native protein. The data indicate that one of the haem propionic acid substituents of ferricytochrome c has a pKa of less than 4.5, whereas the other has a pKa of greater than 9.     

Influence of several perturbants on the rate of autoxidation of horse heart ferrocytochrome c

The effect of several different types of perturbants and pH on the rate of autoxidation of horse heart ferrocytochrome c was investigated. The kinetic behavior is unique to each perturbant used. Rates of autoxidation followed first-order kinetics over the time span (0-180 min) studied. The Cl- and Br- anions exhibit an initial increase in the rate of autoxidation up to 100 mM, followed by a decrease in kinetics at 500 mM anion concentration. The ClO4- anion exhibits only an increase in the rate of autoxidation with increasing ionic strength, where as, propylurea, a hydrophobic perturbant, is not effective in altering the rate of autoxidation at equivalent concentrations. These studies suggest that the perturbations of the reduced form of cytochrome c in solution involve mainly non-specific solvent effects.     

Conversion of oxyhaemoglobin into methaemoglobin by ferricytochrome b5.

Ferricytochrome b5 was found to convert oxyhaemoglobin into methaemoglobin under conditions previously found to be optimal for complex-formation between ferricytochrome b5 and methaemoglobin [Mauk & Mauk (1982) Biochemistry 21, 4730-4734]. As this reaction is completely inhibited by CO, it is proposed that oxyhaemoglobin is oxidized after O2 dissociation, as has been suggested for the oxidation of oxyhaemoglobin by inorganic complexes. From the present analysis, ferricytochrome b5 seems unlikely to contribute significantly to methaemoglobin formation in vivo. Nevertheless, this observation provides a relatively convenient means of investigating the mechanism by which these two proteins interact.     

Volume changes of the molten globule transitions of horse heart ferricytochrome c: a thermodynamic cycle.

Volume changes among the unfolded (U), native (N), and molten globule (MG) conformations of horse heart ferricytochrome c have been measured. U to N (pH 2 to pH 7) was determined in the absence of added salt to be -136 +/- 5 mL/mol protein. U to MG (pH 2, no added salt to pH 2, 0.5 M KCl) yielded + 100 +/- 6 mL/mol. MG to N was broken into two steps, N to NClx at pH 7 by addition of buffered KCl to buffered protein lacking added salt (NClx = N interacting with an unknown number, X, of chloride ions), and MG to NClx by jumping MG at pH 2 in 0.5 M KCl to pH7 at the same salt concentration. The delta V of N to NClx was -30.9 +/- 1.4 mL/mol protein, whereas MG to NClx entailed a delta V of -235 +/- 6 mL/mol. Within experimental error, the results add up to zero for a complete thermodynamic cycle. We believe this to be the first volumetric cycle to have been measured for the conformational transitions of a protein. The results are discussed in terms of hydration contributions from deprotonation of the protein, other hydration effects, and the formation and/or enlargement of packing defects in the protein's tertiary structure during the steps of folding.