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 2·10⁷ M^?1·s^?1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20°C). (2) The ionic strength dependence of the cytochrome c-cytochromeaa₃ 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 (?) of the reaction medium on the reaction of cytochrome c with cytochrome aa₃ was investigated. With increasing ? the second-order rate constant decreased.     

The reaction of Euglena gracilis cytochrome c-552 with nonphysio-logical oxidants and reductants.

The reaction of Euglena gracilis cytochrome c-552 (cytochrome f) with the nonphysiological reactants potassium ferrocyanide, potassium ferricyanide, sodium ascorbate, sodium dithionite, and Chromatium vinosum high potential nonheme iron protein was studied by stopped-flow and temperature-jump kinetic methods. The reaction of the purified, water-soluble protein with the reactants was investigated as a function of ionic strength, pH, and temperature. The results demonstrated that reduction and oxidation takes place at a negatively charged site on the cytochrome c-552 surface. Participation of specific amino acid residues in electron transfer is implicated from the pH results. The results obtained for the nonphysiological reactions of cytochrome c-552 are compared with available data for horse heart cytochrome c and Rhodospirillum rubrum cytochrome c₂. The results strongly suggest that Euglena gracilis cytochrome c-552 undergoes nonphysiological oxidation and reduction by a mechanism different from that found for cytochrome c or cytochrome c₂.     

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 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 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.     

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.     

The effect of ionic strength on the kinetics of fluoride binding to cytochrome c peroxidase.

The kinetics of the reaction between cytochrome c peroxidase and fluoride was investigated as a function of ionic strength over the pH range 4 to 8. The ionic strength was varied between 0.01 and 0.10 m. At 0.01 m ionic strength, the reaction rates were determined between pH 2.7 and 9.2. A consideration of the ionic strength and pH dependence of the association rate constant for the fluoride-cytochrome c peroxidase reaction leads to the conclusion that hydrofluoric acid is the only significant reactive form of the ligand between pH 2.5 and 8. Above pH 8, binding of fluoride anion contributes to the apparent association rate even though the pH-independent rate constant for fluoride anion binding is more than 3 × 10⁵ times smaller than the rate constant for hydrofluoric acid binding.     

Electrostatic effects on the kinetics of photoinduced electron-transfer reactions of the triplet state of zinc cytochrome c with wild-type and mutant forms of Pseudomonas aeruginosa azurin

We study, by laser flash photolysis, the effects of ionic strength on the kinetics of the reaction ³Zncyt + az(II) → Zncyt⁺ + az(I), i.e., oxidative quenching of the triplet state of zinc cytochrome c by the wild-type form and the following three mutants of cupriazurin: Met44Lys, Met64Glu, and the double mutant Met44Lys/Met64Glu. Mutations in the hydrophobic patch of azurin significantly affect the reactivity of the protein with the triplet state of zinc cytochrome c. Dependence on the ionic strength of the bimolecular rate constant for the aforementioned reaction is analyzed by several electrosatic models. The two transition-state theories, Brønsted-Debye-Hückel and van Leeuwen theories, allow the best approximation to the experimental data when effective charges of the proteins are used. Protein-protein interactions are also analyzed in terms of local charges on the protein surfaces. The rate constants depend little on ionic strength, and the monopolar and dipolar electrostatic interactions between zinc cytochrome c and azurin are not well resolved. Semiquantitative analysis of electrostatic interactions indicates that azurin uses its hydrophobic patch for contact with zinc cytochrome c.     

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.     

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.     

Isolation of Paracoccus denitrificans cytochrome cd1: comparative kinetics with other nitrite reductases

