pH dependence of the oxidation-reduction potential of cytochrome c2.

The pH dependence of the spectra and of the oxidation-reduction potential of three cytochromes c2, from Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides and Rhodomicrobium vannielii, were studied. A single alkaline pK was observed for the spectral changes in all three ferricytochromes. In Rps. capsulata cytochrome c2 this spectroscopic pK corresponds to the pK observed in the dependence of oxidation-reduction potential on pH. For the other two cytochromes the oxidation-reduction potential showed a complex dependency on pH which can be fitted to theoretical curves involving three ionizations. The third ionization corresponds to the ionization observed in the spectroscopic studies but the first two occur without changes in the visible spectra. The possible structural bases for these ionizations are discussed.     

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.     

Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Several cytochromes c₂ from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (pK_o) and one in the reduced form (pK_r). These cytochromes fall into one of two groups according to the degree of separation of pK_o and pK_r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c₂, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c₂ and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the pK of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised pK for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c₂, the separation between pK_o and pK_r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c₂/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c₂. Using Coulomb's law, it was found that the energy required to separate pK_o and pK_r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.     

Redox potentials of the photosynthetic bacterial cytochromes c2 and the structural bases for variability

The cytochromes c₂ of the Rhodospirillaceae show a much greater variation in redox potential and its pH dependence than the mitochondrial cytochromes c that have been studied. It is proposed that the range of redox potential for cytochromes c₂ functioning as the immediate electron donor to photo-oxidised bacteriochlorophyll may be 345–395 mV at pH 5.Closely related cytochromes c₂ with different redox potentials show patterns of amino acid substitution which are consistent with changes in hydrophobicity near the haem being at least a partial determinant of redox potential. More distantly related cytochromes are difficult to compare because of the large number of amino acid substitutions and the probability that there are subtle changes in overall peptide chain folding.The redox potential versus pH curves can be analysed in terms of either one ionisation in the oxidised form or two in the oxidised form and one in the reduced. The pK in the oxidised form at higher pH values can be correlated with the pK for the disappearance or shift of the near infrared absorption band located near 695 nm. The structural bases of these ionisations are not known but the possible involvement of the haem propionate residues is discussed.     

pH Variation of midpoint potential for three photosynthetic bacterial cytochromes c′. A link between physical and functional properties

Midpoint redox potential (E_M) versus pH curves are reported over the pH range 5 to 10 for the cytochromes c′ from three species of purple photosynthetic bacteria: Rhodospirillum rubrum, Rhodopseudomonas palustris and Chromatium vinosum. In each case, theoretical curves are fitted to the data and pK values for the reduced (pH 5–5.5) and oxidized (pH 8–8.5) forms of the protein are found to influence the midpoint redox potentials. The oxidized form pK values in each case are found to correlate with previously determined pK values for variation in physical and/or spectroscopic properties. This correlation of functional and physical observables is discussed in terms of a possible mechanism of control of midpoint redox potential through heme iron-ligand bonding as moderated by the protein conformation in response to solution conditions. The reduced form pK values are discussed in terms of a mechanism which would alter the polarity of the heme environment, thereby influencing redox potentials.     

In situ characterisation of photosynthetic electron transport in Rhodopseudomonas capsulata

1. The effects of varying the ambient oxidation/reduction potential on the redox changes of cytochromes c, cytochromes b and P605 induced by a laser flash in chromatophores from Rhodopseudomonas capsulata Ala Pho⁺ have been investigated.2. The appearance and attenuation of the changes with varying ambient redox potential show that, of the cytochromes present, cytochromes c with Em₇ = 340 mV and 0 mV, and cytochrome b, Em₇ = 60 mV were concerned with photosynthetic electron flow.3. The site of action of antimycin was shown to be between cytochrome b₆₀ and a component, as yet unidentified, called Z.4. The appearance or attenuation of laser-induced changes of cytochromes c₀ and b₆₀ on redox titration was dependent on pH, but no effect of pH on the cytochrome c₃₄₀ titration was observed.5. The dependence on ambient redox potential of the laser-induced bleaching at 605 nm enabled identification of the mid-point potentials of the primary electron donor (Em₇ = 440 mV) and acceptor (Em₇ = ?25 mV).6. The interrelationship of these electron carriers is discussed with respect to the pathway of cyclic electron flow.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

Equilibrium and kinetic measurements of the redox potentials of cytochromes c2 in vitro and in vivo

