The respiratory electron transport system of heterotrophically-grown Rhodopseudomonas palustris

The enzymatic activities and the cytochrome components of the respiratory chain were investigated with membrane fractions from chemoheterotrophically grown Rhodopseudomonas palustris. Whereas the level of electron transfer carriers was not distinctly affected by a change of the culture conditions, the potential activities of the enzymes were clearly increased when the cells were grown aerobically. Reduced-minus oxidized difference spectra of the membrane fractions prepared from dark aerobically grown cells revealed the presence of three b-type cytochromes b ₅₆₁, b ₅₆₀ and b ₅₅₈, and at least two c-type cytochromes c ₅₅₆ and c ₂ as electron carriers in the electron transfer chain. Cytochrome of a-type could not be detected in these membranes. Reduced plus CO minus reduced difference spectra of the membrane fractions were indicative of cytochrome o, which may be equivalent to cytochrome b ₅₆₀, appearing in substrate-reduced minus oxidized difference spectra. Cytochrome o was found to be the functional terminal oxidase. CO difference spectra of the high speed supernatant fraction indicated the presence of cytochrome c′. Succinate and NADH reduced the same types of cytochromes. However, a considerable amount of cytochrome b ₅₆₁ with associated β and γ bands at 531 and 429 nm, respectively, was reducible by succinate, but not by NADH. A substantial fraction of the membrane-bound b-type cytochrome was non-substrate reducible and was found in dithionite-reduced minus substrate-reduced spectra. Cytochrome c ₂ may be localized in a branch of the electron transport system, with the branch-point at the level of ubiquinone. The separate pathways rejoined at a common terminal oxidase. Two terminal oxidases with different KCN sensitivity were present in the respiratory chain, one of which was sensitive to low concentrations of KCN and was connected with the cytochrome chain. The other terminal oxidase which was inhibited only by high concentrations of cyanide was located in a branched pathway, through which the electrons could flow from ubiquinone to oxygen bypassing the cytochrome chain.     

During Cytochrome c Maturation CcmI Chaperones the Class I Apocytochromes until the Formation of Their b-Type Cytochrome Intermediates

The c-type cytochromes are electron transfer proteins involved in energy transduction. They have heme-binding (CXXCH) sites that covalently ligate heme b via thioether bonds and are classified into different classes based on their protein folds and the locations and properties of their cofactors. Rhodobacter capsulatus produces various c-type cytochromes using the cytochrome c maturation (Ccm) System I, formed from the CcmABCDEFGHI proteins. CcmI, a component of the heme ligation complex CcmFHI, interacts with the heme-handling protein CcmE and chaperones apocytochrome c₂ by binding its C-terminal helix. Whether CcmI also chaperones other c-type apocytochromes, and the effects of heme on these interactions were unknown previously. Here, we purified different classes of soluble and membrane-bound c-type apocytochromes (class I, c₂ and c₁, and class II c′) and investigated their interactions with CcmI and apoCcmE. We report that, in the absence of heme, CcmI and apoCcmE recognized different classes of c-type apocytochromes with different affinities (nm to μm K_D values). When present, heme induced conformational changes in class I apocytochromes (e.g. c₂) and decreased significantly their high affinity for CcmI. Knowing that CcmI does not interact with mature cytochrome c₂ and that heme converts apocytochrome c₂ into its b-type derivative, these findings indicate that CcmI holds the class I apocytochromes (e.g. c₂) tightly until their noncovalent heme-containing b-type cytochrome-like intermediates are formed. We propose that these intermediates are subsequently converted into mature cytochromes following the covalent ligation of heme via the remaining components of the Ccm complex.     

Characteristics of b-type cytochromes in brain microsomes: Comparison with liver microsomes

Biochemical aspects of b-type cytochromes in swine cerebral microsomes were different from those of cytochrome b₅ in liver microsomes, as well as the difference in absorption spectra. First, the kinetic constants, K_m and V_max, in rotenone-insensitive NADH-cytochrome c reductase activity were different from those of liver microsomes, and the activity of cerebral microsomes was higher than that of liver microsomes. Second, midpoint potentials (E_m) of b-type cytochromes in cerebral microsomes were measured and compared with liver microsomal cytochrome b₅. In cerebral microsomes two components of b-type cytochromes were resolved, and showed E_m's of ?30 and +50 mV, respectively, in the presence of 2 mm KCN. On the other hand, the E_m of liver microsomal cytochrome b₅ was ?6 mV. The high-potential component of cerebral microsomal b-type cytochromes was identified as brain-b′₅ [S. Yoshida, T. Yubisui, and M. Takeshita (1983)Biochem. Int. 7, 291–298] and the low-potential component as brain-b₅. The significance of the difference between cerebral and liver microsomal b-type cytochromes was discussed.     

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.     

