Preliminary 1H-NMR studies of the interaction between cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway

The complex formation of two electron transfer proteins, cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway, has been shown by 1H-NMR spectroscopy. Presence of ferredoxin I produces ferricytochrome c3 1H-NMR spectrum modifications. The chemical shift of perturbated heme methyl resonances has been used to determine the stoichiometry of the complex. At pH 7.6 and 20 degrees C, the two proteins were found to form a complex 1:1 with an association constant, KA, of 10(4) M-1. Two of the four hemes are affected by presence of ferredoxin I and may be involved in the electron transfer sites. The heme methyl resonances are average resonances of free and bound cytochrome c3 resonances, indicating a fast exchange process on the NMR time scale.     

Microcalorimetric investigation of the interactions in the ternary complex calmodulin-calcium-melittin

Flow microcalorimetric titrations of calmodulin with melittin at 25 degrees C revealed that the formation of the high-affinity one-to-one complex in the presence of Ca2+ (Comte, M., Maulet, Y., and Cox, J. A. (1983) Biochem, J. 209, 269-272) is entirely entropy driven (delta H0 = 30.3 kJ X mol-1; delta S0 = 275 J X K-1 X mol-1). Neither the proton nor the Mg2+ concentrations have any significant effect on the strength of the complex. In the absence of Ca2+, a nonspecific calmodulin-(melittin)n complex is formed; the latter is predominantly entropy driven, accompanied by a significant uptake of protons and fully antagonized by Mg2+. Enthalpy titrations of metal-free calmodulin with Ca2+ in the presence of an equimolar amount of melittin were carried out at pH 7.0 in two buffers of different protonation enthalpy. The enthalpy and proton release profiles indicate that: protons, absorbed by the nonspecific calmodulin-melittin complex, are released upon binding of the first Ca2+; Ca2+ binding to the high-affinity configuration of the calmodulin-melittin complex displays an affinity constant greater than or equal to 10(7) M-1, i.e. 2 orders of magnitude higher than that of free calmodulin; the latter is even more entropy driven (delta H0 = 7.2 kJ X site-1; delta S0 = 158 J X K-1 X site-1) than binding to free calmodulin (delta H0 = 4.7 kJ X site-1; delta S0 = 112 J X K-1 X site-1), thus underlining the importance of hydrophobic forces in the free energy coupling involved in the ternary complex.     

Microcalorimetric investigation of the interaction of calmodulin with seminalplasmin and myosin light chain kinase

Flow microcalorimetric titrations of calmodulin with seminalplasmin at 25 degrees C revealed that the high affinity one-to-one complex in the presence of Ca2+ (Comte, M., Malnoe, A., and Cox, J. A. (1986) Biochem. J. 240, 567-573) is entirely enthalpy-driven (delta H0 = -50 kJ.mol-1; delta S0 = O J.K-1.mol-1; delta Cp0 = O J.K-1.mol-1) and is not influenced by the proton or Mg2+ concentration. The Sr2+- and Cd2+-promoted high affinity complexes are also exothermic for -49 and -45 kJ.mol-1, respectively. The observed low affinity interaction in the absence of divalent ions displays no enthalpy change. No enthalpy changes are observed when calmodulin and seminalplasmin are mixed in the presence of millimolar concentrations of Mg2+, Zn2+, or Mn2+. Enthalpy titrations of the 1:1 calmodulin-seminalplasmin complex with Ca2+ and of partly Ca2+-saturated calmodulin with seminalplasmin revealed that only the species calmodulin.Can greater than or equal to 2 is fully competent for high affinity interaction with seminalplasmin. Binding of the second Ca2+ is strongly enhanced (K2 greater than or equal to 5 X 10(7) M-1) as compared to that in free calmodulin (K2 = 2.6 X 10(5) M-1). This is essentially due to the concomitant strongly exothermic step of isomerization of the calmodulin-seminalplasmin complex from its low to its high affinity form. Binding of the remaining two Ca2+ to the high affinity seminalplasmin-calmodulin complex displays the same affinity constants and endothermic enthalpy change as in free calmodulin. A microcalorimetric study on the complex formation between Ca2+-saturated calmodulin and turkey gizzard myosin light chain kinase revealed that the interaction is strongly exothermic with an important overall gain of order (delta H0 = -85 kJ.mol-1; delta S0 = -122 J.K-1.mol-1) and occurs with significant proton uptake (0.44 H+ per mol at pH 7.5). The observed low affinity interaction (K = 2.2 X 10(5) M-1) in the absence of Ca2+ (Mamar-Bachi, A., and Cox, J. A. (1987) Cell Calcium 8, 473-482) displays neither a change in enthalpy nor in protonation.     

