Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase.

The light-induced difference spectra of the fully reduced (a2+ a23+-CO) complex and the mixed-valence carboxycytochrome c oxidase (a3+ a23+-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a23+-CO complex. This would confirm that the spectral properties of cytochrome a3 are not affected by the redox state of cytochrome a and CuA. When the absorbance changes after photolysis of cytochrome a23+-CO with a laser flash were followed on a time scale from 1 mus to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7 X 10(3) s-1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a3. Concomitantly, a decrease of the 830 nm band was observed due to reduction of CuA. This demonstrates that in the partially reduced enzyme a pathway is present between CuA and the cytochrome a3-CuB pair, via which electrons are transferred rapidly.

A comparison of three preparations of cytochrome c oxidase. Optical absorbance spectra, EPR spectra and reaction towards ligands.

Three preparations of cytochrome c oxidase, the preparation as traditionally prepared in our laboratory as described by Van Buuren (1992; PhD Thesis, University of Amsterdam), a preparation according to Volpe and Caughey (Biochem. Biophys. Res. Commun. 61 (1974) 502-509) and a preparation of 'fast' cytochrome c oxidase (Brandt, U., Schägger, H. and Von Jagow, G. (1989) Eur. J. Biochem. 182, 705-711), are compared in their reaction with cyanide and carbon monoxide. The reaction with cyanide is nearly as fast for the Van Buuren preparation as for the 'fast' preparation, but much slower for the Volpe-Caughey preparation. Mixed-valence cytochrome c oxidase (cytochrome a3 and CuB reduced with carbon monoxide bound and cytochrome a and CuA oxidized) is prepared by anaerobic incubation with carbon monoxide. With the Van Buuren preparation complete formation of the species takes 4 h, whereas with the Volpe-Caughey preparation it takes 20 h. Longer incubation under CO results in partial reduction of cytochrome a and CuA. With the 'fast' preparation mixed-valence cytochrome c oxidase is formed after more than one day of incubation with CO, but it is stable for at least 3 days. The presence of oxidized cytochrome c did enhance the reactivity towards cyanide and towards carbon monoxide in cytochrome c oxidase of all three preparations. Furthermore, optical and EPR spectra of the preparations of cytochrome c oxidase are compared. The Volpe-Caughey preparation has an intense g' = 12 EPR-signal, the Van Buuren preparation has hardly any g' = 12 signal and the 'fast' preparation has no g' = 12 signal. In the 'fast' preparation the low-spin heme signal is shifted (from g = 3.00 to g = 2.97). The absorbance spectra of the three preparations in the Soret region are similar with a maximum at 424 nm. Only the 'fast' preparation as isolated was completely oxidized, whereas the other preparations were partially reduced. It was concluded that differences in the reaction of cytochrome c oxidase with ligands are determined by the internal or external ligand bound to the cytochrome a3-CuB couple.

Biochemical and biophysical studies on cytochrome c oxidase. XIII. Effect of cholate on the enzymic activity.

(1) Cholate is a mixed-type inhibitor of the enzymic activity of cytochrome c oxidase. The rate equations for mixed-type inhibition of the enzyme have been derived, based on Minnaert's Mechanism IV (1961, Biochim. Biophys. Acta 50, 23-34). The Ki of cholate for the free enzyme (E) and for the complexes of the enzyme with cytochrome c (ES and EP) was determined, being 125 and 190 microM, respectively. (2) Comparison of the properties of cholate and the intrinsic inhibitor of cytochrome c oxidase (Van Buuren et al. (1971) Biochim. Biophys. Acta 243, 468-480) with respect to their type of inhibition and their affinity for enzyme, reveals that they are identical.

The respiratory chain of Azotobacter vinelandii. III. The effect of cyanide in the presence of substrates.

