The reduction mechanism of ferricytochrome c

The second-order rate constant of the reaction between the hydrated electron and ferrinitrocytochrome c exhibits a marked pH dependence that could not be fully ascribed to changes in geometrical parameters and in net charge of the protein molecule.

The correlation between the pH dependence of the rate constant, the 695-nm absorbance and the ionization state of the nitrated tyrosyl-67 residue indicates that tyrosine-67 is of importance in maintaining the specific structure for the electron transfer mechanism in ferricytochrome c upon reduction.     

Reduction of cytochrome c by thioglycolate: Participation of catalysts

The reduction of cytochrome c by thioglycolic acid was found to be extremely sensitive to metal catalysis. The rate of the uncatalyzed reaction was negligible and independent of pH, indicating that thioglycolic acid cannot reduce cytochrome c directly. Both copper and iron act as catalysts with copper being superior to iron. The metal-catalyzed reaction appears to be independent of pH and the presence of oxygen but is sensitive to the presence of chelating agents. The reduction of cytochrome c by thioglycolic acid is also catalyzed by impurities present in oxidized glutathione. The rate of this reaction is sensitive to changes in pH and oxygen concentration but insensitive to changes in ionic strength. Chelating agents have no effect on the rate of this reaction. The data, therefore, suggest that the reduction of cytochrome c by thioglycolic acid can proceed via distinct mechanisms which are dependent on the nature of the catalyst.     

Dimerization of hydroxylated species of m-aminophenol by cytochrome c with hydrogen peroxide

The cytochrome c and hydrogen peroxide-dependent oxidation of m-aminophenol was investigated by electrochemistry and spectrophotometry. The results indicated that the hydroxylated species of m-aminophenol have at least two conjugated substituted groups on the ring system (most possibly, its oxidized form 2-hydroxy-4-iminoquinone), and that the degradation of cytochrome c by hydrogen peroxide can also be prevented in the presence of m-aminophenol. The hydroxyl radical scavengers, mannitol and sodium benzoate, almost completely eliminate the hydroxylation of m-aminophenol. But oxo-heme species scavenger, uric acid, does not inhibit the hydroxylation. Combining the results of mass spectrum, nuclear magnetic resonance and element analysis with that of spectrophotometry, electrochemistry and chemical scavengers, it is suggested that cytochrome c may act as a peroxidase, which facilitates the hydroxylation and subsequent dimerization of m-aminophenol.     

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.     

Electron transfer reactions of cytochrome c confined within erythrocyte ghosts

We have prepared and characterized resealed erythrocyte ghosts in which the only discernible pigment is cytochrome c. The resealed ghosts have the normal orientation and are free of ‘leaky’ species; they are stable and can be maintained at 4°C for many days without lysis.

The internal cytochrome c participates in redox reactions with both soluble and insolubilized cytochrome c present externally, and with external cytochrome b₅. No reaction was observed with plastocyanin, cytochrome c oxidase or NADPH-cytochrome c reductase.

A study has been made of the reaction of the internal cytochrome c with the low molecular weight reductants, ascorbate and glutathione. Complex kinetics are observed with both reagents: with ascorbate the results are best explained by assuming the existence, in the membrane, of a redox-active species able to undergo dedimerization. A protein bound disulfide bond would satisfy the requirement.     

Kinetic studies on redox reactions of hemoproteins. I. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ferrocyanide

The oxidation-reduction reaction of horse heart cytochrome c and cytochrome c (552, Thermus thermophilus), which is highly thermoresistant, was studied by temperature-jump method. Ferrohexacyanide was used as reductant.

Thermodynamic and activation parameters of the reaction obtained for both cytochromes were compared with each other. The results of this showed that (1) the redox potential of cytochrome c-552,+0.19 V, is markedly less than that of horse heart cytochrome c. (2) ?H_ox³ of cytochrome c-552 is considerably lower than that of horse heart cytochrome c. (3) ?H_ox³ and ?S_red³ of cytoochrome c-552 are more negative than those of horse heart cytochrome c. (4) k_red of cytochrome c-552 is much lower than that of horse heart cytochrome c at room temperature.     