The equilibrium oxidation-reduction mipoint potential (E_m) of isolated Rhodopseudomonas sphaeroides cytochrome c₂ exhibits a pH-dependent behavior which can be ascribed to a pK on the oxidized form at pH 8.0 (Pettigrew et al. (1975) Biochim. Biophys. Acta 430, 197–208). However, as with mammalian cytochrome c (Brandt, K.G., Parks, P.C., Czerlinski, G.H. and Hess, G.P. (1966) J. Biol. Chem. 241, 4180–4185) this pK can more properly be attributed to the combination of a pK beyond pH 11, and a slow conformational change of the ferricytochrome. This has been demonstrated by resolving the E_m of cytochrome c₂ before and after the conformational change. The E_m of the unaltered form is essentially pH independent between pH 7 and 11.5, and the lower equilibrium E_m is due solely to the conformational change. In vivo the conformational change is prevented by the binding of the cytochrome c₂ to the photochemical reaction center, and the cytochrome exhibits an essentially pH-independent E_m from pH 5 to 11. The alkaline transition thus has little physiological significance, and it is unlikely that the redox reactions of cytochrome c₂ in vivo involve protons.     

A thermodynamic characterisation of the cytochromes of chromatophores from Rhodopseudomonas capsulata

1. The cytochromes of chromatophores from photosynthetically grown Rhodopseudomonas capsulata have been characterised both spectrally, using the carotenoid free mutant Ala Pho⁺, and thermodynamically, using the technique of redox titrations. Five cytochromes were present; two cytochromes b, E′₀ = 60 mV at pH 7.0; and three cytochromes c, E′₀ = 340 mV, $\text{E}\text{t}\text{?}{}_0\text{= 120}\text{mV}$, E′₀ = 0 mV at pH 7.0.2. Redox titrations at different values of pH indicated that the mid point potentials of all the cytochromes varied with pH over some parts of the range between pH 6 and 9, with the possible exception of cytochrome c₃₄₀.3. The effects of succinate and NADH on the steady state reduction of the cytochromes are reported. Succinate could reduce cytochromes c₃₄₀, c₁₂₀ and b₆₀; NADH could reduce cytochromes c₃₄₀, c₁₂₀, b₆₀ and b_?25. Cytochrome c₀ could be reduced by dithionite but not by the other substrates tested.     

The two cytochromes c in the facultative methylotroph Pseudomonas AM1

It was previously suggested that there is only one soluble cytochrome c in Pseudomonas AM1, having a molecular weight of 20000, a redox midpoint potential of about +260mV and a low isoelectric pint [Anthony (1975) Biochem. J. 146, 289–298; Widdowson & Anthony (1975) Biochem. J. 152, 349–356]. A more thorough examination of the soluble fraction of methanol-grown Pseudomonas AM1 has now revealed the presence of two different cytochromes c. These were both purified to homogeneity by acid treatment, ion-exchange chromatography, gel filtration, chromatography on hydroxyapatite and preparative isoelectric focusing. Molecular weights were determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; midpoint redox potentials were determined directly by using platinum and calomel electrodes; isoelectric points were estimated by electrophoresis and by the behaviour of the two cytochromes on ion-exchange celluloses. The more abundant cytochrome c_H (λ_max. 550.5nm) had a low molecular weight (11000), a midpoint potential of about +294mV and a high isoelectric point, not being adsorbed on DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The less abundant cytochrome c_L (λ_max. 549nm) was about 30% of the total; it had a high molecular weight (20900), a midpoint potential of about +256mV and a low isoelectric point, binding strongly to DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The pH-dependence of the midpoint redox potentials of the two cytochromes c were very similar. There were four ionizations affecting the redox potentials in the pH range studied (pH4.0–9.5), two in the oxidized form (pK values about 3.5 and 5.5) and two in the reduced form (pK values about 4.5 and 6.5), suggesting that the ionizing groups involved may be the two propionate side chains of the haem. Neither of the cytochromes c was present in mutant PCT76, which was unable to oxidize or grow on C₁ compounds, although still able to grow well on multicarbon compounds such as succinate. Whether or not these two cytochromes c have separate physiological functions is not yet certain.     

