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.     

A covalent complex between horse heart cytochrome c and yeast cytochrome c peroxidase: kinetic properties

The kinetic properties of a 1:1 covalent complex between horse-heart cytochrome c and yeast cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) have been investigated by transient-state and steady-state kinetic techniques. Evidence for heterogeneity in the complex is presented. About 50% of the complex reacts with hydrogen peroxide with a rate 20–40% faster than that of native enzyme; 20% of the complex exists in a conformation which does not react with hydrogen peroxide but converts to the reactive form at a rate of 20 ± 5 s⁻¹; 30% of the complex does not react with hydrogen peroxide to form the oxidized enzyme intermediate, cytochrome c peroxidase Compound I. Intramolecular electron transfer between covalently bound ferrocytochrome c and an oxidized site in cytochrome c peroxidase Compound I is too fast to measure, but a lower limit of 600 s⁻¹ can be estimated at 5°C in a 10 mM potassium phosphate buffer at pH 7.5. Free ferrocytochrome c reduces cytochrome c peroxidase Compound I covalently bound to ferricytochrome c at a rate 10⁻⁴ to 10⁻⁵-times slower than for free Compound I. The transient-state ferrocytochrome c reduction rates of Compound I covalently linked to ferricytochrome c are about 70-times too slow to account for the steady-state catalytic properties of the 1:! covalent complex. This indicates that hydrogen peroxide can interact with the 1:1 complex at sites other than the heme of cytochrome c peroxidase, generating additional species capable of oxidizing free ferrocytochrome c.     

One-electron reactions in biochemical systems as studied by pulse radiolysis. VI. Stages in the reduction of ferricytochrome c

When ferricytochrome c at pH about 9 is reduced by hydrated electrons and/or CO₂^?, it gives rise to an unstable form of ferrocytochrome c whose absorption spectrum, particularly in the Soret region, differs from that of normal ferrocytochrome c. This form changes intramolecularly (life-time about 0.1 s at ambient temperature) to yield normal ferrocytochrome c, and by 0.5 s the change in absorption spectrum in the range 225–600 nm produced by e^?_aq and/or CO₂^? is identical to the final change produced by reduction with an equivalent amount of sodium dithionite. This shows that both e^?_aq and CO^?₂ reduce cytochrome c with practically 100% efficiency. In the range 600–800 nm the spectrum of the unstable form is the same as that of normal ferrocytochrome c, both having small absorptions at 695 nm as compared with ferricytochrome c. As the unstable form disappears however a further loss of absorption at 695 nm occurs. This is taken to imply that the unstable form decays to a second unstable form which then rapidly donates an electron to the unchanged neutral form of ferricytochrome c, so reducing absorption in the 695 nm band. Subsequent to this process the absorption in the 695 nm band increases over a period of minutes owing to re-equilibration between the neutral and alkaline formes of ferricytochrome c. Between pH 7 and 10 the effect of pH on the absorption changes is consistent with the hypothesis of a second unstable form of ferrocytochrome c. Additional phenomena arise in more alkaline solutions. The rates of the various unimolecular processes are thought to be determined by the rates of change of conformation of the protein parts of the molecule following the change in oxidation state.     

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.     

Bound cytochrome C as proton donor and acceptor during enzymic oxidoreduction

When ferrocytochrome $\text{c}$ reacts with delipidated cytochrome oxidase under conditions which prevent oxidation, one proton is taken up per molecule of ferrocytochrome $\text{c}$ bound to cytochrome oxidase. When ferricytochrome $\text{c}$ reacts with delipidated Complex III, one proton is released per molecule of ferricytochrome $\text{c}$ bound to Complex III. From these data it can be concluded that the oxidation of ferrocytochrome $\text{c}$ by cytochrome oxidase leads to the release of a proton and an electron, whereas the reduction of ferricytochrome $\text{c}$ by Complex III leads to the uptake of a proton and an electron. Thus ferrocytochrome $\text{c}$ like QH₂ and NADH is both an electron and proton donor, and ferricytochrome $\text{c}$ like Q and O₂ is both an electron and proton acceptor. The pattern for the three mitochondrial electron transfer sequences NADH → Q, QH₂ → ferricytochrome $\text{c}$ and ferrocytochrome $\text{c}$ → O₂ involves separation of an electron and proton on the side of the membrane where electron transfer is initiated and recombination of an electron and a proton in the terminal acceptor on the side of the membrane where electron transfer terminates.     