(1) Cyanide (100 microM) causes a rapid disappearance of the band (648 nm) of oxidized cytochrome d in particles of Azotobacter vinelandii oxidizing NADH. The rate of disappearance of the band can be related to the rate of inhibition of the oxygen consumption. (2) The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide in the presence of substrates deviate from first-order, indicating that at least two conformations of the enzyme are involved. (3) The rate of binding of cyanide to cytohrrome d increases the larger the rate of turnover of the oxidase. From this it is concluded that cyanide binds preferentially to the enzymically active oxidized conformation of cytochrome d. (4) The instantaneous inhibition of the oxidation of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) by cyanide is related neither to the binding of cyanide to cytochrome d as determined spectrophotometrically, nor to the rate of inhibition of the oxidation of NADH. This indicates that different oxidases are involved in the oxidation of NADH and of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), in line with the conclusion of Jones and Redfearn (1967) Biochim. Biophys. Acta 143, 340-353. (5) From experiments in which the change in redox states of cytochrome b1 and c-551 were related to the rate of binding of cyanide to cytochrome d, it is concluded that the cytochromes b1 and d are located in the main pathway to oxygen, whereas cytochrome c-551 functions in the branch via cytochrome o. (6) After prolonged incubation of particles with cyanide in the presence of NADH a residual activity (5%) was found in which a b-type cytochrome is involved. This shows the existence of a third but minor pathway to oxygen.

The interaction between heme and protein in cytochrome c1.

The optical spectrum of reduced bovine cytochrome c1 at 77 K shows a fine splitting of the beta-band, which is indicative of the native conformation of the protein. At room temperature, this conformation is reflected in an absorbance band at 530 nm. The exposure of the heme of ferrocytochrome c1, investigated by means of solvent-perturbation spectroscopy, appears to be extremely sensitive to temperature and SH reagents bound to the oxidized protein. Addition of combinations of potential ligands to the isolated tryptic heme peptide of cytochrome c1 reveals that only a mixture of methionine and cysteine (or their equivalents) generates a beta-band at 77 K which is identical in shape to that of native cytochrome c1. In the EPR spectrum of a complex of ferrocytochrome c1 and nitric oxide at pH 10.5, no hyperfine splitting derived from a second ligated nitrogen atom could be detected. The results indicate that methionine and cysteine are the axial ligands of heme in cytochrome c1. The EPR spectrum of isolated ferricytochrome c1 is that of a low-spin heme iron compound with a gz value of 3.36 and a gy value of 2.04.

The absorbance coefficient of beef heart cytochrome c1.

Isolated cytochrome c1 contains endogenous reducing equivalents. They can be removed by treating the protein with sodium dithionite followed by chromatography. This treatment has no effect on the reaction with cytochrome c, nor does it alter the optical spectrum, or the polypeptide or amino acid composition of the protein. Both the titration of dithionite-treated ferrocytochrome c1 with potassium ferricyanide and the anaerobic titration of dithionite-treated ferricytochrome c1 with NADH in the presence of phenazine methosulphate lead to the same value for the absorbance coefficient of cytochrome c1: 19.2 mM-1 . cm-1 at 552.4 nm for the reduced-minus-oxidised form. This value was also obtained when the haem content was determined by comparing the spectra of the reduced pyridine haemochromes of cytochrome c and cytochrome c1. Comparison of the optical spectra of cytochrome c and cytochrome c1 by integration shows equal transition moments for the transitions in the porphyrin systems of both proteins. A set of equations with which the concentration of the cytochromes aa3, b, c and c1 can be calculated from one reduced-minus-oxidised difference spectrum of a mixture of these proteins.

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+.

The effect of pH and ionic strength on the pre-steady-state reaction of cytochrome c and cytochrome aa3.

