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 c1, 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 degrees 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 c1 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 Em of greater than 300 mV was found in potentiometric measurements of cytochrome c1 at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c1 but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by ubiquinone.     

Respiratory chain of the alkalophilic bacterium Bacillus firmus RAB and its non-alkalophilic mutant derivative

The membrane-bound respiratory chain components of alkalophilic Bacillus firmus RAB were studied by difference spectroscopy and oxidation-reduction potentiometric titrations. Cytochromes with the following midpoint potentials were identified at pH 9.0: a-type cytochromes, +110 and +210 mV; b-type cytochromes, +20, -120, -280, and -400 mV; and cytochrome c, +60 mV. Only the higher-potential cytochrome a showed an upward shift in midpoint potential when titrated at pH 7.0. Parallel studies of a non-alkalophilic mutant derivative of B. firmus RAB, strain RABN, revealed the presence of only one species each of a-, b-, and c-type cytochromes which exhibited midpoint potentials of +110, -150, and +160 mV, respectively, at pH 7.0. Membranes of both strains were found to contain menaquinone. At pH 9.0, NADH caused the reduction of essentially all of the cytochromes that were seen in fully reduced preparations of wild-type B. firmus RAB membranes. By contrast, at pH 7.0, NADH failed to appreciably reduce the b-type cytochromes. These findings may relate to our recent proposal that an inadequacy in energy transduction (production of a proton motive force) by the alkalophilic respiratory chain at pH 7.0 is what precludes the growth of B. firmus RAB at a neutral pH.     

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.     

The active site of ribonuclease A from the crystallographic studies of ribonuclease-A-inhibitor complexes

Several members of the genus Methanosarcina were investigated by room-temperature and low-temperature difference spectroscopy for the presence of cytochromes. In combination with potentiometric titrations two membrane-bound b-cytochromes and one membrane-bound c-cytochrome could be detected in cells grown on methanol or trimethylamine. Very probably acetate-grown cells contained an additional cytochrome b. The midpoint potentials of the two b-type cytochromes were Em1 = -325 mV and Em2 = -183 mV, respectively. The additional b cytochrome formed during growth on acetate exhibited a midpoint potential of Em3 = -250 mV.     

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 microM for the high and low affinity site, respectively.     

Characterization and phenotypic control of the cytochrome content of Escherichia coli

1. Electron-transport particles derived from Escherichia coli grown aerobically contain three b-type cytochromes with mid-point oxidation-reduction potentials at pH7 of +260mV, +80mV and -50mV, with n=1 for each. The variation of these values with pH was determined. 2. E. coli develops a different set of b-type cytochromes when grown anaerobically on glycerol with fumarate or nitrate as terminal electron acceptor. Electron-transport particles of fumarate-grown cells contain b-type cytochromes with mid-point potentials at pH7 of +140mV and +250mV (n=1). These two cytochromes are also present in cells grown with nitrate as terminal acceptor, where an additional cytochrome b with a mid-point potential of +10mV (n=1) is developed. 3. The wavelengths of the alpha-absorption-band maxima of the b-type cytochromes at 77K were: (a) for aerobically grown cells, cytochrome b (E(m7) +260mV), 556nm and 563nm, cytochrome b (E(m7) +80mV), 556nm and cytochrome b (E(m7)-50mV), 558nm; (b) for anaerobically grown cells, cytochrome b (E(m7) +250mV), 558nm, cytochrome b (E(m7) +40mV), 555nm and cytochrome b (E(m7) +10mV), 556nm. 4. Cytochrome d was found to have a mid-point potential at pH7 of +280mV (n=1). 5. Cytochrome a(1) was resolved as two components of equal magnitude with mid-point potentials of +260mV and +160mV (n=1). 6. Redox titrations performed in the presence of CO showed that one of the b-type cytochromes in the aerobically grown cultures was reduced, even at the upper limits of our range of electrode potentials (above +400mV). Cytochrome d was also not oxidizable in the presence of CO. Neither of the cytochromes a(1) was affected by the presence of CO.     

