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.     

Localization and concentration of hydroxylamine oxidoreductase and cytochromes c-552, c-554, cm-553, cm-552 and a in Nitrosomonas europaea

Two membrane-associated cytochromes, cytochrome c_m-553 and cytochrome c_m-552, were derived from Nitrosomonas europaea. The major c-type cytochrome, cytochrome c_m-553, accounted for 92% of the c heme found in the membrane. It had absorption maxima at 410 nm in the oxidized form and at 417, 523 and 553 nm in the dithionite reduced form. Cytochrome c_m-552 possessed absorption maxima at 409 nm in the oxidized form, at 421, 522 and 552 in the dithionite reduced form, and at 418 in the dithionite reduced plus CO form. The concentration and cellular distribution of the two c-type membrane cytochromes, hydroxylamine oxidoreductase and cytochromes c-552, c-554, and a were determined. Over 95% of the soluble cytochromes (hydroxylamine oxidoreductase cytochromes and c-552 and c-554) were periplasmic, whereas cytochrome c_m-553, cytochrome c_m-552 and cytochrome a were associated with the cell membrane. The outer membrane and cytoplasm were devoid of cytochromes. The extracytoplasmic location of the proton-yielding hydroxylamine oxidizing system (NH₂OH ™ HNO + 2H⁺ + 2e⁻) may contribute to an energy-linked proton gradient. The heme concentrations of hydroxylamine oxidoreductase and cytochromes c-552, c-554, c_m-553, c_m-552 and a were approx. 2.4, 1.2, 0.3, 1.3, 0.1 and 1.1 nmol/mg cell protein, respectively. The corresponding molar ratios of heme were 22:11:2.9:12:1.0:10. The enzyme or cytochrome concentrations for hydroxylamine oxidoreductase and cytochromes c-552, c-554, c_m-553, c_m-552 and a were approx. 0.13, 1.05, 0.09, 0.63, 0.055 and 0.56 nmol/mg cell protein, respectively. The corresponding molar ratios were 0.24:2.2:0.16:1.2:0.1:1.0.     

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.     

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.     

Reconstitution of photosynthetic electron transport and photophosphorylation in cytochrome-c2-deficient membrane preparation of Rhodopseudomonas capsulata.

Cytochrome c₂ was removed by washing from heavy chromatophores prepared from Rhodopseudomonas capsulata cells. The easy removal of the cytochrome could indicate that it was attached on the outside of the membrane. Therefore, the membrane was probably oriented inside out in relation to the membrane of regular chromatophores, from which cytochrome c₂ could not be removed. Washing of the heavy chromatophores caused loss of photphosphorylation activity. The activity was restored to the resolved heavy chromatophores by the supernatant obtained during the washing or by the native cytochrome c₂, which was found to be the active component in this supernatant. The activity could not be restored by other c-type cytochromes. Ascorbate, which enhanced photophosphorylation activity in the heavy chromatophores at the optimal concentration of 8 mm, restored this activity to the washed heavy chromatophores, but at an optimum concentration of 50 mm. Cytochrome c₂ and dichlorophenol indophenol reduced the optimum of the ascorbate concentration to 7 mm. This might indicate that the effect of ascorbate is mediated through cytochrome c₂. Washing the heavy chromatophores caused 70% loss of the light-induced electron transport from ascorbate and from ascorbate-reduced dichlorophenol indophenol to O₂. However, this effect was only observed with the lower concentrations of ascorbate and the dye. The activity was restored either by the supernatant obtained from the washing or by various c-type cytochromes, reduced by ascorbate. Washing the heavy chromatophores did not affect succinate oxidation in the dark. It is suggested that cytochrome c₂ is one of the cytochromes catalyzing the photosynthetic cyclic electron transport, as has been seen from its high specificity in the reconstitution experiments. Light can induce oxidation of various c-type cytochromes and other redox reagents. However, reduction was specific for cytochrome c₂ from Rps. capuslata, since it was the only one which could be both reduced and oxidized as required from a component which is part of a cyclic electron transport chain. It is also suggested that cytochrome c₂ was not part of the succinate oxidase system.     