The dissimilatory nitrite reductase of the cytochrome cd₁ type was purified from Paracoccus denitrificans (ATCC 13543) by a novel procedure that avoided conventional ion-exchange techniques. The characterization of this enzyme was extended to include amino acid composition, extinction coefficients, and kinetic properties not previously reported. Cytochromes cd₁ from Alicaligenes faecalis and Pseudomonas aeruginosa were also isolated and assayed with electron donor proteins. The enzymes from all three sources were shown to obey the same integrated rate law. Cross-reactivities were measured in which a reduced donor protein from one strain was assayed with cytochrome cd₁ from another strain using nitrite as ultimate acceptor. Donors included c-type cytochromes and azurins. In general, the enzymes showed specificity for a donor from the same strain; interspecies cross-reactions were typically slower on the order of 10-fold than corresponding native rates. Notable exceptions were Paracoccus cytochrome cd₁, which alone reacted with eukaryotic horse cytochrome c at appreciable rates, and the Pseudomonas cd₁-Alcaligenesc554 reaction, which was 4-fold faster than the native Alcaligenes cd₁-Alcaligenesc554 reaction. For all three enzymes, competitive kinetics were measured in which the alternative substrates, nitrite and oxygen, competed for enzyme in the same assay. It was found that the competitive kinetics were dominated by nonenzymatic reactions involving an enzyme product, nitric oxide.     

Microsomal electron transport. I. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and cytochrome P-450 as electron carriers in microsomal NADPH-peroxidase activity

An electron transport system that catalyzes the oxidation of NADPH by organic, hydroperoxides has been discovered in microsomal fractions. A tissue distribution study revealed that the microsomal fraction of rat liver was particularly effective in catalyzing the NADPH-peroxidase reaction whereas microsomes from adrenal cortex, lung, kidney, and testis were weakly active. The properties of the hepatic microsomal NADPH-peroxidase enzyme system were next examined in detail.The rate of NADPH oxidation by hydroperoxides was first-order with respect to microsomal protein concentration and a K_m value for NADPH of less than 3 μm was obtained. Examination of the hydroperoxide specificity revealed that cumene hydroperoxide and various steroid hydroperoxides were effective substrates for the enzyme system. Using cumene hydroperoxide as substrate, the reaction rate showed saturation kinetics with increasing concentrations of hydroperoxide and an apparent K_m of about 0.4 mm was obtained. The NADPH-peroxidase reaction was inhibited by potassium cyanide, half-maximal inhibition occurring at a cyanide concentration of 2.2 mm. NADH was able to support the NADPH-dependent peroxidase activity synergistically.Evidence compiled for the involvement of NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) in the NADPH-peroxidase reaction included: (1) an identical pH optimum for both activities; (2) stimulation of NADPH-peroxidase activity by increasing ionic strength; (3) inhibition by 0.05 mm, p-hydroxymercuribenzoate with partial protection by NADPH; (4) inhibition by NADP⁺; and (5) inactivation by antiserum to NADPH-cytochrome c reductase. In contrast, antibody to cytochrome b₅ did not inhibit the NADPH-peroxidase activity. Evidence for the participation of cytochrome P-450 in the NADPH-peroxidase reaction included inhibition by compounds forming type I, type II, and modified type II difference spectra with cytochrome P-450; inhibition by reagents converting cytochrome P-450 to cytochrome P-420; and marked stimulation by in vivo phenobarbital administration. The NADPH-reduced form of cytochrome P-450 was oxidized very rapidly by cumene hydroperoxide under a CO atmosphere.It was concluded that the NADPH-peroxidase enzyme system of liver microsomes is composed of the same electron transport components which function in substrate hydroxylation reactions.     

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.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Kinetics of cytochrome c oxidase from yeast. Membrane-facilitated electrostatic binding of cytochrome c showing a specific interaction with cytochrome c oxidase and inhibition by ATP (With an appendix on the occurrence of two-dimensional diffusion of cytochrome c on the membrane surface)

