Midpoint Potential Of Cytochrome Research Articles

Direct electrochemistry of engineered cytochrome b562 molecules with a ligand binding pocket

The rapid and reversible electron transfer reaction of cytochrome b 562 was observed at an In 2O 3 electrode. The estimated heterogeneous electron transfer rate constant ( k 0′) was k 0′ ≥ 5.0 × 10 −3 cm s −1 at pH 6.5. When the methionine-7 (Met-7) residue, which coordinates to the heme iron as an axial ligand, of the wild-type cytochrome b 562 was replaced by an Ala or Gly residue, a water molecule bound to the heme iron and the electron transfer rate constants decreased to 1.3 × 10 −3 and 1.8 × 10 −3 cm s −1, respectively. This decrease in the electron transfer rate would be due to the larger reorganization energy for the structural change at the redox site. The midpoint potential of cytochrome b 562 was shifted negatively by ∼135 mV by replacing Met-7 with Ala or Gly. Similar dissociation kinetics of cyanide for the mutated molecules as compared to native myoglobin was obtained.

Two Sites of Interaction of Anions with Cytochrome a in Oxidized Bovine Cytochrome c Oxidase

An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.

Spectroelectrochemical Determination of Redox Potential of Cytochrome c′ in Rhodospirillum rubrum Chromatophores

Abstract A spectroelectrochemical technique has been used for redox study of cytochrome c′ in chromatophores in the presence of Toluidine Blue O as a mediator. The midpoint potential of cytochrome c′ thus determined was 0 mV (n=1) at pH 7.0, which agreed well with the value determined by redox titration.

Cytochrome c1 and b-type cytochrome with two heme centers in the photosynthetic membranes from Rhodopseudomonas palustris

The photosynthetic membranes from Rhodopseudomonas palustris contained one species of membrane-bound c-type cytochrome, presumably cytochrome c 1, and a b-type cytochrome with two heme centers. The molecular weight and midpoint potential of cytochrome c 1 were 30000 and 275 mV, respectively. The α peak of the reduced-minus-oxidized difference spectrum of cytochrome c 1 was at 552 nm. Molecular weight of the b-type cytochrome was 32000 and the cytochrome had two midpoint potentials of 60 mV and −55 mV. The α peaks of the reduced-minus-oxidized difference spectra of the high and low midpoint potential heme centers were at 560 and 562 nm, respectively. These results suggested that there was a cytochrome b- c 1 complex in Rps. palustris.

Rapid redox equilibrium between the mitochondrial Q pool and cytochrome b during triphasic reduction of cytochrome b by succinate

(1) The reliability of monitoring the redox reactions of cytochrome b using the different wavelengths employed by different authors has been reexamined. It was found that 562-575 nm is suitable in succinate: cytochrome c reductase but not in mitochondria, in which case 562-540 nm is a better pair. (2) Direct optical measurements of the redox reaction kinetics of the mitochondrial Q pool using a commercial dual-wavelength spectrophotometer are possible when succinate is used as the electron donor. (3) Using the correct wavelength pair, and with malonate to slow down the electron input, the reduction course of cytochrome b was still triphasic but a plateau or a turn replaced the oxidation phase previously reported by several authors. At the same time, the reduction course of the Q pool was also triphasic, and in perfect match with that of cytochrome b. Destruction of the Rieske iron-sulfur cluster by British anti-Lewisite (BAL) + Q 2 treatment or prereduction of the high-potential components made the reduction of both Q and b monophasic. (4) The plot of log ( Q QH 2 ) against log ( b 3+ b 2+ ) gave a straight line with an n value of 1.7 for cytochrome b at pH 7.4. This n value rose to 2.0 at pH 6.5 and dropped to 1.4 at pH 8.5. On the other hand, the mid-point potential of cytochrome b relative to that of the Q pool remained essentially unchanged between pH 6.5 and 8.4. BAL treatment had a small effect on the midpoint potential of cytochrome b relative to that of the Q pool and had no effect on the n value. (5) Addition of quinone homologues and analogues extended the plateau phase in the reduction of cytochrome b, but exogenous quinones did not equilibrate rapidly with cytochrome b. (6) It was concluded that the appearance of the plateau between the two reduction phases of Q and b is caused by the rapid delivery of electrons to the high-potential components of the respiratory chain as envisaged in the Q cycle; the unexpected n value for cytochrome b suggests a concerted reduction by QH 2 of two species of cytochromes b-562.