(1) In the pH range between 5.0 and 8.0, the rate constants for the reaction of ferrocytochrome c with both the high- and low-affinity sites on the cytochrome aa3 increased by a factor of approx. 2 per pH unit. (2) The pre-steady-state reaction between ferrocytochrome c and cytochrome aa3 did nt cause a change in the pH of an unbuffered medium. Furthermore, it was found that this reaction and the steady-state reaction are equally fast in H2O and 2H2O. From these results it was concluded that no protons are directly involved in a rate-determining reaction step. (3) Arrhenius plots show that the reaction between ferrocytochrome c and cytochrome aa3 requires a higher enthalpy of activation at temperatures below 20 degrees C (15--16 kcal/mol) as compared to that at higher temperature (9 kcal/mol). We found no effect of ionic strength on the activation enthalpy of the pre-steady-state reaction, nor on that of the steady-state reaction. This suggests that ionic strength does not change the character of these reactions, but merely affects the electrostatic interaction between both cytochromes.

An EPR study of the photodissociation reactions of oxidised cytochrome c oxidase-nitric oxide complexes.

Complexes of oxidised cytochrome c oxidase with NO in the absence and presence of ligands such as formate, fluoride and cyanide are photodissociable. After photodissociation at 10 K the EPR spectrum of the high-spin cytochrome a3+3 in the absence of ligands or in the presence of fluoride or formate disappears - as does the EPR spectrum of the low-spin cytochrome a3+3 in the presence of cyanide. The action spectra of the photodissociation reaction of these complexes show slight differences but all have maxima at 640-660 nm and below 400 nm, and are assigned to a diamagnetic Cu+B-NO+ complex. The differences in the action spectra in the presence of various ligands are due to binding of these anions to the cytochrome (a3-CuB) couple. The disappearance of the cytochrome a3 signal upon photodissociation of the Cu+B-NO+ complex is explained by a magnetic interaction between cytochrome a3+3 and Cu2+B in the photodissociated complex. The temperature at which NO recombines with Cu2+B is about 30 K and slightly affected by the presence of added ligands. It is suggested that in the oxidised ligand-cytochrome c oxidase complexes the coupling ligand between cytochrome a3+3 and Cu2+B is cyanide, fluoride and formate. The observation that two ligands may bind simultaneously to the cytochrome a3-CuB couple leads to further support for the notion that during turnover of cytochrome c oxidase both metal ions are involved in binding and reduction of oxygen.

Effects of cytochrome c on the oxidation of reduced cytochrome c oxidase by hydrogen peroxide.

The oxidation of the redox centres in reduced cytochrome c oxidase by hydrogen peroxide was studied by stopped-flow spectrophotometry in the absence and presence of reduced cytochrome c. The oxidation rate of cytochrome a decreased in the presence of cytochrome c. This effect was more pronounced at low than at high ionic strength. Cytochrome c did not influence the time-course of the oxidation of CuA or cytochrome a3. The oxidation of cytochrome c itself was faster at low ionic strength. The results suggest that the effect of cytochrome c is caused by re-reduction of cytochrome a by cytochrome c, the rate of which is dependent upon the ionic strength. We conclude that cytochrome a and cytochrome c are in equilibrium and that the equilibrium constant depends on the ionic strength. At low ionic strength, as a complex is formed between cytochrome c and cytochrome c oxidase, cytochrome a is more reduced than at high ionic strength conditions, when no such complex exists. Since CuA is oxidized at the same rate whether cytochrome c is present or not, we conclude that electron transfer from cytochrome a or cytochrome c to CuA is slower than electron transfer from CuA to cytochrome a or/and to the cytochrome a2-CuB couple.

The cytochrome c oxidase-azide-nitric oxide complex as a model for the oxygen-binding site.