Electron-transport chains of phototrophically and chemotrophically grown Chloroflexus aurantiacus

The membrane-bound electron-transfer chain components of both phototrophically and chemotrophically grown Chloroflexus aurantiacus have been characterized. Membranes isolated from chemotrophically grown Chloroflexus have been shown to contain at least three c-type cytochromes and at least three b-type cytochromes. In addition, these cells appear to lack a photochemical reaction center and the high potential (E_m = +260 mV) cytochrome c-554 that serves as the immediate donor to the reaction center in phototrophically grown Chloroflexus. Phototrophically grown cells contain a CO-binding c-type cytochrome, apparently absent in the chemotrophically grown cells. However, a different CO-binding component, which may function as the terminal oxidase, is present in chemotrophically grown cells.     

The electron transport system of the anaerobic Propionibacterium shermanii

1. Electron transport particles obtained from cellfree extracts of Propionibacterium shermanii by centrifugation at 105000xg for 3 hrs oxidized NADH, d,l-lactate, l-glycerol-3-phosphate and succinate with oxygen and, except for succinate, with fumarate, too.
2. Spectral investigation of the electron transport particles revealed the presence of cytochromes b, d and o, and traces of cytochrome a ₁ and a c-type cytochrome. Cytochrome b was reduced by succinate to about 50%, and by NADH, lactate or glycerol-3-phosphate to 80–90.
3. The inhibitory effects of amytal and rotenone on NADH oxidation, but not on the oxidation of the other substrates, indicated the presence of the NADH dehydrogenase complex, or “site I region”, in the electron transport system of P. shermanii.
4. NQNO inhibited substrate oxidations by oxygen and fumarate, as well as equilibration of the flavoproteins of the substrate dehydrogenases by way of menaquinone. The inhibition occurred at low concentrations of the inhibitor, and reached 80–100%, depending on the substrate tested. The site of inhibition of the respiratory activity was located between menaquinone and cytochrome b. In addition, inhibition of flavoprotein equilibration suggested that NQNO acted upon the electron transfer directed from menaquinol towards the acceptor to be reduced, either cytochrome b or the flavoproteins, which would include fumarate reductase.
5. In NQNO-inhibited particles, cytochrome b was not oxidized by oxygen-free fumarate, but readily oxidized by oxygen. It was concluded from this and the above evidence that the branching-point of the electron transport chain towards fumarate reductase was located at the menaquinone in P. shermanii. It was further concluded that all cytochromes were situated in the oxygen-linked branch of the chain, which formed a dead end of the system under anaerobic conditions.
6. Antimycin A inhibited only oxygen-linked reactions of the particles to about 50% at high concentrations of the inhibitor. Inhibitors of terminal oxidases were inactive, except for carbon monoxide.

     