The Cytochromes of Prototheca zopfii

The respiratory pigments of Prototheca zopfii include seven cytochromes: two c-type cytochromes, a soluble c(549) and a membrane bound c(551); three b-type cytochromes, b(555), b(559) and b(564); and cytochromes a and a₃. Cytochromes a and a₃ could be resolved spectrally in the α-band region by reducing the cells in the presence of methanol and cyanide. Methanol shifted the absorption maximum of cytochrome a from 598 to 603 nanometers and permitted dithionite (or substrate) to reduce the cyanide-cytochrome a₃ complex to give a well defined 595-nanometer absorption band. Methanol did not interfere with CO binding by cytochrome a₃, and CO did not alter the methanol effect on cytochrome a. Azide and cyanide, which partially inhibited exogenous respiration, stimulated endogenous respiration. Frozen steady states of the electron transport chain in the presence of cyanide and azide indicated that the stimulation by these inhibitors was due to an increased autooxidation of one of the b-type cytochromes, possibly b(564).     

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.     

Development and endocrine regulation of mitochondrial cytochrome biosynthesis in the insect fat body: δ-[14C]aminolevulinic acid incorporation

The rate of incorporation of [¹⁴C]aminolevulinic acid (ALA) into cytochrome hemes was used to measure mitochondrial cytochrome synthesis in the fat body of adult male Blaberus discoidalis cockroaches. The hemes of cytochromes aa₃+b and c+c₁, were chemically separated to observe differential rates in their synthesis and regulation. [¹⁴C]ALA was linearly incorporated into cytochrome hemes for at least 8 h. No significant pool of endogenous ALA was detected relative to the amount of administered [¹⁴C]ALA. Peak cytochrome synthesis occurred 4 to 6 days after adult emergence. Endocrine disruption by corpora cardiaca-corpora allata extirpation or cervical ligation eliminated the 4-day developmentally related increase in the rate of cytochrome aa₃+b synthesis but had no effect on the production of cytochromes c+c₁. Injections of corpora cardiaca extracts into cervically ligated animals stimulated the rate of production of cytochromes aa₃+b by 2.5 times but did not affect cytochromes c+c₁. By comparison, juvenile hormone injections did not affect the rate of synthesis of either cytochrome fraction. These findings indicate that a neurohormone regulates the rate of synthesis of cytochromes a+b in insect fat body mitochondria.     

Crystal structure at 1.5 Å resolution of the PsbV2 cytochrome from the cyanobacterium Thermosynechococcus elongatus

PsbV2 is a c-type cytochrome present in a very low abundance in the thermophilic cyanobacterium Thermosynechococcus elongatus. We purified this cytochrome and solved its crystal structure at a resolution of 1.5 Å. The protein existed as a dimer in the crystal, and has an overall structure similar to other c-type cytochromes like Cytc₆ and Cytc₅₅₀, for example. However, the 5th and 6th heme iron axial ligands were found to be His51 and Cys101, respectively, in contrast to the more common bis-His or His/Met ligands found in most cytochromes. Although a few other c-type cytochromes were suggested to have this axial coordination, this is the first crystal structure reported for a c-type heme with this unusual His/Cys axial coordination. Previous spectroscopic characterizations of PsbV2 are discussed in relation to its structural properties.     

The oxidation mechanisms of thiosulphate and sulphide in Chlorobium thiosulphatophilum: Roles of cytochrome c-551 and cytochrome c-553

A thiosulphate-cytochrome c reductase was highly purified from Chlorobium thiosulphatophilum and its properties were studied. The enzyme catalyses reduction with Na₂S₂O₃ of c cytochromes, including cytochrome c-551 of the bacterium. Cytochrome c (555, C. thiosulphatophilum) does not react directly with the enzyme at an appreciable rate but stimulates greatly the reduction by the enzyme of cytochrome c-551 with Na₂S₂O₃. The reduction of c cytochromes catalysed by the enzyme is strongly inhibited by cyanide and sulphite.Cytochrome c (553, C. thiosulphatophilum), a c-type cytochrome with covalently bound flavin, was found to catalyse reduction with sulphide of c cytochromes, including cytochrome c-555. The reaction is strongly inhibited by cyanide. Cyanide seems to combine strongly with cytochrome c-553 probably at the flavin moiety. Thus, the absorption spectrum attributable to flavin of the haemoprotein is changed on addition of cyanide, and neither the original spectrum nor the activity reappears even after the cyanide-treated cytochrome has been subjected to gel filtration with a Sephadex G-25 column or to isoelectric focusing.     

The use of photoactivated hetero-bifunctional reagents to cross-link cytochrome c to proteins that bind it by electrostatic interactions.

