The role of the quinone pool in the cyclic electron-transfer chain of Rhodopseudomonas sphaeroides A modified Q-cycle mechanism

(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c₂ oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c₁ and c₂, the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a low-potential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0–3 mol ubiquinol/mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c₁ and c₂, and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycin-sensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 ix oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 became completely reduced following one flash in the presence of antimycin. The results are interpreted as showing that at potentials higher than 180 mV, ubiquinol stoichiometric with cytochrome b-561 reaches the complex from the reaction center. The increased extent of the carotenoid change, when one extra ubiquinol is available in the pool, is interpreted as showing that the ubiquinol oxidase site turns over twice, and the ubiquinone reductase sites turns over once, for a complete turnover of the ubiquinol:cytochrome c₂ oxidoreductase complex, and the net oxidation of one ubiquinol/complex. (7) The antimycin-sensitive reduction of cytochrome c₁ and c₂ is shown to reflect the second turnover of the ubiquinol oxidase site. (8) We suggest that, in the presence of antimycin, the ubiquinol oxidase site reaches a quasi equilibrium with ubiquinol from the pool and the high- and low-potential chains, and that the equilibrium constant of the reaction catalysed constrains the site to the single turnover under most conditions. (9) The results are discussed in the context of a detailed mechanism. The modified Q-cycle proposed is described by physicochemical parameters which account well for the results reported.     

Mechanism of electron transport during thiosulfate oxidation in an obligately mixotrophic bacterium Thiomonas bhubaneswarensis strain S10 (DSM 18181T)

This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181^T). Whole-genome sequence analysis revealed the presence of complete sox (sulfur oxidation) gene cluster (soxCDYZAXB) including the sulfur oxygenase reductase (SOR), sulfide quinone reductase (SQR), sulfide dehydrogenase (flavocytochrome c (fcc)), thiosulfate dehydrogenase (Tsd), sulfite dehydrogenase (SorAB), and intracellular sulfur oxidation protein (DsrE/DsrF). In addition, genes encoding respiratory electron transport chain components viz. complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (ubiquinone-cytochrome c reductase), and various types of terminal oxidases (cytochrome c and quinol oxidase) were identified in the genome. Using site-specific electron donors and inhibitors and by analyzing the cytochrome spectra, we identified the shortest thiosulfate-dependent electron transport chain in T. bhubaneswarensis DSM 18181^T. Our results showed that thiosulfate supports the electron transport activity in a bifurcated manner, donating electrons to quinol (bd) and cytochrome c (Caa ₃ ) oxidase; these two sites (quinol oxidase and cytochrome c oxidase) also showed differences in their phosphate esterification potential (oxidative phosphorylation efficiency (P/O)). Further, it was evidenced that the substrate-level phosphorylation is the major contributor to the total energy budget in this bacterium.

     

Structure/function relationships in mitochondrial cytochrome b revealed by the kinetic and circular dichroic properties of two yeast inhibitor-resistant mutants.

The kinetic and circular dichroic properties of two yeast mutants that are resistant towards specific inhibitors of the mitochondrial cytochrome bc1 complex have been characterized. Both of these mutants have an altered cytochrome b gene in which aromatic residues are exchanged with non-polar residues in a highly conserved region of the protein. The mutant resistant to myxothiazol and mucidin that contains the substitution Phe129----Leu is not greatly affected either in its ubiquinol:cytochrome c reductase or in the spectral properties of cytochrome b. On the other hand, the mutant resistant to stigmatellin that contains the substitution Ile147----Phe shows a large decrease of the catalytic efficiency for ubiquinol and of the maximal turnover of its reductase activity. This stigmatellin mutant also shows an altered circular-dichroic spectrum of the low-potential haem of cytochrome b. This study provides biochemical and biophysical information for identifying a region in mitochondrial cytochrome b that may fulfill a crucial role in the binding of ubiquinol to the bc1 complex. The results are discussed also in terms of the structural model of cytochrome b having a core of four transmembrane helices.     

On the impermeability of the outer mitochondrial membrane to cytochromec

Summary Outer mitochondrial membranes isolated by the swelling-shrinkage sonication procedure of Sottocasaet al. [19, 20] forms small sealed vesicles. If cytochromec is present during the procedure it is trapped inside these vesicles and can not be washed out nor is accessible to external enzymes, e.g., cytochrome oxidase (EC 1.9.3.1) or succinate-cytochromec reductase present as contamination by the inner membrane, but is fully accessible to rotenone-insensitive NADH-cytochromec reductase of the outer membrane. This indicates the impermeability of the outer mitochondrial membrane to cytochromec.A modification of the original procedure for the separation of the outer mitochondrial membrane is described.     

Cytochrome c biogenesis in Campylobacter jejuni requires cytochrome c6 (CccA; Cj1153) to maintain apocytochrome cysteine thiols in a reduced state for haem attachment

The microaerophilic food‐borne pathogen Campylobacter jejuni uses complex cytochrome‐rich respiratory chains for growth and host colonisation. Cytochrome c biogenesis requires haem ligation to reduced apocytochrome cysteines, catalysed by the cytochrome c synthase, CcsBA. While ccsBA could not be deleted, we showed that the thiol reductase DsbD and the CcsX homologue Cj1207 are involved in, but not essential for, cytochromes c biogenesis. Mutant phenotypic analyses and biochemical studies with purified proteins revealed that the mono‐haem c‐type cytochromes Cj1153 (CccA) and Cj1020 (CccB) and the di‐haem Cj0037 (CccC) are electron donors to the cb‐oxidase (CcoNOQP), with CccC being more efficient than CccA. Remarkably, cccA deletion or site‐directed mutagenesis resulted in an almost complete loss of all other c‐type cytochromes. Cytochrome c structural and biogenesis genes were still transcribed in the cccA deletion mutant and the quinol oxidase genes (cioAB) were up‐regulated. Cytochrome c production could be rescued in this mutant by growth with exogenous dithiothreitol or L‐cysteine, suggesting that in the absence of CccA, apocytochrome c haem binding motifs become oxidised, preventing haem attachment. Our results identify CccA, the most abundant periplasmic c‐type cytochrome in C. jejuni, as a novel and unexpected protein required for cytochrome c biogenesis in this pathogen.     