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.     

Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions.

Two potentiometric methods have been used to study the pH-dependent changes in proton binding that accompany complex formation between cytochrome c and cytochrome b5. With one method, the number of protons bound or released upon addition of one cytochrome to the other has been measured as a function of pH. The results from these studies are correlated with the complexation-induced difference titration curve calculated from the titration curves of the preformed complex and of the individual proteins. Both methods demonstrate that complex formation at acid pH is accompanied by proton release, that complex formation at basic pH is accompanied by proton uptake, and that the change in proton binding at neutral pH, where stability of complex formation is maximal, is relatively small. Under all conditions studied, the stoichiometry of cytochrome c-cytochrome b5 complex formation is 1:1 with no evidence of higher order complex formation. Although the dependence of complex formation on pH for interaction between different species of cytochrome c and cytochrome b5 are qualitatively similar, they are quantitatively different. In particular, complex formation between yeast iso-1-cytochrome c and lipase-solubilized bovine cytochrome b5 occurs with a stability constant that is 10-fold greater than observed for the other two pairs of proteins under all conditions studied. Interaction between these two proteins is also significantly less dependent on ionic strength than observed for complexes formed by horse heart cytochrome c with either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic analysis of the interaction of cytochrome c with native and dimethyl ester heme substituted cytochrome b5

The stability of the complex formed between cytochrome c and dimethyl ester heme substituted cytochrome b5 (DME-cytochrome b5) has been determined under a variety of experimental conditions to evaluate the role of the cytochrome b5 heme propionate groups in the interaction of the two native proteins. Interaction between cytochrome c and the modified cytochrome b5 was found to produce a difference spectrum in the visible range that is very similar to that generated by the interaction of the native proteins and that can be used to monitor complex formation between the two proteins. At pH 8 [25 degrees C (HEPPS), I = 5 mM], DME-cytochrome b5 and cytochrome c form a 1:1 complex with an association constant KA of 3 (1) X 10(6) M-1. This pH is the optimal pH for complex formation between these two proteins and is significantly higher than that observed for the interaction between the two native proteins. The stability of the complex formed between DME-cytochrome b5 and cytochrome c is strongly dependent on ionic strength with KA ranging from 2.4 X 10(7) M-1 at I = 1 mM to 8.2 X 10(4) M-1 at I = 13 mM [pH 8.0 (HEPPS), 25 degrees C]. Calculations for the native, trypsin-solubilized form of cytochrome b5 and cytochrome c confirm that the intermolecular complex proposed by Salemme [Salemme, F. R. (1976) J. Mol. Biol. 102, 563] describes the protein-protein orientation that is electrostatically favored at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reaction between the superoxide anion radical and cytochrome c.

1. The superoxide anion radical (O2-) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O2- and ferrocytochrome c. 2. At 20 degrees C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4-10(6) M-1. S -1 and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O2- and the form of cytochrome c which exists above pH approximately 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O2- reacts with the form present below pH 7.45 with k = 1.4-10(6) M-1 - S-1, the form above pH 7.45 with k = 3.0- 10(5) M-1 - S-1, and the form present above pH 9.2 with k = 0. 3. The reaction has an activation energy of 20 kJ mol-1 and an enthalpy of activation at 25 degrees C of 18 kJ mol-1 both above and below pH 7.45. It is suggested that O2- may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex. 4. No reduction of ferricytochrome c by HO2 radicals could be demonstrated at pH 1.2-6.2 but at pH 5.3, HO2 radicals oxidize ferrocytochrome c with a rate constant of about 5-10(5)-5-10(6) M-1 - S-1.     