Periplasmic c Cytochromes and Chlorate Reduction in Ideonella dechloratans

The aim of this study was to clarify the pathway of electron transfer between the inner membrane components and the periplasmic chlorate reductase. Several soluble c-type cytochromes were found in the periplasm. The optical difference spectrum of dithionite-reduced periplasmic extract shows that at least one of these components is capable of acting as an electron donor to the enzyme chlorate reductase. The cytochromes were partially separated, and the fractions were analyzed by UV/visible spectroscopy to determine the ability of donating electrons to chlorate reductase. Our results show that one of the c cytochromes (6 kDa) is able to donate electrons, both to chlorate reductase and to the membrane-bound cytochrome c oxidase, whereas the roles of the remaining c cytochromes still remain to be elucidated. Peptide extracts of the c cytochromes were obtained by tryptic in-gel digestion for matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Peptide sequences obtained indicate that the 6-kDa cytochrome c protein is similar to c cytochromes from the chlorate-reducing bacterium Dechloromonas aromatica.Oxyanions of chlorine (ClO₃⁻ and ClO₄⁻) occur in the environment mainly as by-products from human activities (6, 7). The decomposition of chlorate by microbial respiration is important in the treatment of industrial effluents and has been known since the beginning of the 20th century (2). One of the chlorate-respiring bacteria, the gram-negative Ideonella dechloratans, was isolated by Malmqvist and coworkers (8).Chlorate metabolism takes place in the periplasmic space between the inner and outer membranes and involves the soluble enzymes chlorate reductase and chlorite dismutase. The reaction takes place in two steps. First, chlorate is reduced to chlorite by chlorate reductase in a two-electron transfer reaction. The second step is the decomposition of chlorite into chloride ions and molecular oxygen, which is catalyzed by chlorite dismutase. Both enzymes have been isolated and characterized, and their genes have been sequenced (4, 5, 15). Chlorate reduction is coupled to cell growth, suggesting that chlorate reductase is part of a respiratory chain that generates an electrochemical gradient, which can serve as the driving force for ATP synthesis. The aim of this study was to investigate the pathways of electron transfer, in particular the route between membrane-bound components of the respiratory chain and the soluble periplasmic enzymes, in I. dechloratans. One interesting aspect is the finding that a gene encoding a soluble c-type cytochrome is located downstream of the gene for chlorate reductase (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU768872","term_id":"190607446","term_text":"EU768872"}}EU768872) (J. Bohlin, A. Smedja Bäcklund, N. Gustavsson, S. Wahlberg, and J. Nilsson, unpublished data).Although the electron transport pathways in bacteria differ, two major strategies for the transfer of electrons to soluble enzymes seem to occur. One strategy is the oxidation of quinol by cytochrome bc₁ complex, followed by electron transfer to a soluble c-type cytochrome. In the other strategy, where the bc₁ complex is absent or not involved, electron transfer is mediated by a membrane-anchored periplasmic c-type cytochrome belonging to the NapC/NirT family (13).The chlorate reductase in I. dechloratans shows similarity to molybdopterin-containing members of the type II subgroup of the dimethyl sulfoxide reductase family (10). One member of the family, dimethyl sulfoxide dehydrogenase (Ddh) from the phototrophic Rhodovulum sulfidophilum, utilizes a soluble cytochrome c for transfer of electrons, but in the reverse direction. The β subunit in Ddh donates electrons to the membrane-bound photochemical center, mediated by the soluble cytochrome c₂ (9). Another member of the dimethyl sulfoxide reductase family, the closest known relative to chlorate reductase in I. dechloratans, is selenate reductase from Thauera selenatis (14). The quaternary structure of this enzyme is very similar to that of Ddh in R. sulfidophilum, and it has been suggested that the enzyme may interact with a periplasmic c cytochrome that receives electrons from the bc₁ complex (10). Several other (per)chlorate-reducing bacteria, such as Dechloromonas agitata (1), Dechloromonas aromatica strain RCB (3), and strain GR-1 (12), have been isolated. In D. aromatica, several genes encoding NapC/NirT-like cytochromes have been found, but the physiological roles of the corresponding proteins are not known (3). The electron transfer pathways in D. agitata and strain GR-1 are unknown.The present study aims at investigating the role of soluble c-type cytochromes as electron mediators between the bc₁ complex in the inner membrane and the periplasmic chlorate reductase in I. dechloratans. We have found that at least one of the periplasmic c-type cytochromes is capable to act as a electron donor to the enzyme chlorate reductase.     

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.

     

Quinol-cytochrome c Oxidoreductase and Cytochrome c4 Mediate Electron Transfer during Selenate Respiration in Thauera selenatis

Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (E_m + 234 ± 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified (∼24 and ∼6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatography-tandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c₄ family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of +282 ± 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytc-Ts4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c₄ is a novel route for a member of the DMSO reductase family of molybdoenzymes.     

Purification and characterization of low potential c cytochromes from Desulfovibrio desulfuricans membranes

Desulfovibrio desulfuricans grown in a lactate-sulfate medium produces, in addition to soluble cytochromes, c-type cytochromes which appear to be integral membrane proteins. Two cytochromes can be separated, an abundant 15 kDa cytochrome and a 22 kDa cytochrome. Both have optical spectra characteristics of c-type cytochromes. The 15 kDa cytochrome shows two n = 1 components in potentiometric redox titrations with midpoint potentials at −130 and −270 mV in the membrane; both were slightly lower in detergent-solubilized preparations. We suggest a designation of cytochrome cc_m for this species. Its properties suggest a function as a transmembrane electron carrier between hydrogen and sulfate.     

A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in Complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with α-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, ?75 and 187 mV, respectively. In addition, two very small contributions to the α-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c₁ (Λ_m(25°C) = 553.5 nm; E′₀ = 238 mV) and four cytochromes bΛ_m(25°C) = 558.6, 561.2, 562.1, 566.1 nm and E′₀ = ?83, 26, 85, ?60 mV).     