Characterization of cytochrome b in the isolated ubiquinol-cytochrome c2 oxidoreductase from Rhodopseudomonas sphaeroides GA

Extinction coefficients for cytochrome b and c 1 in the isolated cytochrome bc 1 complex from Rhodopseudomonas sphaeroides GA have been determined. They are 25 mM −1.cm −1 at 561 nm for cytochrome b and 17.4 mM −1.cm −1 at 553 nM for cytochrome c 1 for the difference between the reduced and the oxidized state. Cytochrome b is present in two forms in the complex. One form has an E m7 of 50 mV, an α-peak of 557 nm at liquid N 2 temperature and of 561 nm at RT, which is red-shifted by antimycin A. The other form has an E m7 of −90 mV, a double α-peak of 555 and 561 nm at liquid N 2 temperature corresponding to 559 and 566 nm at RT. The absorption at 566 nm is red-shifted by myxothiazol. The two shifts are independent of each other. Both midpoint potentials of cytochromes b are pH-dependent. The redox center compositions of the cytochrome bc 1 complexes from Rhodopseudomonas sphaeroides and from mitochondria are identical.

Potentiometric studies on yeast Complex III

Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR. A value of 270 mV was obtained for cytochrome c 1, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and −20 mV at room temperature (MCD) compared to 116 and −4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 mV. An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape. The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23°C. Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature. The midpoint potential of cytochrome c 1 was found to be pH independent. The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycholate buffers, while a variation of −30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers. These two detergents also produced different MCD contributions of the two b cytochromes. A decrease in E m of greater than 300 mV was found in potentiometric measurements of cytochrome c 1 at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c 1 but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by ubiquinone.

Midpoint potentials of cytochromes in vesicles of anaerobically-grown Paracoccus denitrificans determined by the indirect coulometric titration method

1. 1. Multiplicity of redox components with spectral properties similar to b-type cytochromes was established in vesicles derived from anaerobically-grown Paracoccus denitrificans. 2. 2. Multiplicity of c-type cytochromes was not apparent either from low temperature spectroscopy or potentiometric titrations. 3. 3. Cytochromes a + a 3 and a component, only observable at liquid nitrogen temperature, with a spectral maximum at 582.5 nm were detected. 4. 4. Redox cycling of electron transport components using the indirect coulometric titration method was a convenient means of pairing redox potentials and was reproducible in total absorbance changes, midpoint potentials and spectral maxima.

Electron transfer reactions of cytochrome f from Brassica komatsuna with hexacyanoferrate.

The electron transfer reactions of membrane-bound monomeric cytochrome f from Brassica komatosuna (Brassica rapa L. var. perviridis Bailey) with hexacyanoferrate (II)-(III) have been studied as a function of pH, ionic strength and temperature. The second-order rate constant for the oxidation of cytochrome f by Fe(CN)6(3-) at pH 7.0, mu 0.1 M, and 20 degrees C is 1.7 X 10(5) M-1 X S-1, which is similar to the value of oligomeric cytochrome f from parsley. The activation parameters obtained were delta H not equal to = -0.87 kcal/mol and delta S not equal to - -38 cal/mol x deg. Respective rat constant and activation parameters obtained for the reduction of cytochrome f by Fe(CN)6(4-) were k = 1.7 X 10(4) M-1 x S-1, delta H not equal to = +6.7 kcal/mol, and delta S not equal to -16 cal/mol x deg. Both the rate constants for the oxidation and the reduction of cytochrome f markedly decreased with increasing ionic strength. The results indicate that the oxidation and the reduction take place at a positively charged site on the cytochrome f surface, and electrostatic interactions are important for these reactions. The participation of protons and specific amino acid residues in electron transfer reactions of cytochrome f is implied from the pH results. Alkaline isomerization of ferricytochrome f was not observed. The midpoint potential of cytochrome f has a constant value of 360 mV between pH 5.0-8.9, and decreases by about 55 mV per pH unit above 8.9. The results are compared with the data for horse heart cytochrome c and Euglena gracilis cytochrome c-552. These data are discussed in relation to the theories of electrostatic corrected outer-sphere electron transfer of Marcus and multiphonon nonadiabatic electron tunneling of Jortner and Hopfield.