The steady-state kinetics of purified yeast cytochrome c oxidase were investigated at low ionic strength where the electrostatic interaction with cytochrome c is maximized. In 10 mM cacodylate/Tris (pH 6.5) the oxidation kinetics of yeast iso-1-cytochrome c were sigmoidal with a Hill coefficient of 2.35, suggesting cooperative binding. The half-saturation point was 1.14 μM. Horse cytochrome c exhibited Michaelis-Menten kinetics with a higher affinity (K_m = 0.35 μM) and a 100% higher maximal velocity.In 67 mM phosphate the Hill coefficient for yeast cytochrome c decreased to 1.42, and the species differences in Hill coefficients were lessened. Under the latter conditions, a yeast enzyme preparation partially depleted of phospholipids was activated on addition of diphosphatidylglycerol liposomes. When the enzyme was incorporated into sonicated yeast promitochondrial particles the apparent K_m for horse cytochrome c was considerably lower than the value for the isolated enzyme.ATP was found to inhibit both the isolated oxidase and the membrane-bound enzyme. With the isolated enzyme in 10 mM cacodylate/Tris, 3 mM ATP increased the half-saturation point with yeast cytochrome c 3-fold, without altering the maximal velocity or the Hill coefficient. 67 mM phosphate abolished the inhibition of the isolated oxidase by ATP.The increase in affinity for cytochrome c produced by binding the oxidase to the membrane was not observed in the presence of 3 mM ATP, with the result that the membrane-bound enzyme was more sensitive to inhibition by ATP. ADP was a less effective inhibitor than ATP, and did not prevent the inhibition by ATP.It is proposed that non-specific electrostatic binding of cytochrome c to phospholipid membranes, followed by rapid lateral diffusion, is responsible for the dependence of the affinity on the amount and nature of the phospholipids and on the ionic strength.ATP may interfere with the membrane-facilitated binding of cytochrome c by a specific electrostatic interaction with the membrane or by binding to cytochrome c.     

Kinetics of electron transfer between soluble cytochrome c-554 and purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Soluble cytochrome c-554 (M _r 10 kDa) is purified from the green sulfur bacterium Chlorobium tepidum. Its midpoint redox potential is determined to be +148 mV from redox titration at pH 7.0. The kinetics of cytochrome c-554 oxidation by a purified reaction center complex from the same organism were studied by flash absorption spectroscopy at room temperature, and the results indicate that the reaction partner of cytochrome c-554 is cytochrome c-551 bound to the reaction center rather than the primary donor P840. The second-order rate constant for the electron donation from cytochrome c-554 to cytochrome c-551 was estimated to be 1.7×10⁷ M^–1 s^–1. The reaction rate was not significantly influenced by the ionic strength of the reaction medium.This revised version was published online in October 2005 with corrections to the Cover Date.     

Electron transfer kinetics between rhus vernicifera stellacyanin and cytochrome c (horse heart cytochrome c and pseudomonas cytochrome c551)

The electron transfer reactions between Rhus vernicifera stellacyanin and either horse heart cytochrome c or Pseudomonas aeruginosa cytochrome c₅₅₁ were investigated by rapid reaction techniques. The time course of electron transfer is monophasic under all conditions, and thus consistent with a simple formulation of the reaction. Both stopped-flow and temperature-jump experiments yield equilibrium constants in reasonable agreement with values calculated from the redox potentials. The differences in reaction rate between the two cytochromes and stellacyanin are discussed in terms of the Marcus theory.     

The catalytic mechanism of Pseudomonas cytochrome c peroxidase

The catalytic mechanism of Pseudomonas cytochrome c peroxidase has been studied using rapid-scan spectrometry and stopped-flow measurements. The reaction of the totally ferric form of the enzyme with H₂O₂ was slow and the complex formed was inactive in the peroxidatic cycle, whereas partially reduced enzyme formed highly reactive intermediates with hydrogen peroxide. Rapid-scan spectrometry revealed two different spectral forms, one assignable to Compound I and the other to Compound II as found in the reaction cycle of other peroxidases. The formation of Compound I was rapid approaching that of diffusion control. The stoichiometry of the peroxidation reaction, deduced from the formation of oxidized electron donor, indicates that both the reduction of Compound I to Compound II and the conversion of Compound II to resting (partially reduced) enzyme are one-electron steps. It is concluded that the reaction mechanism generally accepted for peroxidases is applicable also to Pseudomonas cytochrome c peroxidase, the intramolecular source of one electron in Compound I formation, however, being reduced heme c.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrateferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the K_m (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the ‘association’ rate but also by increasing the ‘dissociation’ rate for bound cytochrome c converting the ‘primary’ (T) site at high salt concentrations into a site similar kinetically to the ‘secondary’ (L) site at low ionic strength. A finite K_m of 170 μM at very high ionic strength indicates a ratio of $\text{K}{}^{\mathrm{\infty }}{}_{\mathrm{<mtext>M</mtext>}}\text{K}{}^0{}_{\mathrm{<mtext>M</mtext>}}$ of about 5000. It is proposed that anions either modify the E¹₀ of cytochrome c bound at the primary (T) site or that they perturb an equilibrium between two forms of bound c in favour of a less active form.