The complex of cytochrome c oxidase with NO and azide has been studied by EPR at 9.2 and 35 GHz. This complex which shows delta ms = 2 EPR triplet and strong anisotropic signals, due to the interaction of cytochrome a2+3 X NO (S = 1/2) and Cu2+B (S = 1/2), is photodissociable . Its action spectrum is similar to that of cytochrome a2+3 X NO with bands at 430, 560 and 595 nm, but shows an additional band in the near ultraviolet region. The quantum yield of the photodissociation process of cytochrome a2+3 X NO in the metal pair appears to depend on the redox state of CuB. When the photolysed sample was warmed to 77 K, a complex was observed with the EPR parameters of cytochrome a3+3 - N-3 - Cu1 +B (S = 1/2). This process of electron and ligand transfer can be reversed by heating the sample to 220 K. It is suggested that in the triplet species azide is bound to Cu2+B whereas NO is bridged between Cu2+B and the haem iron of the cytochrome a2+3. The complex has a triplet ground state and a singlet excited state with an exchange interaction J = -7.1 cm-1 between both spins. The anisotropy in the EPR spectra is mainly due to a magnetic dipole-dipole interaction between cytochrome a2+3 X NO and Cu2+B. From simulations of the triplet EPR spectra obtained at 9 and 35 GHz, a value for the distance between the nitroxide radical and Cu2+B of 0.33 nm was found. A model of the NO binding in the cytochrome a3-Cu pair shows a distance between the haem iron of cytochrome a3 and CuB of 0.45 nm. It is concluded that the cytochrome a3-CuB pair forms a cage in which the dioxygen molecule is bidentate coordinated to the two metals during the catalytic reaction.

EPR signals of oxidized plastocyanin in intact algae.

(1) An electron paramagnetic resonance (EPR) signal was observed at g = 2.05 in the low temperature spectra of intact cells of green, red and blue-green algae and of spinach chloroplasts. The g-value and the shape of the signal were similar to that of purified, soluble plastocyanin. (2) The amount of the copper protein, determined from the EPR signal height, was estimated to be nearly the same in all the studied organisms on the basis of the concentration of chlorophyll. Furthermore, it was found that the amount of the copper protein, determined from the EPR signal height in spinach chloroplasts corresponds with that of plastocyanin as determined chemically by Katoh, S., Suga, I., Shiratori, I. and Takamiya, A. (1961) Arch. Biochem. Biophys. 94, 136-141. (3) Experiments with far-red and red illumination show that the site of the copper protein in vivo is in the electron transport pathway between Photosystems 1 and 2. Plastocyanin is not oxidized by illumination at 77 degrees K, indicating that no electron transfer occurs between the primary electron donor of Photosystem 1, P700, and plastocyanin at that temperature. Furthermore, the experiments suggest that in the intact cells of the studied algae, plastocyanin is not only reduced by Photosystem 2 but also by cyclic electron transport around Photosystem 1.

The mechanism of reduction of cytochrome c as studied by pulse radiolysis.

1. The reaction of hydrated electrons with ferricytochrome c was studied using the pulse-radiolysis technique. 2. In 3.3 mM phosphate-buffer (pH 7.2), 100 mM methanol and at a concentration of cytochrome c of less than 20 muM the reduction kinetics of ferricytochrome c by hydrated electrons is a bimolecular process with a rate constant of 4.5-10-10 M-1-S-1 (21 degrees C). 3. At a concentration of cytochrome c of more than 20 muM the apparent order of the reaction of hydrated electrons with ferricytochrome c measured at 650 nm decreases due to the occurrence of a rate-determining first-order process with an estimated rate constant of 5-10-6s-1 (pH 7.2, 21 degrees C). 4. At high concentration of cytochrome c the reaction-time courses measured at 580 and 695 nm appear to be biphasic. A rapid initial phase (75% and 30% of total absorbance change at 580 and 695 nm, respectively), corresponding to the reduction reaction, is followed by a first-order change in absorbance with a rate constant of 1.3-10-5 S-1 (pH 7.2, 21 degrees C). 5. The results are interpreted in a scheme in which first a transient complex between cytochrome c and the hydrated electron is formed, after which the heme iron is reduced and followed by relaxation of the protein from its oxidized to its reduced conformation. 6. It is calculated that one of each three encounters of the hydrated electron and ferricytochrome c results in a reduction of the heme iron. This high reaction probability is discussed in terms of charge and solvent interactions. 7. A reduction mechanism for cytochrome c is favored in which the reduction equivalent from the hydrated electron is transmitted through a specific pathway from the surface of the molecule to the heme iron.

