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.     

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.     

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.     

Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b₅, cytochrome c₁ and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, and 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c₁ and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.     

Identification of two different Q-binding sites in QH2-cytochrome c oxidoreductase,using the Q analogue n-heptadecylmercapto-6-hydroxy-5,8-quinolinequinone

The pK and mid-point redox potential of the Q-analogue 7-(n-heptadecyl)mercapto-6-hydroxy-5,8-quinolinequinone (HMHQQ) in aqueous medium are so low that under the experimental conditions used for studying the inhibition of electron transfer in submitochondrial particles only the oxidized, anionic form is present. The K_D of the analogue, determined by comparing its inhibitory effect with that of n-heptyl-4-hydroxyquinolineN-oxide, is (0.003+0.24×mg protein/ml) μM. The inhibition of succinate oxidation is pH dependent, due to a pH-dependent change in the overcapacity of the QH₂-oxidizing system above the Q-reducing system. If the terminal part of the respiratory chain is reduced with ascorbate, the analogue inhibits the reduction of cytochrome b by substrate in the presence of antimycin with a similar K_D value. In the absence of ascorbate the K_D value is 100-times higher. The reduction of cytochrome b by substrate in particles treated with 2,3-dimercaptopropanol (BAL)+O₂ is also sensitive to HMHQQ, with a K_D value in between the two values given above. It is concluded that the QH₂ oxidase system contains two different sites for interaction with ubiquinone. The site responsible for the inhibition of steady-state electron transfer is near the Fe-S cluster, as is shown by the sensitivity to the redox state of this cluster and by the effect of HMHQQ on the EPR signal of the reduced cluster. The second site, which is similar to the antimycin-binding site, is occupied only at higher concentrations of inhibitor. The affinity of HMHQQ for this site is not affected by the redox state of the Fe-S cluster.     

The interaction of the reaction center secondary quinone with the ubiquinone-cytochrome c2 oxidoreductase in Rhodopseudomonas sphaeroides chromatophores

(1) Two populations of reaction centers in the chromatophore membrane can be distinguished under some conditions of initial redox poise (300 mV < E_h < 400 mV): those which transfer a reducing equivalent after the first flash from the secondary quinone (Q_II) of the reaction center to cytochrome b of the ubiquinone-cytochrome c₂ oxidoreductase; and those which retain the reducing equivalent on Q^?_II until a second flash is given. These two populations do not exchange on a time scale of tens of seconds. (2) At redox potentials higher than 400 mV, Q^?_II generated after the first flash is no longer able to reduce cytochrome b-560 even in those reaction centers associated with an oxidoreductase. Under these conditions, doubly reduced Q_II generated by a second flash is required for cytochrome b reduction, so that the Q_II effectively functions as a two-electron gate into the oxidoreductase at these high potentials. (3) At redox potentials below 300 mV, although the two populations of Q_II are no longer distinguishable, cytochrome b reduction is still dependent on only part of the reaction center population. (4) Proton binding does not oscillate under any condition tested.     

Occurrence of cytochrome aa3 in Anacystis nidulans

The cytochrome content of membrane fragments prepared from the bluegreen alga (cyanobacterium) Anacystis nidulans was examined by difference spectrophotometry. Two b-type cytochromes and a hitherto unknown cytochrome a could be characterized. In the reduced-minus-oxidised difference spectra the a-type cytochrome showed an α-band at 605 nm and a γ-band at 445 nm. These bands shifted to 590 and 430 nm, respectively, in CO difference spectra. NADPH, NADH and ascorbate reduced the cytochrome through added horse heart cytochrome c as electron mediator. In presence of KCN the reduced-minus-oxidised spectrum showed a peak at 600 nm and a trough at 604 nm. Photoaction spectra of O₂ uptake and of horse heart cytochrome c oxidation by CO-inhibited membranes showed peaks at 590 and 430 nm. These findings are consistent with cytochrome aa₃ being the predominant respiratory cytochrome c oxidase in Anacystis nidulans.     

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 β-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 β-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 g_z value of 3.36 and a g_y value of 2.04.     