The branched respiratory chain of heterotrophically dark-grown Chloroflexus aurantiacus

The respiratory electron-transport chain of heterotrophically dark-grown Chloroflexus aurantiacus has been investigated. Membranes isolated from these cells have been shown to contain at least three c-type cytochromes (E_{m, 7.0} 255,180, and 10 mV), three b-type cytochromes (E_{m, 7.0} of 210, 60 and −65 mV) and two cytochromes of the a type with E_{m, 7.0} of 330 and 190 mV. Spectroscopic evidence from CO-difference spectra, CN⁻-duference spectra and spectra at fixed oxidation-reduction potentials suggests that the two a-type components may be analogous to cytochromes a and a₃ of mitochondria. The analyses of the effects induced by CN⁻, myxothiazol and antimycin A on both steady-state respiratory activities and semi-rapid oxidation-reduction kinetic patterns of c- and a-type cytochromes indicate the presence of a branched respiratory chain. Growth of Chloroflexus in medium lacking added copper diminished the concentration of the a-type cytochromes but not those of cytochromes of the b and c type.     

Asymmetry of an energy transducing membrane. The location of cytochrome c2 in Rhodopseudomonas spheroides and Rhodopseudomonas capsulata

Monospecific antibodies have been prepared against cytochrome c₂ from Rhodopseudomonas spheroides and Rhodopseudomonas capsulata, and against cytochrome c′ from Rps. capsulata. These antibodies precipitated their respective antigens, but did not cross react with a wide range of procaryotic or eucaryotic cytochromes, or with other bacterial proteins. The cytochromes produced during aerobic growth were immunologically indistinguishable from those produced during photosynthetic growth.Cytochrome c₂ is located in vivo in the periplasmic space between the cell wall and the cell membrane, and when chromatophores are prepared from whole cells the cytochrome becomes trapped inside these vesicles. The implications of these results to energy coupling in the photosynthetic bacteria are discussed.     

Soluble cytochromes ofRhodopseudomonas marina

Three acidicc-type cytochromes (c-552,c-550 andc′) were purified from the soluble fraction ofRhodopseudomonas marina. Cytochromec′ is a high-spin cytochrome capable of binding carbon monoxide reversibly to its reduced form. It occurs as a dimer with anM_r of 36700 (estimated by gel filtration) while the monomer has anM_r of 17800 (determined by SDS-acrylamide gel electrophoresis). Cytochromec′ has a midpoint redox potential of +73 mV and an isoelectric point at pH 4.3. Cytochromesc-550 andc-552 are typical low-spin cytochromes. Cytochromec-550 has anM_r of 12500, an isoelectric point at pH 4.5 and a negative redox potential of −163 mV. The molecular properties of cytochromec-552 are as follows:M_r, 18000; isoelectric point, pH 5.4; redox potential, +283 mV.     

Reduction kinetics of bacterial cytochromes c2.

In a continuing effort to understand the mechanism of electron transfer by c-type cytochromes we have extended our investigations of the oxidation and reduction of Rhodospirillum rubrum cytochrome c₂. We have utilized the oxidant, oxidized azurin, and the reductants SO₂^?, S₂O₄^2?, sodium ascorbate, and reduced azurin. The results of these studies demonstrate that, as found previously with the iron hexacyanides, electron transfer apparently takes place at the exposed heme edge. Furthermore, we report studies on the reduction of ferricytochrome c₂ from Rhodopseudomonas sphaeroides, Rhodopseudomonas capsulata, Rhodomicrobium vannielii, and Rhodopseudomonas palustris by potassium ferrocyanide. Based on the amino acid sequence homology between the various cytochromes c₂ and presumed structural homology, the observed rates of electron transport are analyzed in terms of the structure in the region of the exposed heme edge.     

Properties of Ascaris muscle mitochondria. I. Cytochromes

1. The cytochrome system in Ascaris muscle mitochondria was further characterized using purer preparations.2. Difference spectra (at 22 °C and ?196 °C) of the mitochondrial preparations using succinate and ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine show that Ascaris muscle mitochondria contain cytochromes c₁, c and aa₃, and also at least three b-type cytochromes. The b-type cytochrome is the predominant component.3. Cytochrome c and Ascaris cytochrome b-560 can be extracted from the mitochondrial preparations with 150 mM KCl, leaving the membrane-bound cytochromes c₁, b and aa₃ in the KCl residue.     