Identification of the site of interaction between cytochrome c3 and ferredoxin using peptide mapping of the cross-linked complex.

Structural studies carried out on a cross-linked complex between cytochrome c3 and ferredoxin I, both isolated from Desulfovibrio desulfuricans Norway, allowed the identification of the site of interaction between the two redox proteins. Staphylococcus aureus proteinase and chymotrypsin digestions led to characterization of peptides containing both cytochrome c3 and ferredoxin sequences. The cytochrome c3 sequences involved in the three isolated cross-linked peptides contained several lysine residues localized around the heme 4 crevice. This analysis stressed the peculiar role of lysines 100, 101, 103, 104 and 113, which could be considered as major cross-link sites, as opposed to the lysines 75, 79 and 82, which could be considered as minor cross-link sites. One cross-linked peptide, containing two ferredoxin sequences joined to one cytochrome c3 sequence, had been isolated, suggesting the possibility of more than one cross-link per covalent complex. All these results led to the identification of heme 4 of cytochrome c3 as the site of interaction for the ferredoxin I. This study confirms the proposal that could be deduced from the hypothetical structure of the complex built by computer graphics modelling (Cambillau, C., Frey, M., Mosse, J., Guerlesquin, F. and Bruschi, M. (1988) Proteins: struct., funct. genet. 4, 63-70).     

Spectrophotometric analysis of the interaction between cytochrome b5 and cytochrome c

The interaction between cytochrome c and the tryptic fragment of cytochrome b5 has been found to produce a difference spectrum in the Soret region with a maximum absorbance at 416 nm. The intensity of this difference has been used to determine the stoichiometry of complex formation and the stability of the complex formed. At pH 7.0 [25 degrees C (phosphate), mu = 0.01 M], the two proteins were found to form a 1:1 complex with an association constant, KA, of 8(3) x 10(4) M-1. The stability of the complex was found to be strongly dependent on ionic strength with KA increasing to 4(3) x 10(6) M-1 at mu = 0.001 M [25 degrees C, pH 7.0 (phosphate)]. Analysis of the dependence of KA on pH from pH 6.5 to 8 demonstrated that this complex is maximally stable between pH 7 and 8 or about midway between the isoelectric points of the two proteins. Analysis of the temperature dependence of KA revealed that formation of the complex between the two proteins is largely entropic in origin with delta Ho = 1 +/- 3 kcal/mol and delta So = 33 +/- 11 eu [pH 7.0 (phosphate), mu = 0.001 M]. This result may be explained either by the model of Clothia and Janin [Clothia, C., & Janin, J. (1975) Nature (London) 256, 705] in terms of extensive solvent reorganization upon complexation or by the model of Ross and Subramanian [Ross, P. D., & Subramanian, S. (1981) Biochemistry 20, 3096] in which the negative enthalpic and entropic contributions of short-range protein-protein interactions are offset by proton release.     

1H-n.m.r. investigation of the interaction between cytochrome c and cytochrome b5.

The interaction between eukaryotic cytochrome c and the tryptic fragment of bovine liver microsomal cytochrome b5 was studied by 1H-n.m.r. spectroscopy, and a procedure was developed that may be generally applicable to the study of macromolecular interactions by n.m.r. At pH6.3 (27 degrees C, I approx. 0.04) the two ferricytochromes were found to form a 1:1 complex with an association constant of approx. 10(3) M -1. The protein--protein-interaction region was found to encompass the region of the surface of horse cytochrome c that includes Ile-81, Phe-82, Ala-83 and Ile-85, and Lys-13 and Lys-72 of horse cytochrome c were suggested to be involved in two important intermolecular interactions. Me3Lys-72 of Candida krusei cytochrome c was shown to be involved in the interaction.     