Study of redox potential in cytochrome <Emphasis Type="Italic">c</Emphasis> covalently bound to terminal oxidase of alkaliphilic <Emphasis Type="Italic">Bacillus pseudofirmus</Emphasis> FTU

Spectroelectrochemistry was used to determine the midpoint redox potentials of heme cofactors of the caa3-type cytochrome oxidase from the alkaliphilic bacterium Bacillus pseudofirmus FTU. The apparent midpoint potentials (E(m)(app)) for the most prominent transitions of hemes a and a3 (+193 and +334 mV, respectively) were found to be similar to the values reported for other enzymes with high homology to the caa3-type oxidase. In contrast, the midpoint potential of the covalently bound cytochrome c (+89 mV) was 150-170 mV lower than in cytochromes c, either low molecular weight or covalently bound to the caa3 complex in all known aerobic neutralophilic and thermo-neutralophilic bacteria. Such an unusually low redox potential of the covalently bound cytochrome c of the caa3-type oxidase of alkaliphilic bacteria, together with high redox potentials of hemes a and a3, ensures more than twice higher difference in redox potentials inside the respiratory complex compared to the homologous mitochondrial enzyme. The energy released during this redox transition might be stored in the transmembrane H+ gradient even under low Deltap in the alkaline environment of the bacteria at the expense of a significant increase in DeltaG of the coupled redox reaction.     

Midpoint potentials of the mitochondrial cytochromes of Crithidia fasciculata.

The midpoint potentials of the mitochondrial respiratory chain cytochromes of the protozoan Crithidia fasciculata at pH 7.2, Em7.2, show great similarity to those measured in higher organisms. Values of Em7.2 for cytochromes a and a3 are +165 and +340 mV. Both c cytochromes have Em7.2 = +230 mV. There are two b cytochromes with the same spectral characteristics with Em7.2 = -20 and -135 mV. These values are compatible with two sites of energy conservation for oxidative phosphorylation in these mitochondria. All cytochrome components show potentiometric titrations with n = 1. There is a fluorescent flavoprotein in these mitochondria with Em7.2 = -40 mV and n =2, whose function is not known.     

Complete analysis of the cytochrome components of beef heart mitochondria in terms of spectra and redox properties. The c1-cytochromes.

Using newer techniques for conducting and analyzing potentiometric titrations, we have studied the thermodynamic and spectral properties of cytochrome c1 in beef heart mitochondria. We find two species of cytochrome c1, both with n = 2 values for the number of electrons involved in their oxidation or reduction. One has an Em approximately 210 mV and a spectral peak near 555 nm and the other has an Em approximately 255 mV and a spectral peak nearer 553 nm. These Em values are pH-independent in the range of pH 6 to 8. The Em and n values of these two components are indistinguishable from those of two species of cytochrome aa3 (i.e. spectral feature of 605 nm).     

A cytochrome b/c1 complex with ubiquinol--cytochrome c2 oxidoreductase activity from Rhodopseudomonas sphaeroides GA

A cytochrome b/c1 complex which catalyses the reduction of cytochrome c by ubiquinol has been isolated from Rhodopseudomonas sphaeroides GA. It contains two hemes b and substoichiometric amounts of ubiquinone-10 and of the Rieske Fe-S center per cytochrome c1, and is essentially free of reaction center and bacteriochlorophyll. The complex consists of three major polypeptides with apparent molecular masses of 40, 34 and 25 kDa. The 34-kDa polypeptide carries heme. Cytochrome c1 has a midpoint potential of 285 mV. For cytochrome b two midpoint potentials, at 50 and -60 mV, at pH 7.4, can be derived if one assumes two components of equal amount. Ubiquinol--cytochrome c oxidoreductase activity is specific for ubiquinol and bacterial cytochromes c, and is inhibited by antimycin A and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. The complex shows oxidant-induced reduction of cytochrome b.     

Potentiometric study of cytochrome c1aa3 from Thermus thermophilus

We have examined the redox behavior of the cytochrome c1aa3 complex from Thermus thermophilus. In potentiometric titrations the cytochrome c behaves as an independent center having n = 1 and E = 205 mV (NHE). Under the assumption that the individual centers equilibrate independently in this experiment, changes in the absorption band at 603 nm have been resolved into two components: cytochrome a (n = 1, Em = 270 mV, 60% spectral contribution) and cytochrome a3 (n = 2, Em = 360 mV, 40% spectral contribution). The n = 2 process was attributed to strong chemical coupling between cytochrome a3 and CuB. The enzyme was also titrated with a mixture of NADH and PMS, and the results are shown not to conform to a model of intramolecular equilibrium according to the equilibrium constants obtained from the potentiometric titration. It is suggested that a conformational equilibrium within the complex may control electron transfer between cytochromes a and a3.     

Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.

1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.     

The pH dependence of the oxidation-reduction midpoint potential of cytochromes c2 in vivo.

A recent report by Pettigrew et al. [Biochim, Biophys. Acta 430, (1976), 197-208] has examined the pH dependence of the oxidation-reduction midpoint potential of cytochromes c2 in vitro. In media of low ionic strength, these workers identified several pKs on the oxidized forms of the cytochromes, and in some cases there were also pKs on the reduced species. In this work we examine the pH dependence of the midpoint potentials of the cytochromes in situ, attached to the chromatophore membrane. Under these conditions no pK values are detected, and we conclude that in vivo there is no net change in the protonation of cytochrome c2 during oxidation or reduction.     

Controlling the functionality of cytochrome c(1) redox potentials in the Rhodobacter capsulatus bc(1) complex through disulfide anchoring of a loop and a beta-branched amino acid near the heme-ligating methionine.

The cytochrome c(1) subunit of the ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) contains a single heme group covalently attached to the polypeptide via thioether bonds of two conserved cysteine residues. In the photosynthetic bacterium Rhodobacter (Rba.) capsulatus, cytochrome c(1) contains two additional cysteines, C144 and C167. Site-directed mutagenesis reveals a disulfide bond (rare in monoheme c-type cytochromes) anchoring C144 to C167, which is in the middle of an 18 amino acid loop that is present in some bacterial cytochromes c(1) but absent in higher organisms. Both single and double Cys to Ala substitutions drastically lower the +320 mV redox potential of the native form to below 0 mV, yielding nonfunctional cytochrome bc(1). In sharp contrast to the native protein, mutant cytochrome c(1) binds carbon monoxide (CO) in the reduced form, indicating an opening of the heme environment that is correlated with the drop in potential. In revertants, loss of the disulfide bond is remediated uniquely by insertion of a beta-branched amino acid two residues away from the heme-ligating methionine 183, identifying the pattern betaXM, naturally common in many other high-potential cytochromes c. Despite the unrepaired disulfide bond, the betaXM revertants are no longer vulnerable to CO binding and restore function by raising the redox potential to +227 mV, which is remarkably close to the value of the betaXM containing but loop-free mitochondrial cytochrome c(1). The disulfide anchored loop and betaXM motifs appear to be two independent but nonadditive strategies to control the integrity of the heme-binding pocket and raise cytochrome c midpoint potentials.     

Mitochondrial respiratory chain of Tetrahymena pyriformis. The thermodynamic and spectral properties.

The mitochondria isolated from the ciliate protozoon Tetrahymena pyriformis carry an oxidative phosphorylation with P/O ratio of 2 for succinate oxidation and P/O ratio of 3 for the oxidation of the NAD-linked substrates. The respiration is more than 90% inhibited with 1 mM cyanide while antimycin A and rotenone inhibit at concentrations of 1000-fold higher than those effective in mammalian mitochondria. Using a combination of spectral studies and potentiometric titrations, the components of the respiratory chain were identified and characterized with respect to the values of their half-reduction potentials. In the cytochrome bc1 region of the chain a cytochrome c was present with an Em7.2 of 0.225 V and two components with absorption maxima at 560 nm and the half-reduction potential values of -0.065 and -0.15 V at pH 7.2. The cytochrome with the more positive half-reduction potential was identified as the analogue of the cytochrome(s) b present in mitochondria of higher organisms, while the cytochrome with the more negative half-reduction potential was tentatively identified as cytochrome o. In addition ubiquinone was present at a concentration of approx. 4 nmol per mg mitochondrial protein. In the spectral region where cytochromes a absorb at least three cytochromes were found. A cytochrome with an absorption maximum at 593 nm and a midpoint potential of -0.085 V at pH 7.2 was identified as cytochrome a1. The absorption change at 615-640 nm, attributed usually to cytochrome a2, was resolved into two components with Em7,2 values of 0,245 and 0.345 V. It is concluded that the terminal oxidase in Tetrahymena pyriformis mitochondria is cytochrome a2 which in its two component structure resembles cytochrome aa3.     