Lysine residues of horse heart cytochrome c have been modified with N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) and ethyl N-5-azido-2-nitrobenzoylaminoacetimidate (ANB-AI), reagents that attach nitroaryl azides onto the surface of proteins by amide and amidine linkages, respectively. When acting as an electron acceptor for yeast cytochrome b₂, modification of cytochrome c with ANB-NOS increases the K_m for the reaction by 2-fold, while modification with ANB-AI has little effect on the K_m. The V_max for the reduction of cytochrome c by cytochrome b₂ is reduced by the attachment of both compounds to cytochrome c. When the modified cytochromes c were illuminated with phosvitin, cytochrome b₅, and cytochrome c peroxidase, cross-linked species were formed which could be resolved by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. In each case the amidine derivatives of cytochrome c modified with ANB-AI showed more cross-linking than the amide derivatives of cytochrome c modified with ANB-NOS. When the modified cytochromes c were present in a 3-fold excess of phosvitin, cross-linked products containing 1, 2, and 3 molecules of cytochrome c covalently attached to phosvitin were observed. Photolysis of the modified cytochromes c in the presence of cytochrome b₅, resulted in the formation of a cross-linked 1:1 complex between the two cytochromes as well as higher order aggregates containing up to 5 molecules of cytochrome c plus cytochrome b₂. When cytochrome c peroxidase was illuminated with the modified cytochromes c, the predominant cross-linked product was a 1:1 complex between the two heme proteins. However, a cross-linked species was detected in small amounts with the apparent composition of 2 molecules of cytochrome c and 1 of the peroxidase. Also, a procedure is described for the synthesis of ANB-AI with ¹⁴C in the imidocarbon which is ultimately derived from ¹⁴CN.     

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.     

Characterization and expression of the co-transcribed cyc1 and cyc2 genes encoding the cytochrome c4 (c552) and a high-molecular-mass cytochrome c from Thiobacillus ferrooxidans ATCC 33020

The sequence of the cyc1 gene encoding the Thiobacillus ferrooxidans ATCC 33020 c₅₅₂ cytochrome, shows that this cytochrome is a 21-kDa periplasmic c₄-type cytochrome containing two similar monohaem domains. The kinetics of reduction and the fact that cytochromes c₄ are considered to be physiological electron donors of cytochrome oxidases suggest that the last steps of the iron respiratory chain are: rusticyanin→cytochrome c₄→cytochrome oxidase. In Thiobacillus ferrooxidans, cyc1 is co-transcribed with the cyc2 gene, encoding a high-molecular-mass monohaem cytochrome c. This suggests that the cytochromes encoded by these genes belong to the same electron transfer chain.     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

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.     

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.     

Effect of membrane potential on equilibrium poise between cytochromea and cytochromec 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+c ₁) 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+a ₃) but not of cytochromes (c+c ₁) 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 cytochromesc ₁ andc in contact with the outer phase and cytochromesa anda ₃ plugged through, so that the equilibrium distribution of electrons between thec anda cytochromes is influenced by the electric field across the membrane.

     

Cytochromes and prenylquinones in preparations of cytoplasmic and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans

The cytochrome and prenylquinone compositions were compared for cytoplasmic membranes and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans. Reduced-minus-oxidized difference absorption spectra at ?196°C indicated that the thylakoid membranes contained photosynthetic cytochromes such as cytochrome ?, cytochrome b-559 and cytochrome b₆, while cytochromes c-549 and c-552 were detected spectrophotometrically only after their release by sonic oscillation. The cytoplasmic membrane preparation contained one or two low-potential cytochrome(s) with α-band maxima at 553 and 559 nm at ?196°C, which differed from the cytochromes in the thylakoid membranes. A cytochrome specific to the cytoplasmic membranes was also found by heme-staining after lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Both types of membranes contained the three prenylquinones plastoquinone-9, phylloquinone and 5′-monohydroxyphylloquinone, but in different proportions.     

13th International Congress of Biochemistry

Cytochrome caa₃ (cytochrome oxidase) from the thermophilic bacterium PS3 can exhibit full catalytic activity in the presence of ascorbate and TMPD or other electron donors and in the absence of added soluble c-type cytochromes. It appears to possess only a low-affinity and not a high-affinity site for the soluble cytochromes. Proteoliposomal cytochrome caa₃ develops an effective membrane potential in the presence of ascorbate and TMPD or PMS, in the absence of added soluble cytochrome c. Reduction of the a₃ centre is blocked in the presence of cyanide. During reductive titrations of the cyanide-inhibited enzyme, electrons initially equilibrate among three centres, the c haem, the a haem and one of the associated Cu atoms. During steady-state turnover, electrons probably enter the complex via the bound c haem; the a haem and perhaps an associated Cu_A atom are reduced next. It is concluded that, despite its size and hydrophobic association with the aa₃ complex, the haem c-containing subunit can behave in an analogous way to that of mammalian cytochrome c, bound at the high-affinity site of the eucaryotic enzyme.