Flash-induced proton release in Rhodopseudomonas sphaeroides spheroplasts

Proton release by flash excitations was measured with right-side-out vesicles prepared from Rhodopseudomonas sphaeroides by lysozyme-EDTA treatment followed by hypotonic treatment. Absorbance change at 586 nm in the presence of bromcresol purple was measured to monitor the pH change. In the presence of horse heart cytochrome c, which catalyzes the electron transfer from the cytochrome b-c1 complex to the primary electron donor, the single-turnover flash elicited release of about two protons per primary electron donor, which was rereduced rapidly by the added cytochrome c. The halftime of the proton release was about 70 ms at pH 6.3 and at a redox potential of about 150 mV. The rate was considerably lower than that of the electron transfer from the cytochrome b-c1 complex to cytochrome c. However, multiple flashes with intervals of 60 ms caused release of the same amount of protons as that by flashes with longer intervals. This indicated that the proton release itself was rapid, but delocalization was slower. Antimycin A inhibited the proton release, and myxothiazol almost completely abolished it.     

Spectroelectrochemical study of the cytochrome a site in carbon monoxide inhibited cytochrome c oxidase

The reduction potential of the cytochrome a site in the carbon monoxide derivative of beef heart cytochrome c oxidase has been studied under a variety of conditions by thin-layer spectroelectrochemistry. The reduction potential exhibits no ionic strength dependence and only a 9 mV/pH unit dependence between pH 6.5 and 8.5. The weak pH dependence indicates that protonation of the protein is not stoichiometrically linked to oxidoreduction over the pH range examined. The temperature dependence of the reduction potential implies a relatively large standard entropy of reduction of cytochrome a. The measured thermodynamic parameters for reduction of cyctochrome a are (all relative to the normal hydrogen electrode) delta Go'(25 degrees C) = -6.37 kcal mol-1, delta Ho' = -21.5 kcal mol-1, and delta So' = -50.8 eu. When cytochrome c is bound to the oxidase, the reduction potential of cytochrome a and its temperature dependence are not measurably affected. Under all conditions studied, the cytochrome a site did not exhibit simple Nernstian n = 1 behavior. The titration behavior of the site is consistent with a moderately strong anticooperative interaction between cytochrome a and CuA [Wang, H., Blair, D. F., Ellis, W. R., Jr., Gray, H. B., & Chan, S. I. (1985) Biochemistry (following paper in this issue)].     

The mechanism of proton translocation by the cytochrome system of mitochondria. Characterization of proton-transfer reactions associated with oxidoreductions of terminal respiratory carriers.

A direct kinetic analysis is presented of rapid proton-releasing reactions at the outer or C-side of the membrane, in ox heart and rat liver mitochondria, associated with aerobic oxidation of reduced terminal respiratory carriers in the presence of antimycin. Valinomycin plus K+ enhances the rate of cytochrome c oxidation and the rate and extent of H+ release. In the presence of valinomycin the leads to H+/e- ratio, computed on the basis of total electron flow from respiratory carriers to oxygen, varies with pH, remaining always lower than 1, and is unaffected by N-ethylmaleimide. 2-Heptyl-4-hydroxyquinoline N-oxide and 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, at concentrations which inhibit in the presence of antimycin the oxygen-induced reduction of b cytochromes, cause also a marked depression of the H+ release associated with aerobic oxidation of terminal respiratory carriers. Aerobic oxidation of the cytochrome system in mitochondria and of isolated b-c1 complex and cytochrome c oxidase results in scalar proton release from ionizable groups (redox Bohr effects). In mitochondria and submitochondrial particles, about 70% of the oxidoreductions of the components of the cytochrome system are linked to scalar proton transfer by ionizable groups. In isolated b-c1 complex scalar proton transfer, resulting from redox Bohr effect, amounts to 0.9H+ per Fe-S protein (190 muT). In isolated cytochrome c oxidase, Bohr protons amount to 0.8 per haem a + a3. The results presented indicate that the H+ release from mitochondria during oxidation of terminal respiratory carriers derives from residual antimycin-insensitive electron flow in the quinone-cytochrome c span and from redox Bohr effects in the b-c1 complex and cytochrome c oxidase. There is no sign of proton pumping by cytochrome oxidase during its transition from the reduced to the active 'pulsed' state and the first one or two turnovers.     

