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-cytochromeaa₃ 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 · 10⁸ M^?1 · s^?1 and about 10⁷ M^?1 · s^?1 (I = 8.8 mM, pH 7.0, 10° 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 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 · 10⁷ M^?1 · s^?1 at low ionic strength (I = 223 mM, 10°C). The value of this rate constant decreases to 1.8 · 10⁵ 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 · 10⁵ M^?1 · s^?1 and k_?1 = 3.3 · 10⁵ M^?1 · s^?1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10°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⁺ai cytochrome c³⁺₁ + cytochrome c²⁺.     

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.     

Kinetics of flash-induced electron transfer between bacterial reaction centres,mitochondrial ubiquinol: Cytochrome c oxidoreductase and cytochrome c

Ascorbate-reduced horse heart cytochrome c reduces photo-oxidized bacterial reaction centres with a second-order rate constant of (5–8) · 10⁸ M^?1 · s^?1 at an ionic strength of 50 mM. In the absence of cytochrome c, the cytochrome c₁ in the ubiquinol:cytochrome c oxidoreductase is oxidized relatively slowly (k = 3.3 · 10⁵ M^?1 · s^?1). Ferrocytochrome c binds specifically to ascorbate-reduced reductase, with a K_d of 0.6 μM, and only the free cytochrome c molecules are involved in the rapid reduction of photo-oxidized reaction centres. The electron transfer between ferricytochrome c and ferrocytochrome c₁ of the reductase is rapid, with a second-order rate constant of 2.1 · 10⁸ M^?1 · s^?1 at an ionic strength of 50 mM. The rate of electron transfer from the Rieske iron-sulphur cluster to cytochrome c₁ is even more rapid. The cytochrome b of the ubiquinol:cytochrome c oxidoreductase can be reduced by electrons from the reaction centres through two pathways: one is sensitive to antimycin and the other to myxothiazol. The amount of cytochrome b reduced in the absence of antimycin is dependent on the redox potential of the system, but in no case tested did it exceed 25% of the amount of photo-oxidized reaction centres.     

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 μM for the high and low affinity site, respectively.     

Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with K_i values of 4.5, 91, 200 and 330 μM, respectively.2. The inhibition constant K_i of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme.3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k₁ of 33 M^?1 · s^?1 and dissociation constant K_d of 3.9 mM.4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M^?1 · s^?1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes.5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome a₃-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

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 cytochrome aa₃ increase by a factor of approx. 2 per pH unit. (2) The pre-steady-state reaction between ferrocytochrome c and cytochrome aa₃ did not 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°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.     

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 is presented 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.     

Electron transfer between horse heart and Candida krusei cytochromes c in the free and bound states

Electron transfer between horse heart and Candida krusei cytochromes c in the free and phosvitin-bound states was examined by difference spectrum and stopped-flow methods. The difference spectra in the wavelength range of 540–560 nm demonstrated that electrons are exchangeable between the cytochromes c of the two species. The equilibrium constants of the electron transfer reaction for the free and phosvitin-bound forms, estimated from these difference spectra, were close to unity at 20°C in 20 mM Tris-HCl buffer (pH 7.4). The electron transfer rate for free cytochrome c was (2–3) · 10⁴ M^?1 · s^?1 under the same conditions. The transfer rate for the bound form increased with increase in the binding ratio at ratios below half the maximum, and was almost constant at higher ratios up to the maximum. The maximum electron exchange rate was about 2 · 10⁶ M^?1 · s^?1, which is 60–70 times that for the free form at a given concentration of cytochrome c. The activation energy of the reaction for the bound cytochrome c was equal to that for the free form, being about 10 kcal/mol. The dependence of the exchange rate on temperature, cytochrome c concentration and solvent viscosity suggests that enhancement of the electron transfer rate between cytochromes c on binding to phosvitin is due to increase in the collision frequency between cytochromes c concentrated on the phosvitin molecule.     

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 reaction 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 1.4·10⁴, 3.2·10³, 3.8·10⁴ and 1.3·10⁸ M^?1·s^?1 (pH 7.0, I = 0, 10°C), respectively. Application of the Debye-Hückel theory to the 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.     

Ferrocyanide as electron donor to cytochrome aa3. Cytochrome c requirement for oxygen uptake

1. In the absence of cytochrome c, ferrocyanide or ferrous sulphate reduces cytochrome c oxidase (EC 1.9.3.1), but no continuous oxygen uptake ensues, as it does with N,N,N′,N′-Tetramethyl-p-phenylenediamine or reduced phenazine methosulphate as reductants, unless a substoichiometric amount of cytochrome c or an excess of clupein is present. Cytochrome c cannot be replaced by porphyrin cytochrome c.2. Cytochrome c, porphyrin cytochrome c and clupein all stimulate the reduction of cytochrome aa₃ by ferrocyanide.3. A model is proposed to explain these findings in which a high-affinity site for cytochrome c on the oxidase regulates the access of hydrophilic electron donors to a low-affinity site, and reduction via the high-affinity site is required for continuous oxygen uptake.4. Furthermore, it is shown that upon reaction of oxidase with ferrocyanide, cyano-oxidase is formed.     

