The reactivity of cytochrome c with soft ligands.

The spectral changes caused by binding soft ligands to the cytochrome c iron and their correlation to ligand affinities support the hypothesis that the iron—methionine sulfur bond of this heme protein is enhanced by delocalization of the metal l₂, electrons into the empty 3d orbitals of the ligand atom. These findings also explain the unique spectrum of cytochrome c in the far red.     

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa3 were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa3. The bimolecular rate constant for this reaction is 2 x 10(7) M-1 . s-1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20 degrees C). (2) The ionic strength dependence of the cytochrome c-cytochrome aa3 interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa3 complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (epsilon) of the reaction medium on the reaction of cytochrome C with cytochrome aa3 was investigated. With increasing epsilon the second-order rate constant decreased.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Kinetics of glucose repression of yeast cytochrome c.

The kinetics of glucose repression of cytochrome c synthesis was measured by a radioimmune assay. When 5 or 10% glucose was added to a derepressed culture, the rate of cytochrome c synthesis was reduced to the repressed level with a half-life of 2 min. The addition of 1 or 0.5% glucose repressed the rate of cytochrome c synthesis to the same level as high glucose concentrations but with a longer half-life of 3 min. Glucose repression had no effect on the stability or function of the cytochrome c protein. Cellular levels of active cytochrome c mRNA during glucose repression were measured by translation of total cellular polyadenylic acid-containing RNA and immunoprecipitation cytochrome c from the translation products. The results of these measurements indicate that glucose represses the rate of cytochrome c synthesis through a reduction in the level of translatable cytochrome c mRNA.     

Ordered complexes of cytochrome c fragments. Kinetics of formation of the reduced (ferrous) forms

The kinetics of formation of noncovalently bound ferrous complexes derived from fragments of horse heart cytochrome c have been investigated. When the reactions are initiated by combining ferrous heme fragments with an appropriate apofragment, in the presence of 50 mM imidazole, second order rate processes are observed with rate constants essentially the same as those reported with ferric heme fragments (Parr, G. R., and Taniuchi, H. (1979) J. Biol. Chem. 254, 4836-4842). An additional, probably consecutive, kinetic process is also demonstrated. If imidazole is not present in the reaction buffer, the kinetic profiles are dramatically altered. While this is partially due to aggregation (dimerization) of the ferrous heme fragments, it can nevertheless be demonstrated that the complementation reactions with apofragments are much faster than those observed with the corresponding ferric heme fragments (in the absence of imidazole). These results reflect the effect of the oxidation state of the heme iron on the folding mechanism and, thus, the manifold nature of protein folding pathways. The rate of reduction of productive ferric complexes by sodium ascorbate was investigated and biphasic reactions were found in all cases. The data indicate an equilibrium between two forms of the ferric complexes. The results of an experiment in which the complementation of ferric (1-25)H and (23-104) was carried out in the presence of sodium ascorbate indicate that the intermediate complex (Parr, G. R., and Taniuchi, H. (1980) J. Biol. Chem. 255, 8914-8918) is not reducible by ascorbate. Thus, the increase in oxidation-reduction potential occurring on formation of the productive complex from the unbound heme fragment occurs at a late stage of the overall reaction, possibly coinciding with ligation of methionine 80 to the heme iron.     

The reaction of Pseudomonas aeruginosa cytochrome c oxidase with sodium metabisulphite (Short Communication)

Spectrophotometric evidence is presented for the formation of a complex between metabisulphite and reduced Pseudomonas aeruginosa cytochrome c oxidase. The effects of metabisulphite on the recombination of CO with the reduced enzyme are discussed in terms of alternate binding sites for S(2)O(5) (2-) and CO.     

Kinetics and mechanism of the folding of cytochrome c.

The reversible folding of cytochrome c in urea at pH 4.0 was investigated by repetitive pressure perturbation kinetics and by equilibrium spectroscopic methods. Two folding reactions were observed in the 1 ms to 10 s time range. The rates and amplitudes of these reactions depend on urea concentration in a complex manner, which is different for each process. The absorbance spectra of the kinetic amplitudes of the two reactions also differ from each other. A model with a three-state mechanism can quantitatively account for all of the kinetic and equilibrium data, and it enables us to determine the rate constants and volume changes of the two steps. If a rapid protonation step is added to the mechanism, the analysis can be extended to calculate the pH dependence of the rate and amplitude of the faster folding step. This pH dependence is in excellent agreement with previously published data [Tsong, T. Y. (1977) J. Biol. Chem. 252, 8778-8780]. Kinetic experiments in the 695-nm band show clearly that the axial ligand methionine-80 is involved in the slow folding process and the other axial ligand, histidine-18, is involved in the fast process. Additional experiments with a cyanogen bromide fragment of the protein, and fluorescence detection of the folding kinetics of the intact protein, support an interpretation of the model in terms of known structural elements of cytochrome c. This work provides new information about the mechanism of the folding of cytochrome c, resolves conflicts in earlier interpretations, and demonstrates the applicability of the repetitive pressure perturbation kinetics method to protein folding.     

Interaction of cytochrome c with cytochrome c oxidase. Photoaffinity labeling of beef heart cytochrome c oxidase with arylazido-cytochrome c.