Structure of cytochrome c551 from Pseudomonas aeruginosa refined at 1.6 Å resolution and comparison of the two redox forms

The molecular structures of ferri- and ferrocytochrome c₅₅₁ from Pseudomonas aeruginosa have been refined at a resolution of 1.6 Å, to an R factor of 19.5% for the oxidized molecule and 18.7% for the reduced. Reduction of oxidized crystals with ascorbate produced little change in cell dimensions, a 10% mean change in F_obs, and no damage to the crystals. The heme iron is not significantly displaced from the porphyrin plane. Bond lengths from axial ligands to the heme iron are as expected in a low-spin iron compound. A total of 67 solvent molecules were incorporated in the oxidized structure, and 73 in the reduced, of which four are found inside the protein molecule. The oxidized and reduced forms have virtually identical tertiary structures with 2 ° root-mean-square differences in main-chain torsion angles φ and ψ, but with larger differences along the two edges of the heme crevice. The difference map and pyrrole ring tilt suggest that a partially buried water molecule (no. 23) in the heme crevice moves upon change of oxidation state.Pseudomonas cytochrome c₅₅₁ differs from tuna cytochrome c in having: (1) a water molecule (no. 23) at the upper left of the heme crevice; that is, between Pro62 and the heme pyrrol 3 ring on the sixth ligand Met61 side, where tuna cytochrome c has an evolutionary invariant Phe82 ring; (2) a string of hydrophobic side-chains along the left side of the heme crevice, and fewer positively charged lysines in the vicinity; and (3) a more exposed and presumably more easily ionizable heme propionate group at the bottom of the molecule. A network of hydrogen bonds in the heme crevice is reminiscent of that inside the heme crevice of tuna cytochrome c. As in tuna, a slight motion of the water molecule toward the heme is observed in the oxidized state, helping to give the heme a more polar microenvironment. The continuity of solvent environment between the heme crevice and the outer medium could explain the greater dependence of redox potential on pH in cytochrome c₅₅₁ than in cytochrome 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.     

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.     

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 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.     

Redox state of cytochrome C in the presence of the 6-hydroxydopamine/oxygen couple: Oscillations dependent on the presence of hydrogen peroxide or superoxide

The reduction of ferricytochrome c in the presence of 6-hydroxydopamine/O₂ mixtures was examined under various reaction conditions. As the autoxidation of 6-hydroxy-dopamine progressed to completion, there were fluctuations in the net redox reactivity between reducing and oxidizing steady states. This was reflected in a sequence of damped oscillations in the redox state of cytochrome c. Corresponding to the time when 6-hydroxydopamine was 75–100% exhausted, reoxidation of the ferrocytochrome c occurred (prevented by catalase or catalase plus Superoxide dismutase). After the H₂O₂, in turn, was mostly consumed, the next phase commenced in which the cytochrome c became reduced for a second time. This reductive phase was 52% inhibited by superoxide dismutase. In the subsequent and final phase of the process, a progressive oxidation of cytochrome c lasting at least 24 h was observed. Of the initial reduction of ferricytochrome c, at most 37% can be attributed to direct reduction by 6-hydroxydopamine or its semiquinone. This initial net reduction of cytochrome c was inhibited 51% by superoxide dismutase and 41% by catalase. However, since either catalase or superoxide dismutase inhibited the autoxidation of 6-hydroxydopamine by at least as much as it slowed the reduction of cytochrome c, their effects in slowing the reduction of cytochrome c resulted largely from the decreased production of those free radicals which reduce ferricytochrome c, and only in part from accelerated removal. Elimination of the actions of transition metal ions (whether by passage of the buffer solutions through Chelex 100 resins or by addition of desferrioxamine to the reaction medium) slowed both the reoxidation and rereduction by up to 96%. Addition of mannitol decreased the rate of the first reoxidation by 25% and increased the rate of the rereduction by 7%. In general, the oscillations are explicable in terms of changes in the steady state levels of O₂ and H₂O₂, with metal ions playing a major role and hydroxyl radicals a minor role in both the reoxidation and rereduction.     