Biochemical and biophysical studies on cytochrome c oxidase. XX. Reaction with sulphide.

1. Upon addition of sulphide to oxidized cytochrome c oxidase, a low-spin heme sulphide compound is formed with an EPR signal at gx = 2.54, gy = 2.23 and gz = 1.87. Concomitantly with the formation of this signal the EPR-detectable low-spin heme signal at g = 3 and the copper signal near g = 2 decrease in intensity, pointing to a partial reduction of the enzyme by sulphide. 2. The addition of sulphide to cytochrome c oxidase, previously reduced in the presence of azide or cyanide, brings about a disappearance of the azido-cytochrome c oxidase signal at gx = 2.9, gy = 2.2, and gz = 1.67 and a decrease of the signal at g = 3.6 of cyano-cytochrome c oxidase. Concomitantly the sulphide-induced EPR signal is formed. 3. These observations demonstrate that azide, cyanide and sulphide are competitive for an oxidized binding site on cytochrome c oxidase. Moreover, it is shown that the affinity of cyanide and sulphide for this site is greater than that of azide.

Inhibition of pig kidney diamine oxidase by nazlinin and nazlinin derivatives.

Nazlinin (1-(4-butylamino)-1,2,3,4-tetrahydro-beta-carboline) (1), an alkaloid recently isolated from Nitraria schoberi, and its two derivatives, 1-(4-butylamino)-3,4-dihydro-beta-carboline (2) and 1-(4-butylamino)-beta-carboline (3), were synthesized and their interaction with pig kidney diamine oxidase (PKDO) was studied. Nazlinin appeared to be a very poor substrate while 3 was a good substrate with an apparent Km of 9.3-10(-5) M. The enzyme was inhibited by 1 and 2. With both compounds the mode of inhibition found was non-competitive and inhibition constants calculated from the slopes and intercepts of double-reciprocal plots show that 2 is a much more potent inhibitor than the natural product. The relationship between the structure of these compounds and the results found is discussed.

The oxidation-reduction kinetics of the reaction of cytochrome c1 with non-physiological redox agents.

The kinetics of the oxidation-reduction reactions of cytochrome c1 with ascorbate, ferricyanide, triphenanthrolinecobalt(III) and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) have been examined using the stopped-flow technique. The reduction of ferricytochrome c1 by ascorbic acid is investigated as a function of pH. It is shown that at neutral and alkaline pH the reduction of the protein is mainly performed by the doubly deprotonated form of ascorbate. From the ionic-strength-dependence studies of the reactions of cytochrome c1 with ascorbate, ferricyanide and triphenanthrolinecobalt(III), it is demonstrated that the reactions rate is governed by electrostatic interactions. The second-order rate constants for the reaction of cytochrome c1 with ascorbate, ferricyanide, TMPD and triphenanthrolinecobalt(III) are 1.4 . 10(4), 3.2 . 10(3), 3.8 . 10(4) and 1.3 . 10(8) M-1 . s-1 (pH 7.9, I = 0, 10 degrees C), respectively. Application of the Debye-Hückel theory to the data of the ionic-strength-dependence studies of these redox reactions of cytochrome c1 yielded for ferrocytochrome c1 and ferricytochrome c1 a net charge of --5 and --4, respectively. The latter value is close to that of --3 for the oxidized enzyme, calculated from the amino acid sequence of the protein. This implies that not a local charge on the surface of the protein, but the overall net charge of cytochrome c1 governs the reaction rate with small redox molecules.

The pre-steady state reaction of ferrocytochrome c with the cytochrome c-cytochrome aa3 complex.