Active Site of Cytochrome cbb3

Cytochrome cbb₃ is the most distant member of the heme-copper oxidase family still retaining the following major feature typical of these enzymes: reduction of molecular oxygen to water coupled to proton translocation across the membrane. The thermodynamic properties of the six redox centers, five hemes and a copper ion, in cytochrome cbb₃ from Rhodobacter sphaeroides were studied using optical and EPR spectroscopy. The low spin heme b in the catalytic subunit was shown to have the highest midpoint redox potential (E_m,7 +418 mV), whereas the three hemes c in the two other subunits titrated with apparent midpoint redox potentials of +351, +320, and +234 mV. The active site high spin heme b₃ has a very low potential (E_m,7 -59 mV) as opposed to the copper center (Cu_B), which has a high potential (E_m,7 +330 mV). The EPR spectrum of the ferric heme b₃ has rhombic symmetry. To explain the origins of the rhombicity, the Glu-383 residue located on the proximal side of heme b₃ was mutated to aspartate and to glutamine. The latter mutation caused a 10 nm blue shift in the optical reduced minus oxidized heme b₃ spectrum, and a dramatic change of the EPR signal toward more axial symmetry, whereas mutation to aspartate had far less severe consequences. These results strongly suggest that Glu-383 is involved in hydrogen bonding to the proximal His-405 ligand of heme b₃, a unique interaction among heme-copper oxidases.The heme-copper oxidases form a family of enzymes that have structural homology of the catalytic subunit in common (1). This family of proteins, characterized by six conserved histidine ligands of the redox cofactors, ranges from classical, mitochondrial terminal oxidases to nitric-oxide reductases, and the members have been classified according to evolutionary relationships of their sequences (2–4). The bacterial cbb₃-type cytochrome c oxidases form a distinct, divergent subfamily within the heme-copper oxidases (5). Terminal oxidases share the catalytic activity of four-electron reduction of molecular oxygen to water coupled to translocation of protons across the membrane (6, 7). Cytochrome cbb₃, expressed in some bacteria as a sole terminal oxidase, is characterized by its ability to maintain catalytic activity under low oxygen tension (8), and it has also been shown to have the capacity to translocate protons (9).The Rhodobacter sphaeroides cytochrome cbb₃ is encoded by the ccoNOQP operon composed of four genes (10). The catalytic subunit CcoN homes a binuclear active site composed of a high spin heme b₃ and a nearby copper ion (Cu_B). There are altogether four low spin hemes in the enzyme. In addition to a protoheme (heme b) residing in the vicinity of the active site in subunit CcoN, there are three hemes c present in the soluble domains of the two other transmembrane subunits, a monoheme subunit CcoO and a diheme subunit CcoP (11). There is yet one more membrane-spanning subunit, CcoQ, without bound cofactors (12). Although the catalytic subunit shows homology to the other heme-copper oxidases (13), the other three subunits bear no resemblance to subunits of other types of terminal oxidases. However, subunit CcoO has been shown to have sequence homology with the nitric-oxide reductase subunit NorC (14).The crystal structures of a few heme-copper oxidases have been resolved (15–19), but only structural homology models are currently available for cytochromes cbb₃ (20–23). Apart from the signatures common to all heme-copper oxidases, the sequence alignments have revealed only very few other conserved residues when terminal oxidases are compared. Even though some amino acids, absent from cytochrome cbb₃, have been shown to be of critical importance to the function of the classical heme-copper oxidases, the major functions still remain the same in all of these enzymes.The thermodynamic properties of the cbb₃-type oxidases have been investigated sparsely. Apart from work yielding partial information about the properties of the hemes (11, 24, 25), two more complete studies have been carried out (5, 26). All the hemes in cytochrome cbb₃ were proposed to have high redox potentials, both in the Pseudomonas stutzeri and Bradyrhizobium japonicum enzymes (5, 26). This is also the case in all other studies, except for the enzyme from Rhodothermus marinus, where two low potential redox centers were reported (25). However, little is known about the copper center in the active site (Cu_B). Early Fourier transform infrared (FTIR)² spectroscopic measurements identified the presence of a heme/copper binuclear center in R. sphaeroides cytochrome cbb₃ (11), and more recent resonance Raman and FTIR studies have given additional information about the structure of the active site (27–29).In the absence of deconvoluted spectral components and thereby clear assignments of the redox centers in the cbb₃-type oxidases, and the lack of consensus about their thermodynamic properties, a complete study was required. In this work we have set out to investigate the thermodynamic properties of all the redox centers in cytochrome cbb₃ from R. sphaeroides using a combination of optical and EPR redox titrations with the main focus on the details of the catalytic site. This effort will form a basis for further mechanistic studies.     

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.     

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).     

Partial purification and characterization of two soluble c-type cytochromes from Chromatium vinosum

Two c-type cytochromes from Chromatium vinosum have been partially purified and characterized. Cytochrome c₅₅₀, which appears to function as an electron carrier in the cyclic electron transport chain of this photosynthetic purple sulfur bacterium, has a molecular weight of approximately 15,000 and an oxidation-reduction midpoint potential (E_m) of + 240 mV at pH 7.4. It has (in the reduced form) an α band at 550 nm and a β band at 520 nm. Cytochrome c₅₅₁ is characterized by absorbance maxima at 354 and 409 nm in the oxidized form and 418, 523, and 551 nm in the reduced form. The reduced cytochrome reacts with CO. Cytochrome c₅₅₁ is a monomeric protein with a molecular weight of 18,800 ± 700 and E_m = ?299 ± 5 mV (pH independent between pH 6.3 and 8.0). It appears to lack a methionine axial ligand as indicated by the absence of an absorbance band at 695 nm in the oxidized form.