Cytochrome c-556, a di-heme protein from Pseudomonas aeruginosa

A procedure is described for the purification of cytochrome c-556 from Pseudomonas aeruginosa. The isolated hemoprotein exists as a dimer with a molecular weight of approximately 77 200. The dimer can be dissociated into a monomeric species (or single polypeptide chain) of 40 500 molecular weight by means of sodium dodecyl sulfate or 4 M urea. The amino acid composition demonstrates the presence of four half-cystine residues per 43 000 molecular weight. Heme and iron analyses indicate that two c-type hemes are covalently linked to each polypeptide chain. The absorption spectrum of ferrocytochrome c-556 has a double α-band with a peak at 556 nm and a shoulder at 552 nm; the β-band appears at 521 nm and the Soret band at 420 nm.The electron paramagnetic resonance spectrum of ferricytochrome c-556 contains the elements of two ferric iron species, one a low spin and the other a high spin form.The function of cytochrome c-556 is obscure. The purified cytochrome does not react with Pseudomonas cytochrome oxidase nor with the Pseudomonas cytochrome c-551 or copper protein.The properties of cytochrome c-556 indicate that it is probably not the same species as the cytochrome c-554 previously isolated from the same organism.     

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 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.     

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⁺.     

Conformation change of cytochrome c. I. Ferrocytochrome c structure refined at 1.5 A resolution

Tuna ferrocytochrome c has been crystallographically refined at a resolution of 1.5 Å using the Diamond real-space method followed by Jack-Levitt restrained energy and reciprocal space refinement, monitoring progress continuously with superimposed Fourier and difference Fourier maps: The final R factor for cytochrome plus 53 solvent molecules, using 13,840 reflections with intensities greater than 2 σ, is 17·3%. The overall structure remains as described earlier (Takano et al., 1977), but structural details have been clarified to the point where meaningful comparison can be made with the oxidized molecule (following paper). Main and side-chain flexibility as judged by isotropic temperature parameters correlate with position in the molecule, with greatest flexibility at external chain loops. The haem group is held tightly in place by its attachments and neighbours, and is deformed slightly into a saddle shape. The iron does not deviate significantly from the best mean plane of the haem, and bond lengths to ligands are as expected from model compounds.A water molecule buried in the haem crevice is bonded to Asn52, Tyr67 and Thr78, the latter two being bonded also to Met80 and the outer haem propionate. It is proposed that this buried water molecule is involved in the reduction of ferricytochrome c by chromous ion, and the reactions of Tyr67 with KI₃ and tetranitromethane. Two other buried water molecules occur beneath the 20's loop at the right, and within the 40's loop at the bottom. Reasonable if tentative functional assignments can be made for all 24 of the evolutionarily invariant residues in the cytochrome molecule.     

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 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.     

Cytochrome b oxidation and reduction reactions in the ubiquinone-cytochrome b/c2 oxidoreductase from Rhodopseudomonas Sphaeroides

1. The kinetics of cytochrome b reduction and oxidation in the ubiquinone-cytochrome b/c₂ oxidoreductase of chromatophores from Rhodopseudomonas sphaeroides Ga have been measured both in the presence and absence of anti-mycin, after subtraction of contributions due to absorption changes from cytochrome c₂, the oxidized bacteriochlorophyll dimer of the reaction center, and a red shift of the antenna bacteriochlorophyll.2. A small red shift of the antenna bacteriochlorophyll band centered at 589 nm has been identified and found to be kinetically similar to the carotenoid bandshift.3. Antimycin inhibits the oxidation of ferrocytochrome b under all conditions; it also stimulates the amount of single flash activated cytochrome b reduction 3- to 4-fold under certain if not all conditions.4. A maximum of approximately 0.6 cytochrome b-560 (E_m(7) = 50 mV, n = 1, previously cytochrome b₅₀) hemes per reaction center are reduced following activating flashes. This ratio suggests that there is one cytochrome b-560 heme functional per ubiquinone-cytochrome b/c₂ oxidoreductase.5. Under the experimental conditions used here, only cytochrome b-560 is observed functional in cyclic electron transfer.6. We describe the existence of three distinct states of reduction of the ubiquinone-cytochrome b/c₂ oxidoreductase which can be established before activation, and result in markedly different reaction sequences involving cytochrome b after the flash activation. Poising such that the special ubiquinone (Q_z) is reduced and cytochrome b-560 is oxidized yields the conditions for optimal flash activated electron transfer rates through the ubiquinone-cytochrome b/c₂ oxidoreductase. However when the ambient redox state is lowered to reduce cytochrome b-560 or raised to oxidize Q_z, single turnover flash induced electron transfer through the ubiquinone-cytochrome b/c₂ oxidoreductase appears impeded; the points of the impediment are tentatively identified with the electron transfer step from the reduced secondary quinone (Q_II) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Q_z, respectively.     

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.