1. Using stopped-flow technique we have investigated the electron transfer form cytochrome c to cytochrome aa3 and to the (porphyrin) cytochrome c-cytochrome aa3 complex. 2. In a low ionic strength medium, the pre-steady state reaction occurs in a biphasic way with rate constants of at least 2.10(8) M-1.s-1 and about 10(7) M-1.S-1 (I=8.8 mM, pH 7.0, 10 degrees C), respectively. 3. A comparison of the rate constants, determined in the presence of an excess of cytochrome c with those found in the presence of an excess of cytochrome aa3 reveals the existence of two slower reacting sites on the functional unit (2 hemes and 2 coppers) of cytochrome aa3. On basis of these results we discuss various models. If no site-site interactions are assumed (non-cooperative model) cytochrome aa3 has 2 high and 2 low affinity sites available for the reaction with ferrocytochrome c. If negative cooperativity occurs, cytochrome aa3 has 2 high affinity sites which change into 2 low affinity sites upon binding of one cytochrome c molecule. The latter model is favoured.

The effect of pH and ionic strength on the steady-state activity of isolated cytochrome C oxidase.

1. The turnover number and apparent Km of isolated beef-heart cytochrome c oxidase were found to increase continuously when the pH was lowered from 8.6 to 4.6 (turnover number 32-630 s-1). In this pH range neither irreversible denaturation of the enzyme nor an optimum for the turnover number was observed. 2. The turnover number of cytochrome c oxidase was found to be independent of ionic strength. It was concluded that the dependence of the activity of cytochrome c oxidase on ionic strength is caused by a change in the value of Km for cytochrome c. 3. The pH dependence of the turnover number of cytochrome c oxidase can be described by a simple model in which at least three sites on the complex of cytochrome c oxidase with cytochrome c (pKa 8.0, 6.5 and 4.8) can take up a proton.

The reduction of porphyrin cytochrome c by hydrated electrons and the subsequent electron transfer reaction from reduced porphyrin cytochrome c to ferricytochrome c.

1. Hydrated electrons, produced by pulse radiolysis react with porphyrin cytochrome c with a bimolecular rate constant of 3-10(10) M-1 S-1 at 21 degrees C and pH 7.4. 2. After the reduction step an absorbance change with a half-life of 5 microns is observed with the spectral range of 430-470 nm. A relatively stable intermediate then decays with a half-life of 15 s. 3. The spectrum of the intermediate observed 50 microns after the generation of hydrated electrons shows a broad absorption band between 600 and 700 nm and a peak at 408 nm. The spectrum is attributed to the protonated form of an initially produced porphyrin anion radical. 4. Reduced porphyrin cytochrome c reacts with ferricytochrome c with a bimolecular constant of 2-10(5) M-1- S-1 in 2 mM phosphate pH 7.4, at 21 degrees C and of 2 - 10(6) M-1-S-1 under the same conditions but at 1 M ionic strength. It is proposed that electron transfer in an analogous exchange reaction between ferrocytochrome c and ferricytochrome c occurs via the exposed part of the haem.

Ionic strength effects on cytochrome aa3 kinetics.

1. The occurrence of an optimal ionic strength for the steady-state activity of isolated cytochrome aa3 can be attributed to two opposite effects: upon lowering of the ionic strength the affinity between cytochrome c and cytochrome aa3 increases, whereas in the lower ionic strength region the formation of a less active cytochrome c-aa3 complex limits the ferrocytochrome c association to the low affinity site. 2. At low ionic strength, the reduction of cytochrome c-aa3 complex by ferrocytochrome c1 proceeds via non-complex-bound cytochrome c. Under these conditions the positively charged cytochrome c provides the electron transfer between the negatively charged cytochromes c1 and aa3. 3. Polylysine is found to stimulate the release of tightly bound cytochrome c from the cytochrome c-aa3 complex. This property points to the existence of negative cooperativity between the two binding sites. We suggest that the stimulation is not restricted to polylysine, but also occurs with cytochrome c. 4. Dissociation rates of both high and low affinity sites on cytochrome aa3 were determined indirectly. The dissociation constants, calculated on the basis of pre-steady-state reaction rates at an ionic strength of 8.8 mM, were estimated to be 0.6 nM and 20 microM for the high and low affinity site, respectively.