Autocatalytic peroxidation of ferrocytochrome c

1. Ferricytochrome c acts as a catalyst in the peroxidation of ferrocytochrome c thereby giving rise to an autocatalytic reaction.

2. The rate of the peroxidation reaction is proportional to the concentration of H₂O₂ and ferricytochrome c but is independent of the concentration of ferrocytochrome c in the concentration ranges studied.

3. Integration of the rate equation, d[c³⁺]/dt = k[c³⁺][H₂O₂], gives a theoretical expression which fits the experimental time courses for the ferrocytochrome c peroxidation reaction.

4. No direct spectral evidence was found for the formation of a catalytically active ferricytochrome c-H₂O₂ derivative. Kinetic evidence is presented, however, which indicates the existence of such an intermediate.

5. Ferricytochrome c was more susceptible than ferrocytochrome c to an apparent degradation reaction caused by excess H₂O₂, thus supporting the idea that the cytochrome c heme iron is more accessible in the oxidized form.     

High Yield of Methylophilus methylotrophus Cytochrome c″ by Coexpression with Cytochrome c Maturation Gene Cluster from Escherichia coli

Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c″ from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c″ isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS–PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and ¹H-NMR spectroscopy. The success in expressing the mature cytochrome c″ in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship.     

Characterization of cytochrome c from Nitrobacter Agilis

Cytochrome c from Nitrobacter agilis was isolated and purified approx. 60-fold. Absorption spectra of both the oxidized and the reduced Nitrobacter cytochrome c and the oxidized minus reduced difference spectrum of this cytochrome were essentially identical to the corresponding spectra of horse-heart cytochrome c. The redox potential of this cytochrome was determined by spectrophotometric titration with ferrocyanide/ferricyanide and found to be +0.282 V over the pH range 6.0 to 8.7, while a potential of +0.265 V was determined in the same manner for horse-heart cytochrome c. The titration also indicated that the Nitrobacter ferrocytochrome is oxidized by a single electron transfer.     

Components of cytochrome c oxidase detectable by EPR spectroscopy

A procedure for the preparation from frozen beef heart mitochondria of cytochrome c oxidase (EC 1.9.3.1) of high heme ( 14 μmoles/mg protein) and low extraneous copper ( 1.1 atoms Cu/mole heme) and low lipid ( 0.05 g phospholipid/g protein) content is described. EPR signals observed with the enzyme between 6 and 100 °K at various states of oxidation and at different conditions of pH and presence of solutes are described in detail. The quantities of paramagnetic species represented by these signals are estimated. Under no conditions does the sum of the EPR detectable species represent more than approx. 50% of the potentially paramagnetic components of the enzyme. Comparisons are made to the corresponding signals as observed in whole tissue, mitochondria and submitochondrial particles from a number of species. The assignment of the observed signals to known components of cytochrome c oxidase is discussed briefly.     

Persistence of the competent state in mouse fibroblasts is independent of c-fos and c-myc expression

Induction of competence in quiescent fibroblasts by platelet-derived growth factor (PDGF) is accompanied by a dramatic increase in the expression of c-fos and c-myc genes. However, the maintenance of the competent state and progression through G1 does not require high expression of these proto-oncogenes. These results suggest that the induction of c-fos and c-myc by growth factors in quiescent fibroblasts may be required to render the cells competent for progression.     

Biochemical and biophysical studies on cytochrome c oxidase XIV. The reaction with cytochrome c as studied by pulse radiolysis

1. The reduction of cytochrome c oxidase by hydrated electrons was studied in the absence and presence of cytochrome c.

2. Hydrated electrons do not readily reduce the heme of cytochrome c oxidase. This observation supports our previous conclusion that heme a is not directly exposed to the solvent.