Electrogenic events in the ubiquinone-cytochrome bc2 oxidoreductase of rhodopseudomonas sphaeroides

The reductant of ferricytochrome c₂ in Rhodopseudomonas sphaeroides is a component, Z, which has an equilibrium oxidation-reduction reaction involving two electrons and two protons with a midpoint potential of 155 mV at pH 7. Under energy coupled conditions, the reduction of ferricytochrome c₂ by ZH₂ is obligatorily coupled to an apparently electrogenic reaction which is monitored by a red shift of the endogeneous carotenoids. Both ferricytochrome c₂ reduction and the associated carotenoid bandshift are similarly affected by the concentrations of ZH₂ and ferricytochrome c₂, pH, temperature the inhibitors diphenylamine and antimycin, and the presence of ubiquinone. The second-order rate constant for ferricytochrome c₂ reduction at pH 7.0 and at 24°C was 2 · 109 M^?1 · s^?1, but this varied with pH, being 5.1 · 10⁸ M^?1 · s^?1 at pH 5.2 and 4.3 · 10⁹ M^?1 · s^?1 at pH 9.3. At pH 7 the reaction had an activation energy of 10.3 kcal/mol.     

Oxidation of c-Type Cytochromes by the Membrane-Bound Cytochrome Oxidase (Cytochrome aa(3)) of Blue-Green Algae

Respiratory particles containing an aa₃-type cytochrome oxidase were prepared from Anacystis nidulans, Synechocystis 6714, Synechococcus lividus, Anabaena variabilis, Nostoc sp. strain MAC, Nostoc muscorum, and Mastigocladus laminosus. Oxidation of c-type cytochromes by membrane preparations of the different blue-green algae was observed using purified cytochromes from horse heart, Candida krusei, tuna, Saccharomyces oviformis, Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas palustris, Rhodocyclus purpureus, Paracoccus denitrificans, Anacystis nidulans, Anabaena variabilis, Euglena gracilis, and Scenedesmus obliquus. Rapid oxidations were consistently observed with the mitochondrial c-type cytochromes (horse heart cytochrome c reacts most rapidly) and with cytochromes c₂ from Rhodopseudomonas palustris and Rhodocyclus purpureus; in contrast, the cytochrome c₂ from Rhodospirillum rubrum and the plastidic cytochromes from E. gracilis and Scendesmus obliquus were inactive with all membrane preparations. All reactions were inhibited by low concentrations of KCN, NaN₃, and CO, and they were activated by Tween 80, thus indicating participation of the terminal oxidase. The results are discussed in view of the spectral similarities between the terminal oxidase of blue-green algae and the mitochondrial aa₃-type cytochrome oxidase of plants and other eukaryotes.     

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, lysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 ± 10 mV (b₅₆₁), and 0 ± 10 mV (b₅₆₆) and cytochrome c₁ (E_{m 7.2} = +280±5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40–60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b₅₆₆ is pH-dependent above pH 7.0 (?60 mV/pH unit) while that of b₅₆₁ is essentially pH-independent from pH 6.7–8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b₅₆₆ (b_T), b₅₆₁ (b_K) and c₁ respectively is made on the basis of redox midpoint potentials. No significant amounts of oxidized high-spin heme-iron are detectable. In addition, the preparation contains four distinct types of iron-sulfur centers: S₁ and S₂ (E_{m 7.4} = ?260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c₁ complex: Rieske's iron-sulfur protein (E_{m 7.4} = +280 mV) and Ohnishi's Center 5 (E_{m 7.4} = +35 mV).     

Investigation of cobalt(II) substituted carboxypeptidase a interacting with azide and cyanate ions

Cobalt(II)-substituted carboxypeptidase A has been found to reversibly bind N3^? and NCO^?, but not NCS^?, in the pH range 5–10, thus including the pH range of activity of the enzyme. The pH dependence of the anion binding constant is affected by two ionizations, which are assigned as those regulating k_cat and K_M. The electronic and ₁H NMR spectra are consistent with a substantially pseudotetrahedral geometry of the anion derivatives.     

Detection of two further b-type cytochromes in Rhodopseudomonas spheroides

The photosynthetically-incompetent mutant V-2 of Rhodopseudomonas spheroides which is incapable of synthesising bacteriochlorophyll was grown aerobically under conditions of both high and low aeration. Potentiometric titration at 560 nm minus 570 nm revealed the presence of several different components tentatively identified as b-type cytochromes. Two such components of oxidation-reduction midpoint potentials of +390 mV ± 10 mV and +255 mV ± 7 mV have not previously been detected in membranes of Rps. spheroides. These components have also been resolved by difference spectra at controlled oxidation-reduction potentials and fourth derivative spectra. Neither component appeared to react with CO. With increasing aeration of the culture medium the relative concentration of these two b-type cytochromes diminished, whilst that of the a-type oxidase increased.