Near infrared spectral changes of cytochrome aa3 during potentiometric titrations.

Singular value decomposition (SVD) was used to deconvolute the spectral changes occurring in the near infrared region during potentiometric titrations of cytochrome aa3. Overall oxidized minus reduced difference spectra revealed a broad absorbance feature centered near 830 nm with an apparent Em near 250 mV. However, SVD did not isolate any spectral species with an absorbance centered near 830 nm. It was found that the spectral changes occurring in the wavelength region from 650 to 950 nm were associated mainly with cytochromes a and a3. It was concluded that the absorbance at 830 nm should not be used as an independent measure of the concentration of CuA in cytochrome aa3.     

Surface potential changes of mitoplasts in the presence of pyridoxal phosphate modified cytochromes c.

1. The addition of native cytochrome c to mitoplasts leads to a decrease of surface potential of the mitoplast membrane. However the surface potential is slightly decreased (approximately 3 mV) when PLP(Lys 86)-cytochrome c and PLP(Lys 79)-cytochrome c were added. 2. The native and PLP-modified cytochromes c do not influence the order parameters S and isotropic constant a when both spin probe I and probe II were used. It is shown that cytochrome c binding to the membrane does not affect the hydrophobic intermembrane area as well as the lipid arrangements of the mitoplast membrane. 3. At low ionic strength there was observed a significant difference in the membrane potential when PLP-cytochromes c were added to the mitoplasts. 4. At high ionic strength the addition of native or PLP-modified cytochromes c does not change the membrane potential.     

Energy tranduction 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 (b148, b56, 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/N2 mixture, the apparent E'0 of cytochrome b270 shifts markedly towards higher potentials (+355mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 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 c2 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 c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 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 C0-difference spectra and with an alpha band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome "0" and considered the alternative CO-sensitive oxidase.     

Oxidation-reduction potentials of cytochromes in Ascaris muscle mitochondria: high-redox-potential cytochrome b558 in complex II (succinate-ubiquinone reductase)

Oxidation-reduction midpoint potentials (Ems) were determined at pH 7.0 for cytochromes in the anaerobic respiratory chain of Ascaris mitochondria by redox titration techniques. Cytochrome b558, which is associated with complex II that functions as fumarate reductase in the terminal step of the respiratory chain, was shown to have an Em of -34 mV in the isolated complex II and -54 mV in mitochondria. These values are much higher than the value of Ascaris cytochrome b558. In contrast, Ems of cytochromes C + C1 and cytochrome b559.5 were determined in situ to be 235 mV and 78 mV, respectively, which are comparable to those of their mammalian counterparts.     

Activity of cyclic-AMP phosphodiesterase in permeabilised cells of Bakers'' yeast

1. Pulse-radiolysis experiments were performed in the presence of methyl viologen and cytochrome c3. After the pulse, methyl viologen radicals are formed and the kinetics of these radicals with cytochrome c3 are studied, The reaction between cytochrome c3 and methyl viologen radicals (MV+) is diffusion controlled. The ionic strength dependence and the pH-dependence of this reaction were studied. From the ionic strength dependence (at pH 7.8) we found that the net charge of the fully oxidized cytochrome c3 molecule was Z = + 4.7 +/- 0.7. 2. After the pulse an equilibrium is reached for the reaction of MV+ with cytochrome c3. From this equilibrium an apparent midpoint potential can be obtained. The apparent midpoint potential of this multihaem molecule was found to depend on the degree of reduction, alpha. With the help of the Nernst equation an empirical equation is obtained to describe this dependence of the midpoint potential: E0 = - 0.250 - 0.088 alpha (in V). 3. An estimation is made of the energy of interaction between the haems due to electrostatic interactions (delta epsilon less than 32 mV) and due to ionic strength effects (- 12 mV less than delta epsilon less than 26 mV). The results suggest that the redox properties of the individual haems in the cytochrome c3 molecule are dependent on the degree of reduction of the other haems in the molecule. 4. The reaction of cytochrome c3 with MV+ or with ethanol radicals (EtOH) has been compared with the reactions of horse-heart cytochrome c and of metmyoglobin with the same radicals. The reaction of MV+ or EtOH with horse-heart cytochrome c is found to be diffusion controlled; the reactions with metmyoglobin on the other hand are most probably controlled by an activation energy.