Electron transfer through the isolated mitochondrial cytochrome b-c1 complex

(1) A kinetic analysis of electron donation into and through the cytochrome b-c₁ complex isolated from bovine heart mitochondria has been undertaken, using trimethylquinol as the donor. (2) Rate constants of two routes of redox equilibration with quinols have been defined by kinetic measurements and with the use of the inhibitors antimycin A and myxothiazol. (3) A model of electron transfer based upon the original Q-cycle formulation is presented to explain these and related results.     

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.     

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.     

Spontaneous inactivation of the Ca2+-sensitive K+ channels of human red cells at high intracellular Ca2+ activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a $\text{(}\text{dCa}{}^{\mathrm{2+}}\text{/}\text{d}\text{t)-}\text{sensitive}$ process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about ?65 mV was observed. At a membrane potential of about ?70 mV and an intracellular concentration of about 2·10^?4M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

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.     

Flash-induced turnover of the cytochrome bc1 complex in chromatophores of Rhodobacter capsulatus: binding of Zn decelerates likewise the oxidation of cytochrome b, the reduction of cytochrome c1 and the voltage generation

The effect of Zn²⁺ on the rates of electron transfer and of voltage generation in the cytochrome bc₁ complex (bc₁) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn²⁺ retarded the oxidation of cytochrome b and allowed to monitor (at 561-570 nm) the reduction of its high potential heme b_h (in the absence of Zn²⁺ this reaction was masked by the fast re-oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c₁ reduction (as monitored at 552-570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn²⁺ <100 μM the reduction of heme b_h remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc₁ turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme b_h is the main electrogenic reaction in the cytochrome bc₁ complex, (2) imply an allosteric link between the site of heme b_h oxidation and the site of cytochrome c₁ reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc₁ complex.     

A reevaluation of the events leading to the electrogenic reaction and proton translocation in the ubiquinol-cytochrome c oxidoreductase of Rhodopseudomonas sphaeroides

Chromatophore membranes from Rhodopseudomonas sphaeroides activated by light display a carotenoid band shift (phase III) that occurs in response to the electrogenic event (charge separation) in the ubiquinol-cytochrome c oxidoreductase. The rate of formation of this electrogenic event has previously been shown to be strongly dependent on the initial redox state of a bound ubiquinone species (designated Q_z) associated with the oxidoreductase. When Q_z is reduced (quinol form; Q_zH₂) the electrogenic event takes place in less than 5 ms. When Q_z is oxidized (quinone form; Q_z) it is much slower; under these conditions the fact that it occurs has been ignored. In this report, we address this issue and describe events that lead to the generation of carotenoid band shift phase III when the total population of Q_z of the chromatophore is oxidized before flash activation. The following characteristics are apparent: (1) When oxidized Q_z is present before activation, the half-time of formation of carotenoid band-shift phase III is 10–20-times slower than when Q_zH₂ is present before activation. (2) When oxidized Q_z is present, the measured full extent of phase III generated by a single-turnover flash is diminished by about one-half of that observed when Q_zH₂ is present before activation. (3) The rate of formation of the carotenoid band shift phase III when Q_z is initially oxidized corresponds closely to the rate of completion of the flash-activated electron-transfer cycle. This can be seen under two different conditions: (a) as the partial reduction of cytochrome c₁ + c₂ (at redox potentials of 200–300 mV) or (b) as the partial reduction of flash-oxidized bacteriochlorophyll dimer, (BChl)₂⁺ (at redox potentials above 300 mV). (4) At the higher redox potentials (above 300 mV), antimycin-sensitive proton binding shares a common, rate-limiting step with the carotenoid band shift phase III and (BChl)₂⁺ reduction. (5) However, proton binding at redox potentials above 300 mV is not observed at all unless valinomycin (K⁺) is present. Thus, proton binding occurs only when the carotenoid band shift is collapsed in milliseconds, whereas, conversely, the carotenoid band shift is stably generated when proton binding is not observed. These and other observations are the basis of a reevaluation of our current views on the coupling of electron transfer and proton translocation in photosynthetic bacteria.     