Electron transfer mechanism and interaction studies between cytochrome C3 and ferredoxin

Ferredoxin, cytochrome c3 and hydrogenase are specific partners of the sulfate reduction pathway of Desulfovibrio desulfuricans Norway and might be exemplary for electron exchange mechanism studies. Cytochrome c3 contains four low redox potential haems for 13 000 molecular weight. Two ferredoxins isolated from the same bacteria are dimers of 6 000 molecular weight per subunit (Ferredoxin I: one (4 Fe-4S) cluster per subunit, ferredoxin II: two (4 Fe-4 S) clusters per subunit). The amino acid sequence of ferredoxin I is reported and compared to the ferredoxin II sequence. The structural characteristics of ferredoxins and cytochrome c3 should allow a discussion on the nature of the interaction. 1H-NMR spectra of ferredoxin I and cytochrome c3 in the absence and presence of ferredoxin are presented.     

The type I/type II cytochrome c3 complex: an electron transfer link in the hydrogen-sulfate reduction pathway

In Desulfovibrio metabolism, periplasmic hydrogen oxidation is coupled to cytoplasmic sulfate reduction via transmembrane electron transfer complexes. Type II tetraheme cytochrome c3 (TpII-c3), nine-heme cytochrome c (9HcA) and 16-heme cytochrome c (HmcA) are periplasmic proteins associated to these membrane-bound redox complexes and exhibit analogous physiological function. Type I tetraheme cytochrome c3 (TpI-c3) is thought to act as a mediator for electron transfer from hydrogenase to these multihemic cytochromes. In the present work we have investigated Desulfovibrio africanus (Da) and Desulfovibrio vulgaris Hildenborough (DvH) TpI-c3/TpII-c3 complexes. Comparative kinetic experiments of Da TpI-c3 and TpII-c3 using electrochemistry confirm that TpI-c3 is much more efficient than TpII-c3 as an electron acceptor from hydrogenase (second order rate constant k = 9 x 10(8) M(-1) s(-1), K(m) = 0.5 microM as compared to k = 1.7 x 10(7) M(-1) s(-1), K(m) = 40 microM, for TpI-c3 and TpII-c3, respectively). The Da TpI-c3/TpII-c3 complex was characterized at low ionic strength by gel filtration, analytical ultracentrifugation and cross-linking experiments. The thermodynamic parameters were determined by isothermal calorimetry titrations. The formation of the complex is mainly driven by a positive entropy change (deltaS = 137(+/-7) J mol(-1) K(-1) and deltaH = 5.1(+/-1.3) kJ mol(-1)) and the value for the association constant is found to be (2.2(+/-0.5)) x 10(6) M(-1) at pH 5.5. Our thermodynamic results reveal that the net increase in enthalpy and entropy is dominantly produced by proton release in combination with water molecule exclusion. Electrostatic forces play an important role in stabilizing the complex between the two proteins, since no complex formation is detected at high ionic strength. The crystal structure of Da TpI-c3 has been solved at 1.5 angstroms resolution and structural models of the complex have been obtained by NMR and docking experiments. Similar experiments have been carried out on the DvH TpI-c3/TpII-c3 complex. In both complexes, heme IV of TpI-c3 faces heme I of TpII-c3 involving basic residues of TpI-c3 and acidic residues of TpII-c3. A secondary interacting site has been observed in the two complexes, involving heme II of Da TpII-c3 and heme III of DvH TpI-c3 giving rise to a TpI-c3/TpII-c3 molar ratio of 2:1 and 1:2 for Da and DvH complexes, respectively. The physiological significance of these alternative sites in multiheme cytochromes c is discussed.     

An investigation of heavy meromyosin-ADP binding equilibria by proton release measurements.