The oxidation-reduction potentials of cytochrome o + c4 and cytochrome o purified from Azotobacter vinelandii.

Oxidation-reduction titrations of Azotobacter vinelandii cytochrome o + c4 and cytochrome o were performed with simultaneous potential and absorbance measurements under anaerobic conditions. Cytochrome c4 has a midpoint potential (Em, 7.4) of 260mV and purified cytochrome o has an Em, 7.4 of -18mV. Little change in the midpoint potential of cytochrome o was observed when titrated in the pH range 6.2--9.8.

On the Mechanism of the Cytochrome c Oxidase Reaction

The anaerobic‐aerobic transition of the CO‐inhibited ascorbate → cytochrome c→ cytochrome aa3 system is investigated using purified oxidase. In this system the presence of CO in the medium facilitates the measurements of the oxygen kinetics and at the same time allows a spectral resolution of cytochromes a and a3. Measurements of the oxygen consumption, and the steady‐state reduction of the cytochromes c, a, and a3 as a function of the oxygen concentration are reported. A double reciprocal plot of oxygen consumption rate versus oxygen concentration is nonlinear at low concentration of cytochrome c but becomes linear as the cytochrome concentration is increased. The plot of oxygen consumption rate against [O2] × [a2+3· CO], where a2+3· CO is the CO complex of the oxidase measured at 590 nm, is a straight line through the origin. This suggests that the binding of CO to the oxidase does not depend on the redox state of cytochrome a but is described by a single dissociation constant and also that the reaction between reduced cytochrome a3 and O2 is a second‐order reaction, k+1= 1.4 · 108 M−1 s−1. The results indicate that transfer of electrons from cytochrome a to cytochrome a3 during turnover is an irreversible second‐order reaction. But a similar simple relation seems not to be valid for the transfer of electrons from cytochrome c to a or from cytochrome c to a3. The assumption of a quasi‐equilibrium between cytochrome c and cytochrome a leads to the conclusion that the midpoint potential of cytochrome a changes with the redox state of cytochrome a3, probably as a consequence of allosteric interaction between the hemes.

Energy transduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the CO-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type ( E′ 0 at pH 7.0 +413±5, +270±5, +148±5, +56±5 and −32±5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes ( b 148, b 56, and b −32) vary as a function of pH with a slope of 30 mV per pH unit. 2. In the presence of a Co/N 2 mixture, the apparent E′ 0 of cytochrome b 270 shifts markedly towards higher potentials (+355 mV); a similar but less pronounced shift is apparent also for cytochrome b 150. The effect of CO on the midpoint potential of cytochrome b 270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain. 3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c 2 and of virtually all cytochrome cc′. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c 2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c 2 nor the CO-binding cytochrome cc′ are involved in this pathway. 4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in CO-difference spectra and with an α band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b 270, can be classified as a typical cytochrome “ o” and considered the alternative CO-sensitive oxidase.