Cytochrome c derivatives labeled with a 3-nitrophenylazido group at lysine 13, at lysine 22, or at both residues have been prepared. The interaction of the cytochrome c derivatives with beef heart cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) in the presence of ultrviolet light results in formation of a covalent complex between cytochrome c and the oxidase. Using the lysine 22 derivative, the polypeptide composition of the oxidase is not modified, nor is its catalytic activity, whereas with the lysine 13 derivative, the gel electrophoretic pattern is altered and the catalytic activity of the complex diminished. The data are consisten with a specfic covalent interaction of the lysine 13 derivative of cytochrome c with the polypeptide of molecular weight 23,700 (Subunit II) of cytochrome c oxidase.     

Kinetics of electron transfer between cytochrome c and laccase.

Rate constants have been determined for the electron-transfer reactions between reduced horse heart cytochrome c and resting Rhus vernicifera laccase as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of reduced cytochrome c was determined to be k = 125 M-1 s-1 at 25 degrees C in 0.2 M phosphate buffer at pH 6.0 with the activation parameters delta H++ = 16.2 kJ mol-1 and delta S++ = 28.9 J mol-1 K-1. The rate constants increased with decreasing buffer concentration, indicating that electron transfer from cytochrome c to laccase is favored by the local electrostatic interaction (ZAZB = -0.9 at pH 6 and -1.3 at pH 4.8) between the basic proteins with positive net charges. From the increase of the rate of electron transfer with decreasing pH, one of the driving forces of the reaction was suggested to be the difference in the redox potentials between the type 1 copper in laccase and the central iron in cytochrome c. Further, on addition of one hexametaphosphate anion per cytochrome c molecule, the rate of the electron transfer was increased, probably because the association of both proteins became more favorable.     

Interactions of sulphide and other ligands with cytochrome c oxidase. An electron-paramagnetic-resonance study.

The effect of sulphide on resting oxidized cytochrome c oxidase was studied by both e.p.r. and optical-absorption spectroscopy. Excess sulphide causes some reduction of cytochrome a, CuA and CuB, and the formation of the cytochrome a3-SH complex after about 1 min. After several hours in the presence of excess sulphide only the e.p.r. signals due to low-spin ferricytochrome a3-SH persist, giving a partially reduced species. Re-oxidation of this partially reduced sulphide-bound enzyme by ferricyanide makes all of the metal centres except CuB detectable by e.p.r. We conclude that sulphide has reduced and binds to CuB as well as to ferricytochrome a3. Sulphide binding to cuprous CuB may raise its mid-point potential and make re-oxidation difficult. Addition of reductant (ascorbate + NNN'N'-tetramethyl-p-phenylenediamine) and sulphide together to the oxidized resting enzyme produces a species in which cytochrome a and CuA are nearly completely reduced and cytochrome a3 is e.p.r.-detectable as approx. 80% of one haem in the low-spin sulphide-bound complex. The g = 12 signal of this partially reduced derivative is almost unchanged in magnitude relative to that of the resting enzyme; this suggests that the g = 12 signal may arise from less than 20% of the enzyme and that it may be relatively unreactive to both ligation and reduction. Such a reactivity pattern of the g = 12 form of the oxidase is also demonstrated with the ligands F- and NO, which are thought to bind to cytochrome a3 and CuB respectively.     

The reaction of Rhodospirillum rubrum cytochrome c2 with iron hexacyanides.

The reaction of Rhodospirillum rubrum cytochrome c2 with the nonphysiological reactants, ferrocyanide and ferricyanide has been investigated as a function of ionic strength, temperature and pH, using both stopped-flow and temperature-jump kinetic methods. The results are consistent with a complex reaction mechanism involving the formation of two intermediate complexes. The site of electron transfer appears to be at the front of the cytochrome c2 molecule near the hem e crevice with interacton of both ferri and ferrocyanide with a positively charged region of the molecule. Comparison of the proposed electron transfer mechanism of cytochrome c2 with ferro-ferricyanide is made with the mechanism proposed based upon structural considerations.     

The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide.

The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 X 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 X 10(4)M-1-s-1 and 1.6 X 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.     

Characterization of the reaction between ferrocytochrome c and cytochrome c oxidase.

The reaction between cytochrome c oxidase and ferrocytochrome c has been investigated by the stopped-flow method. It has been found that only one electron acceptor, a heme group, in the oxidase is rapidly reduced by cytochrome c. The presence of N3- does not affect the reduction of the acceptor, which supports the hypothesis that this is identical with cytochrome a. The results are consistent with the existence of a simple equilibrium between cytochrome a and cytochrome c: c-2 + a-3+ in equilibrium c-3+ + a-2+ with an equilibrium constant corresponding to an oxidation-reduction potential of cytochrome a 30 mV higher than that for cytochrome c at pH 7.4. The oxidation-reduction potential of the a-3+ /a-2+ couple, 285 mV (based on a potential of 255 mV for cytochrome c), and the optical properties of the reduced form indicate that it is identical with neither of the reduced hemes seen in potentiometric titrations. The oxidase species resulting from the rapid reduction of cytochrome a by cytochrome c is proposed to represent a metastable intermediate state which, under anaerobic conditions, eventually is transformed into a more stable state characterized by a reduced high-potential heme.