The interaction of magnesium-ADP with skeletal muscle heavy meromyosin has been studied by measuring the accompanying release of protons. Total pH changes of the order of 0.03 were involved, and measurements were performed with a discrimination of some ten-thousandths of a pH unit. At pH 8.0 and 25 degrees C about 0.5 mol of protons per mol of heavy meromyosin is released at saturation. A stoichiometry of binding close to 2 mol of ADP per mol of protein was found, with a binding constant, obtained from the proton release titration curve (pH 8.0, 25 degrees C), of 2 X 10(5) M-1. At 5 degrees C the release of protons per mole is slightly greater, and the binding constant is somewhat increased, reflecting a negative enthalpy of binding. Similar proton release behavior is observed in the presence of manganous ions in place of magnesium. The liberation of protons is thus unrelated to the temperature-dependent isomerization of myosin in the presence of substrate. Alkylation of a reactive thiol group (SH1) does not change the proton liberation at pH 8.0. From the pH dependence of proton release, the association constant of heavy meromyosin with magnesium-ADP at other pH values can be inferred and shows an appreciable rise as the pH increases. The pH-proton release profile also allows the pK of the ionizing groups perturbed by the ligand to be deduced. At least two groups ionizing above pH 7 and one below are involved. Their pK's in the unperturbed state are assigned as 8.5, 9.3, and about 6.6, respectively; they are displaced in the complex to about 8.0, 9.1, and 6.3. A relation to the pH-activity profile of myosin ATPase is indicated. The pH-proton release profile is somewhat changed when the SH1 group is alkylated. Measurements with potassium-ADP, in the absence of magnesium, show that at pH 8.0 there is no proton release but rather a sizeable proton absorption (about 0.5 mol of protons per mol of heavy meromyosin). The association constant derived from the titration curves (pH 8.0, 25 degrees C) is 3 X 10(4) M-1.     

Proton-coupled structural changes upon binding of carbon monoxide to cytochrome cd1: a combined flash photolysis and X-ray crystallography study

We have investigated dynamic events after flash photolysis of CO from reduced cytochrome cd(1) nitrite reductase (NiR) from Paracoccus pantotrophus (formerly Thiosphaera pantotropha). Upon pulsed illumination of the cytochrome cd(1)-CO complex, at 460 nm, a rapid (<50 ns) absorbance change, attributed to dissociation of CO, was observed. This was followed by a biphasic rearrangement with rate constants of 1.7 x 10(4) and 2.5 x 10(3) s(-1) at pH 8.0. Both parts of the biphasic rearrangement phases displayed the same kinetic difference spectrum in the region of 400-660 nm. The slower of the two processes was accompanied by proton uptake from solution (0.5 proton per active site at pH 7.5-8.5). After photodissociation, the CO ligand recombined at a rate of 12 s(-1) (at 1 mM CO and pH 8.0), accompanied by proton release. The crystal structure of reduced cytochrome cd(1) in complex with CO was determined to a resolution of 1.57 A. The structure shows that CO binds to the iron of the d(1) heme in the active site. The ligation of the c heme is unchanged in the complex. A comparison of the structures of the reduced, unligated NiR and the NiR-CO complex indicates changes in the puckering of the d(1) heme as well as rearrangements in the hydrogen-bonding network and solvent organization in the substrate binding pocket at the d(1) heme. Since the CO ligand binds to heme d(1) and there are structural changes in the d(1) pocket upon CO binding, it is likely that the proton uptake or release observed after flash-induced CO dissociation is due to changes of the protonation state of groups in the active site. Such proton-coupled structural changes associated with ligand binding are likely to affect the redox potential of heme d(1) and may regulate the internal electron transfer from heme c to heme d(1).     

Proton stoichiometry of the cytochrome c peroxidase mechanism as a function of pH.

The proton stoichiometry for the oxidation of cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) to cytochrome c peroxidase Compound I by H2O2, for the reduction of cytochrome c peroxidase Compound I to cytochrome c peroxidase Compound II by ferrocyanide, and for the reduction of cytochrome c peroxidase Compound II to the native enzyme by ferrocyanide has been determined as a function of pH between pH 4 and 8. The basic stoichiometry for the reaction is that no protons are required for the oxidation of the native enzyme to Compound I, while one proton is required for the reduction of Compound I to Compound II, and one proton is required for the reduction of Compound II to the native enzyme. Superimposed upon the basic stoichiometry is a contribution due to the perturbation of two ionizable groups in the enzyme by the redox reactions. The pKa values for the two groups are 4.9 +/- 0.3 and 5.7 +/- 0.2 in the native enzyme, 4.1 +/- 0.4 and 7.8 +/- 0.2 in Compound I, and 4.3 +/- 0.4 and 6.7 +/- 0.2 in Compound II.     