The oxidation—reduction potentials of electron carriers in chloroplast Photosystem I fragments

Oxidation-reduction titrations of several electron carriers found in chloroplast Photosystem I fragments have been performed. The midpoint potential of P700 in these fragments and in chloroplasts has been found to be +520 mV by optical absorbance methods or electron paramagnetic resonance spectroscopy. The copper-containing protein plastocyanin is present in Photosystem I fragments and has a midpoint potential of +320 mV, significantly less positive than the midpoint potential of cytochrome f in the same fragments, which was measured to be +375 mV. Photo-system I fragments contain two b cytochromes, a low-potential form of cytochrome b 559 ( E m = +110 mV) and cytochrome b 563 ( E m = −100 mV).

Uncoupler-dependent decrease in midpoint potential of the chloroplast cytochrome b6

1. The midpoint potential of cytochrome b 6 in freshly prepared coupled chloroplasts is approximately +5 mV and the slope of the titration curve is characteristic of a two-electron transition. Subsequent titrations performed one-half hour or more after chloroplast preparation show a slight negative shift of the midpoint potential and a one-electron slope. 2. Titrations of cytochrome b 6 in the presence of carbonylcyanide- p-trifluoromethoxyphenylhydrazone (FCCP) or NH 4Cl show the midpoint potential of approximately half the b 6 complement negatively shifted to about −140 mV. 3. Brief exposure of the chloroplasts to NH 4Cl and actinic illumination under typical uncoupling conditions with controlled potential conditions causes a shift in oxidation state consistent with a lowering of the average midpoint potential to about −100 mV. It is concluded that the midpoint potential of at least half the cytochrome b 6 undergoes a negatively directed shift of 100–150 mV under uncoupling conditions in the presence of NH 4Cl and FCCP.

Spectral study of b cytochromes in yeast mitochondria and intact cells

Three types of b cytochromes are demonstrated in Candida utilis mitochondria. One of these b cytochromes has a symmetrical α-band at 561.5 nm at room temperature. This b cytochrome is readily reduced either by anaerobiosis or by cyanide treatment in the presence of glycerol 1-phosphate or succinate both in coupled and uncoupled mitochondria. The second b cytochrome has a double α-band at 565 nm and 558 nm. This b cytochrome is readily reduced either by anaerobiosis or by cyanide treatment in the presence of glycerol 1-phosphate or succinate in coupled mitochondria, but in uncoupled mitochondria it is slowly reduced after anaerobiosis and this reduction rate is enhanced by antimycin A addition. Thus the oxidation-reduction state of this cytochrome is energy dependent. The first cytochrome is spectroscopically identified as cytochrome b K and the second as cytochrome b T. The third b cytochrome has an α-band around 563 nm ( b 563) and is reduced slowly after anaerobiosis in uncoupled mitochondria but faster than the b T. Further properties of this component are not known. Midpoint potentials of cytochromes b T, b 563 and b K are approximately −50 mV, +5 mV, and +65 mV, respectively. In intact cells, cytochrome b T is reduced immediately after anaerobiosis or cyanide treatment, and rapidly oxidized when uncoupler is added. Addition of antimycin A instead of uncoupler to the anaerobic cells causes oxidation of mainly cytochrome b T while addition of antimycin A to the aerobic cells results in a reduction of the cytochrome b T.

The redox state of cytochrome b559 in spinach chloroplasts

Spinach chloroplasts suspended in a mixture of redox buffers were titrated under anaerobic conditions by adding small amounts of oxidant (potassium ferricyanide) or reductant (reduced methyl viologen). At specific redox potentials, measured with a platinum electrode, samples of chloroplasts were removed, frozen to −196 °;C, and single-beam absorption spectra were measured before and after actinic irradiation at −196 °C. The extent of the low temperature light-induced reduction of C-550 and oxidation of cytochrome b 559 was determined as a function of redox potential. Difference spectra between samples at different redox potentials were used to determine titration curves for the chemically induced changes. With intact chloroplasts at 0 °C the midpoint potential of cytochrome b 559 was found to be + 450 mV. Titration of Tris-washed chloroplasts at 20 °C showed that cytochrome b 559 is oxidized at lower potentials and over a wider range of potentials than in intact chloroplasts. A cytochrome b 559 with a still lower midpoint potential (dithionite reducible but not ascorbate reducible) was also demonstrated in intact chloroplasts which had not been subjected to treatments which modify the normal high potential cytochrome b 559.