Rates of reduced cytochrome c-ferricyanide binding and electron transfer

The oxidation of reduced cytochrome c by ferricyanide has been studied over a wide range of ferricyanide concentrations using a continuous-flow apparatus. The formation of a ferrocytochrome c-ferricyanide complex has been demonstrated and the binding and electron transfer processes separated to give both the oxidation electron transfer rate and the binding rate parameters. The electron transfer rate has been found to be 1.86 · 10³ s^?1 in H₂O buffer and 1.36 · 10³ s^?1 in ²H₂O demonstrating that a deuterium isotope effect of similar magnitude (R = 1.37) to that found in the cytochrome reactions in photosynthetic bacteria [18] is also found in the reaction studied here. The binding association rate parameters also show a similar deuterium isotope effect suggesting that water rotation may be involved in both the binding of ferricyanide to reduced cytochrome c and the subsequent oxidation electron transfer.     

Electron transfer between cytochrome c2 and the tetraheme cytochrome c in Rhodopseudomonas viridis

Kinetics of electron transfer from soluble cytochrome c₂ to the tetraheme cytochrome c have been measured in isolated reaction centers and in membrane fragments of the photosynthetic purple bacterium Rhodopseudomonas viridis by time-resolved flash absorption spectroscopy. Absorbance changes kinetics in the region of cytochrome -bands (540–560 nm) were measured at 21 °C under redox conditions where the two high-potential hemes (c-559 and c-556) of the tetraheme cytochrome were chemically reduced. After flash excitation, the heme c-559 donates an electron to the special pair of bacteriochlorophylls and is then re-reduced by heme c-556. The data show that oxidized heme c-556 is subsequently re-reduced by electron transfer from reduced cytochrome c₂ present in the solution. The rate of this reaction has a non-linear dependence on the concentration of cytochrome c₂, suggesting a (minimal) two-step mechanism involving the f ormation of a complex between cytochrome c₂ and the reaction center, followed by intracomplex electron transfer. To explain the monophasic character of the reaction kinetics, we propose a collisional mechanism where the lifetime of the temporary complex is short compared to electron transfer. The limit of the halftime of the bimolecular process when extrapolated to high concentrations of cytochrome c₂ is 60 ± 20 s. There is a large ionic strength effect on the kinetics of electron transfer from cytochrome c₂ to heme c-556. The pseudofirst-order rate constant decreases from 1.1 × 10⁷ M^-1 s^-1 to 1.3 × 10⁶ M^-1 s^-1 when the ionic strength is increased from 1 to 1000 mM. The maximum rate (1.1 × 10⁷ M^-1 s^-1) was obtained at about 1 mM ionic strength. This dependence of the rate on ionic strength s uggests that attractive electrostatic interactions contribute to the binding of cytochrome c₂ with the tetraheme cytochrome. On the basis of our data and of previous molecular modelling, it is proposed that cytochrome c₂ docks close to the low-potential heme c-554 and reduces heme c-556 via c-554.     

Studies on the reaction of imidazole with cytochrome c3 from Desulfovibrio vulgaris

1. Cytochrome c₃, a unique hemoprotein with a negative redox potential and four heme groups bound to a single polypeptide chain, reacts with imidazole in the reduced state to form a low-spin ferro · imidazole complex which is spectrally characterized by a 3.1 nm blue shift in the α-peak (from 550.5 to 547.4 nm). The spectral imidazole · cytochrome c₃ complex is detectable at 77 but not at 298 K.2. Mammalian ferrocytochrome c did not undergo a spectral interaction with imidazole at either 77 or 298 K, indicating that the imidazole · cytochrome c₃ complex reflects a unique event for cytochrome c₃.3. Formation of the imidazole · cytochrome c₃ complex is strongly dependent on imidazole concentration (apparent K_d of approx. 50 mM), and is abolished in the presence of 100 mM phosphate. This latter effect is attributable to formation of an imidazole · phosphate complex. A pH titration of the imidazole · cytochrome c₃ spectral complex implicates ionization of an imidazole function (pK = 8.5).4. EPR studies at 8.5 K of ferricytochrome c₃ before and after one reduction-oxidation cycle indicate that at least two of the hemes undergo reaction with imidazole forming two different low-spin ferric heme · imidazole complexes, with significant shifts in the g values of two heme signals.5. The spectral and EPR results are consistent with formation as the primary event of a low-spin ferrocytochrome c₃ · imidazole complex in which increased hydrophobicity and protonation-deprotonation effects are contributary to the consequent lability of cytochrome c₃.     

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa₃) is coupled to translocation of H⁺ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa₃ is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational “strain” in the cytochrome aa₃ complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.     