High affinity binding of Bcl-xL to cytochrome c: possible relevance for interception of translocated cytochrome c in apoptosis

The release of cytochrome c from mitochondria and apoptosis relies on several preferential and selective interactions involving the Bcl-2 family of proteins. There is, however, no direct evidence for the interaction of cytochrome c with these proteins at any stage of apoptosis. To investigate if any pro-survival protein from the Bcl-2 family could intercept cytochrome c after its translocation from mitochondria, the interaction of cytochrome c with bacterially expressed human Bcl-x(L) was studied at pH 7. In size-exclusion chromatography, purified full-length His(6)-tagged Bcl-x(L) migrated as both dimer and monomer, of which the monomeric fractions were used for experiments. Coimmunoprecipitation studies show that cytochrome c interacts with Bcl-x(L). The extent of caspase activity in cell lysate elicited by externally added cytochrome c is reduced when a preincubated mixture of Bcl-x(L) and cytochrome c is used instead. Equilibrium binding monitored by optical absorption of cytochrome c as a function of titrating concentrations of Bcl-x(L) yields the association constant, K(ass) = 8.4(+/- 4) x 10(6) M(-1) (binding affinity, K(diss) = 1/K(ass) approximately 120 nM) which decreases at high ionic strength. The rates for binding of Bcl-x(L) to cytochrome c, studied by stopped-flow kinetics at pH 7, show that the bimolecular rate constant for binding, k(bi) = 0.24 x 10(6) M(-1) s(-1). Values of the thermodynamic and kinetic parameters for Bcl-x(L)-cytochrome c interaction are very similar to those known for regulatory protein-protein interactions in apoptosis.     

Rate enhancement of the internal electron transfer in cytochrome c oxidase by the formation of a peroxide complex; its implication on the reaction mechanism of cytochrome c oxidase

The oxidation of reduced cytochrome c oxidase by hydrogen peroxide was investigated with stopped-flow methods. It was reported by us previously (A.C.F. Gorren, H. Dekker and R. Wever (1986) Biochim. Biophys. Acta 852, 81-92) that at low H2O2 concentrations cytochrome a is oxidised simultaneously with cytochrome a3, but that at higher H2O2 concentrations the oxidation of cytochrome a is slower than that of cytochrome a3. We now report that for high peroxide concentrations (10-45 mM) the oxidation rate of cytochrome a increased linearly with the concentration of H2O2 (k = 700 M-1.S-1). Upon extrapolation to zero H2O2 concentration an intercept with a value of 16 s-1 (at 20 degrees C and pH 7.4) was found. A reaction sequence is described to explain these results; according to this model the rate constant (16 S-1) at zero H2O2 concentration represents the true value of the rate of electron transfer from cytochrome a to cytochrome a3 when the a3-CuB site is oxidised and unligated. However, when a complex of hydrogen peroxide with oxidised cytochrome a3 is formed, this rate is strongly enhanced. The slope (700 M-1.S-1) would then represent the rate of cytochrome a3(3+)-H2O2 complex formation. From experiments in which the pH was varied, we conclude that the reaction of H2O2 with cytochrome a3(2+) is independent of pH, whereas the electron-transfer rate from cytochrome a to cytochrome a3 gradually decreases with increasing pH. From the temperature dependence we could calculate values of 23 kJ.mol-1 and 45 kJ.mol-1 for the activation energies of the oxidations by H2O2 of cytochrome a3(2+) and cytochrome a2+, respectively. The similarity of the values that were obtained for cytochrome a oxidation both with H2O2 and with O2 as the electron acceptor suggests that the reactions share the same mechanism. In 2H2O the reactions studied decreased in rate. For the reaction of 2H2O2 with reduced cytochrome a3 in 2H2O, a small effect was found (15% decrease in rate constant). However, the internal electron-transfer rate from cytochrome a to cytochrome a3 decreased by 50%, Our results suggest that the internal electron transfer is associated with proton translocation.