Energy dependence of oxidation-reduction potentials of the [formula omitted] and [formula omitted] cytochromes in beef heart submitochondrial particles

In coupled beef heart submitochondrial particles (Mg-ATP) the addition of ATP causes the measured oxidation-reduction midpoint potential of about 35–40% of the total b -cytochrome complement to assume a value more positive by at least 70 mV. There is no significant effect on the midpoint potentials of cytochromes c + c 1 . Hence, submitochondrial particles which many consider to be “inside out” display qualitatively the same behavior as the intact mitochondrion: under the experimental conditions the location of the membrane-bound carriers with respect to the bulk aqueous phase would appear not to be relevant to the chemical events of the energy conserving mechanism.

Effect of membrane potential on equilibrium poise between cytochrome a and cytochrome c in rat liver mitochondria.

The object of this work was to test the suggestion that the equilibrium poise between cytochromea and cytochromec in mitochondria might be influenced by the membrane potential. 1. The midpoint potentials of cytochromes (c+c1) and cytochromea (CO present) were found to be 250 mV and 245 mV, respectively, by equilibrating rat liver mitochondria with mixtures of ferrocyanide and ferricyanide anaerobically in presence of antimycin A and measuring the redox state of the cytochromes spectrophotometrically. In absence of CO, cytochrome oxidase gave an anomalous redox titration curve with a “midpoint” at about 275 mV. 2. When the mitochondria were equilibrated with ferricyanide/ferrocyanide, the redox poise of cytochromea (CO present) and of cytochromes (a+a3) but not of cytochromes (c+c1) was dependent on the sign and magnitude of the membrane potential developed by treating the mitochondria as follows: by adding ATP, by chaging the composition of the suspension medium so as to vary the Donnan or Nernst potential, by adding valinomycin in a medium of low K+ ion content, or by adding a pulse of acid or alkali when the membrane was made permeable to protons with FCCP. 3. The findings agree with the suggestion that the respiratory chain is arranged across the cristae membrane with cytochromesc1 andc in contact with the outer phase and cytochromesa anda3 plugged through, so that the equilibrium distribution of electrons between thec anda cytochromes is influenced by the electric field across the membrane.

Potentiometric Determination of Interrelationships of Energy Conservation and Ion Gradients in Mitochondria

Abstract The reduction potential of cytochrome c is monitored potentiometrically with N,N,N',N'-tetramethyl-p-phenylenediamine as a mediator of electrons from cytochrome c to a platinum electrode. Rotenone and cyanide are used as respiratory inhibitors in simultaneous observation of reversal of electron transport between cytochrome b measured spectroscopically and cytochrome c measured potentiometrically. Rotenone, malonate, antimycin A, and cyanide are employed to inhibit respiration for observation of reversal of electron transfer between cytochrome a measured spectroscopically and cytochrome c measured potentiometrically. The midpoint potential of cytochrome a at pH 7.4 is estimated to be more than 330 mv. Rotenone alone is used as an inhibitor of electron transfer in a study of the interrelation between the phosphate potential and the reduction potential of cytochrome c in nonrespiring mitochondria; reduction potential changes in cytochrome c are shown to give an accurate estimate of phosphate potential changes in mitochondria. The standard free energy of hydrolysis of bound ATP to bound ADP at pH 7.4 is estimated to be -9.4 kcal. By means of the potentiometric technique with rotenone-inhibited mitochondria to follow phosphate potential changes, the interaction of ion gradient and ion transport with the energy conservation system is investigated by adding calcium, valinomycin, nigericin, or uncoupling agents to alter ion permeability or ion gradients.