3. In a mixture of cytochrome c and cytochrome c oxidase, cytochrome c is first reduced by hydrated electrons (k = 4 · 10¹⁰ M⁻¹ · s⁻¹ at 22 °C and pH 7.2) after which it transfers electrons to cytochrome c oxidase with a rate constant of 6 · 10⁷ M⁻¹ · s⁻¹ at 22 °C and pH 7.2.

4. It was found that two equivalents of cytochrome c are oxidized initially per equivalent of heme a reduced, showing that one electron is accepted by a second electron acceptor, probably one of the copper atoms of cytochrome c oxidase.

5. After the initial reduction, redistribution of electrons takes place until an equilibrium is reached similar to that found in redox experiments of Tiesjema, R. H., Muijsers, A. O. and Van Gelder, B. F. (1973) Biochim. Biophys. Acta 305, 19–28.     

Evidence for a crosslink between c-heme and a lysine residue in cytochrome P460 of Nitrosomonas europaea

Cytochrome P460 and hydroxylamine oxidoreductase (HAO) of Nitrosomonas europaea catalyze the oxidation of hydroxylamine. Cytochrome P460 contains an unidentified heme-like chromophore whose distinctive spectroscopic properties are similar to those for the P460 heme found in HAO. The heme P460 of HAO has previously been shown by protein chemistry and NMR structural analysis to be a c-heme with an additional covalent crosslink between the C2 ring carbon of a tyrosine residue of the polypeptide chain and a meso carbon of the porphyrin [Arciero, D.M. et al. (1993) Biochemistry 32, 9370–9378]. The recent determination of the gene sequence for cytochrome P460 [Bergmann, D.J. and Hooper, A.B. (1994) FEBS Lett. 353, 324–326] indicates that the heme in this protein also possesses a c-heme binding site and provides the basis for determining whether an HAO-like crosslink exists to the porphyrin.Sequence analysis of a purified heme-containing tryptic chromopeptide from cytochrome P460 revealed two predominant amino acid residues per cycle. Two peptides present in the chromopeptide with the sequences NLPTAEXAAXHK and DGTVTVXELVSV. Comparison of the data to the gene sequence for the protein revealed that the gaps in the first peptide (indicated by X's) code for C residues, confirming the prediction of a c-heme binding motif. The gap in the sequence in the second peptide at cycle 7 is predicted by the gene sequence to be a K. The results suggest that the lysine residue is crosslinked in some manner to the porphyrin macrocycle, possibly mimicking the tyrosine crosslink found for the heme P460 of HAO. While a common role for the crosslinked residues in HAO and cytochrome P460 is difficult to ascertain due to the dissimilarities in side chain structure, it may be related to the similar pK_a values for lysine and tyrosine.     

Cytochrome c involved in the reductive decomposition of organic mercurials. Purification of cytochrome c-I from mercury-resistant Pseudomonas and reactivity of cytochromes c from various kinds of bacteria

Cytochrome c-I which was involved in the decomposition of organic mercurials as an electron carrier was purified from the cell-free extract of the mercury-resistant strain, Pseudomonas K62, by means of (NH₄)₂SO₄ precipitation and column chromatography on Sephadex G-150, DEAE-Sephadex and Sephadex G-75. The cytochrome was crystallized in a needle-like form. It showed absorption maxima at 550, 521, and 416.5 nm in the reduced form, and the pyridine ferrohemochrome had absorption maxima at 549, 520, and 413 nm, suggesting it to be a c-type cytochrome.

Cytochromes c prepared from type cultures of bacteria belonging to the genera Aeromonas, Micrococcus, Bacillus, Corynebacterium, Staphylococcus, Aerobacter, and Pseudomonas were all inactive with respect to the decomposition of phenylmercuric acetate. However, cytochrome c prepared from Pseudomonas CF, which was isolated from the activated sludge acclimatized with HgCl₂ and phenylmercuric acetate, as well as the cytochrome c-I of Pseudomonas K62, were active in this respect.