Cytochrome a1 of Nitrosomonas europaea resembles aa3-type cytochrome c oxidase in many respects

Cytochrome c oxidase of Nitrosomonas europaea has been called cytochrome a₁ by Erickson et al. (Erickson, R.H., Hooper, A.B. and Terry, K.R. (1972) Biochim. Biophys. Acta 283, 155–166) because the reduced form of their preparation had the α peak at 595 nm. In the present studies, the enzyme was purified to an electrophoretically almost homogeneous state and some of its properties were studied. The enzyme much resembled cytochrome aa₃-type oxidase although its reduced form showed the α peak at 597 nm. (1) The absorption spectra of the CO compound of the reduced enzyme and CN⁻ compounds of the oxidized and reduced enzyme were similar to those of the respective compounds of cytochrome aa₃, as well as the absorption spectrum of the intact enzyme resembled that of the cytochrome. (2) The enzyme possessed two molecules of haem a and 1–2 atoms of copper in the molecule. (3) The enzyme molecule was composed of two kinds of subunits of M_r 50000 and 33000, respectively, as are other bacterial cytochromes aa₃. Although the enzyme resembled other bacterial cytochromes aa₃ in many properties, it differed greatly in two properties; its CO compound was easily dissociated into the oxidized enzyme and CO in air, and 50% inhibition of its activity by CN⁻ required approx. 100 μM of the reagent. The enzyme oxidized 0.57, 1.6 and 1.8 mol horse, Candida krusei and N. europaea ferrocytochromes c per s per mol haem a, respectively, in 10 mM phosphate buffer, pH 6.0. The turnover numbers with eukaryotic ferrocytochromes c were increased to 32 and 14, respectively, by addition of cardiolipin (14 μ · ml⁻¹).     

Electrocatalytic four-electron reduction of oxygen at the cytochrome c3-adsorbed electrode

The electrocatalytic activity of cytochrome c₃ for the reduction of molecular oxygen was characterized from the studies of the adsorption of cytochrome c₃ and the co-adsorption of cytochrome c₃ with cytochrome c on the mercury electrode by the a.c. polarographic technique. The adsorption of cytochrome c₃ on the mercury electrode is irreversible and is diffusion-controlled. The maximum amount of cytochrome c₃ adsorbed was 0.92 · 10^?11 mol · cm^?2 at ?0.90 V. The amount of cytochrome c₃ in the mixed adsorbed layer with cytochrome c was determined from the differential capacitance measurement. It was shown that the fractional coverage of cytochrome c₃ can be estimated from its bulk concentration and the diffusion coefficient (1.05 · 10^?6 cm² · s^?1). Cytochrome c₃ catalyzes the electrochemical reduction of molecular oxygen from the two-electron pathways via hydrogen peroxide to the four-electron pathway at the mercury electrode in neutral phosphate buffer solution. The catalytic activity varies with the bulk concentration of cytochrome c₃. The highest catalytic activity for the oxygen reduction (no hydrogen peroxide formation) is attained when one-half of the mercury electrode surface is covered by cytochrome c₃. The addition of cytochrome c or bovine serum albumin to the cytochrome c₃ solution inhibits the catalytic activity of cytochrome c₃. The reversible polarographic behavior of cytochrome c₃ through the mixed adsorbed layer of cytochrome c₃ and cytochrome c was also investigated.     

Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles

1. Beef heart mitochondria have a cytochrome c₁ : c : aa₃ ratio of 0.65 : 1.0 : 1.0 as isolated; Keilin-Hartree submitochondrial particles have a ratio of 0.65 : 0.4 : 1.0. More than 50% of the submitochondrial particle membrane is in the ‘inverted’ configuration, shielding the catalytically active cytochrome c. The ‘endogenous’ cytochrome c of particles turns over at a maximal rate between 450 and 550 s^?1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300–400 s^?1, at 28° – 30°C, pH 7.4.2. Ascorbate plus N,N,N′,N′-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c₁ and cytochrome c steady states at 552.5–547 nm and 550–556.5 nm, respectively.3. Cytochrome c₁ is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate+N,N,N′,N′-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c₁ and c in the aerobic steady state, with a rate constant for the c₁ → c reduction step greater than 10³ s^?1.4. The greater apparent response of the $\text{c}\text{aa}{}_3$ electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the $\text{c}{}_1\text{c}$ step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa₃ units in situ. Cytochrome c₁ can either reduce the cytochrome c-cytochrome aa₃ complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.     

Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c₁ of the bc₁-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c₅₅₂ and cytochrome c₅₅₀ have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c₁ were generated and used together with a soluble cytochrome c₅₅₂ module as a model system for interprotein ET reactions. Both c₁ fragments lack the membrane anchor; the c₁ core fragment (c_1CF) consists of only the hydrophilic heme-carrying domain, whereas the c₁ acidic fragment (c_1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c₁ reveals fast bimolecular rate constants (10⁷ to 10⁸ M^− 1 s^− 1) for the ET reaction with its physiological substrates c₅₅₂ and c₅₅₀, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c₁-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c₅₅₀ over c₅₅₂ reacting with cyochrome c₁ was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.