The role of cytochrome b-566 in the electron-transfer chain of Rhodopseudomonas sphaeroides

Spectrophotometric, kinetic, thermodynamic and stoichiometric properties of the low-potential b-type cytochrome of chromatophores from Rhodopseudomonas sphaeroides are reported. Cytochrome b-566 has a double α-band with maxima at 559 and 566 nm. Resolution of the spectrum by full-spectral redox potentiometry showed no indication that the two peaks represent more than one component. The component titrated with E_m,7 ≈ ?80 ± 10 mV. By appropriate choice of wavelength pairs and by subtraction of the contribution due to other components, the kinetics of cytochrome b-566 absorbance changes following flash excitation have been resolved from those of other components. Time-resolved flash spectra corrected for the contributions of other components are consistent with the behavior of both peaks of the α-band as a single kinetic species. The kinetics of cytochrome b-566 in the presence of antimycin show that the reduction of this cytochrome occurred only if cytochrome b-561 was reduced before the flash, either chemically, by poising the ambient redox potential (E_h) below the E_m of cytochrome b-561 (E_m,7 ≈ 50 mV), or photochemically at higher redox potentials by a previous flash. The rate of reduction of cytochrome b-566 varied with E_h. At low E_h (approx. 0 mV) reduction on the first flash showed $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1.25}\text{ms}$; at high E_h (approx. 180 mV) reduction on the second flash showed $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 10}\text{ms}$. In the absence of antimycin at E_h ≈ 0 mV, cytochrome b-566 was observed to become rapidly reduced ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 500 μ}\text{s}$) and then reoxidized ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 2}\text{ms}$) after a single flash. At higher redox potentials (E_h > 80 mV) no kinetic changes which could be unambiguously attributed to cytochrome b-566 were observed following a single flash. The results are interpreted in terms of a Q-cycle mechanism in which the reductant for cytochrome b-566 is the semiquinone formed on oxidation of ubiquinol from the quinone pool. The oxidation of the ubiquinol occurs by a concerted reaction in which one electron is accepted by the Rieske-type FeS center and the other by cytochrome b-566. We suggest that the kinetic characteristics may indicate a pathway for reduction of the b-type cytochromes in which cytochrome b-566 is the immediate electron acceptor and donates to cytochrome b-561 in a serial pathway. The experimental results in the presence of antimycin are compared with data from a computer simulation of the thermodynamic behavior of the chain, and the computer model is shown to provide an excellent fit.     

Inhibition by Sr2+ of specific mitochondrial Ca2+-efflux pathways

The effect of Sr²⁺ on the set point for external Ca²⁺ was studied in rat heart and liver mitochondria with the aid of a Ca²⁺-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca²⁺ influx on the Ca²⁺ uniporter and efflux by various mechanisms. We studied the Ca²⁺-Na⁺ exchange pathway in heart mitochondria and the Δψ-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr²⁺ was found to shift the set points towards lower external Ca²⁺ both in heart mitochondria under conditions of Ca²⁺-Na⁺ exchange and in liver mitochondria under conditions that should promote opening of the Δψ-modulated pathway. The effect on the set point was found to be due to inhibition of Ca²⁺ efflux by Sr²⁺ taken up by the mitochondria, while Sr²⁺ efflux was too slow to be measurable.     

NMR studies of malaria 31P nuclear magnetic resonance of blood from mice infected with Plasmodium berghei

High resolution ³¹P-NMR has been used for the non-invasive observation of metabolites and metabolic rates in blood of normal mice and of mice infected with Plasmodium berghei, the causative agent of malaria. ³¹P-NMR was used to quantitate levels of 2,3-diphosphoglycerate in whole cells as a function of the degree of parasitemia and yielded good agreement with the results of enzymatic assays. The time-dependence of ³¹P metabolites was monitored in both normal and infected erythrocytes, greater rates of decay of 2,3-diphosphoglycerate being observed in malarial blood which correlate with the level of parasitemia. Very high metabolic rates of infected cells render measurement of intracellular pH unreliable on freshly drawn whole blood. When appropriate measures are taken to avoid this complication, no difference is observed in the intracellular pH of parasitized and non-parasitized erythrocytes from infected animals. In both normal and parasitized mice the intraerythrocytic pH is more acidic than that of the suspending medium by 0.15 pH unit at 25°C. Unlike free-living protozoa, the parasitic protozoan Plasmodium does not contain detectable levels of phosphonates or polyphosphates, in either whole cells or perchloric acid extracts thereof.     

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.