Comparison of the binding sites on cytochrome c for cytochrome c oxidase, cytochrome bc1, and cytochrome c1. Differential acetylation of lysyl residues in free and complexed cytochrome c

The isolated complexes of ferricytochrome c with cytochrome c oxidase, cytochrome c reductase (cytochrome bc1 or complex III), and cytochrome c1 (a subunit of cytochrome c reductase) were investigated by the method of differential chemical modification (Bosshard, H.R. (1979) Methods Biochem. Anal. 25, 273-301). By this method the chemical reactivity of each of the 19 lysyl side chains of horse cytochrome c was compared in free and in complexed cytochrome c and binding sites were deduced from altered chemical reactivities of particular lysyl side chains in complexed cytochrome c. The most important findings follow. 1. The binding sites on cytochrome c for cytochrome c oxidase and cytochrome c reductase, defined in terms of the involvement of particular lysyl residues, are indistinguishable. The two oxidation-reduction partners of cytochrome c interact at the front (exposed heme edge) and top left part of the molecule, shielding mainly lysyl residues 8, 13, 72 + 73, 86, and 87. The chemical reactivity of lysyl residues 22, 39, 53, 55, 60, 99, and 100 is unaffected by complex formation while the remaining lysyl residues in positions 5, 7, 25, 27, 79, and 88 are somewhat less reactive in the complexed molecule. 2. When bound to cytochrome c reductase or to the isolated cytochrome c1 subunit of the reductase the same lysyl side chains of cytochrome c are shielded. This indicates that cytochrome c binds to the c1 subunit of the reductase during the electron transfer process.     

Purification of Sulphite-Cytochrome c Reductase of Thiobacillus novellus and Reconstitution of Its Sulphite Oxidase System with the Purified Constituents

Sulphite-cytochrome c reductase (sulphite: ferricytochrome coxidoreductase, EC 1.8.2.1 [EC] ) derived from Thiobacillus novelluswas purified by chromatography on a DEAE-cellulose column andby gel filtration with a Sephadex G-100 column. Although thereductase thus purified moved as a single band both in gel filtrationand in isoelectric focusing it was always split into two bandsby polyacrylamide gel electrophoresis; the one had the enzymaticactivity and showed absorption spectrum of cytochrome, whilethe other had no activity and was colourless, in contrast withthe results reported by Charles and Suzuki [(1966) Biochim.Biophys. Acta 128: 522]. The enzymatic properties of the purifiedreductase were almost the same as those of the enzyme obtainedby Charles and Suzuki. Cytochrome c-551 free of the reductase activity was obtained.Its molecular weight was determined to be 23,000 by polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate.The cytochrome seemed to exist in the organism as a complexwith the reductase or a subunit of the enzyme. In the stateof the complex with the enzyme, the cytochrome was reduced veryquickly on addition of sulphite, while the cytochrome free ofthe reductase activity was hardly reduced by the enzyme withsulphite. A sulphite oxidase system was reconstituted with the reductase,cytochrome c-550 and cytochrome oxidase highly purified fromthe bacterium. ¹ Present address: Water Research Institute, Nagoya University,Nagoya 464, Japan ² Present address: Institute for Biological Science, SumitomoChemical Co., Ltd., Takarazuka, Hyogo 665, Japan (Received January 23, 1981; Accepted March 9, 1981)     

Use of specific trifluoroacetylation of lysine residues in cytochrome c to study the reaction with cytochrome b5, cytochrome c1, and cytochrome oxidase

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b₅, cytochrome c₁ and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, and 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c₁ and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.     

Phyllospadine,a New Flavonoidal Alkaloid from the Sea-Grass Phyllosphadix iwatensis

Attempts to solubilize active ubiquinol: cytochrome c reductase, cytochrome b-c₁ complex, from the submitochondrial particles from sweet potato root tissue ended in failure because all detergents tested caused inactivation of this enzyme complex. Consequently, the complex was isolated with the content of cytochrome b as the marker for purification after solubilization with deoxycholate though it was inactive. Deoxycholate had no effect on two ±-bands at 555 and 558 nm but caused a blue shift of an ±-band at 563 nm in the reduced-minus-oxidized difference spectrum of the submitochondrial particles at low temperature. The purified complex exhibited the same difference spectra at low and room temperatures as the submitochondrial particles in the presence of deoxycholate, which suggests that the complex has three (at least two) cytochrome b components with different spectroscopic properties and that the apparent molar ratio of cytochrome b to c₁ is 1.5. The purified complex consisted of eight subunits: I, 51 kDa; II, 49kDa; III, 33kDa; IV, 32 kDa; V, 27 kDa; VI, 17 kDa; and VII and VIII, 10 kDa. Subunits III and IV were cytochrome c₁ and b, respectively.     

A new photoaffinity-labeled derivative of mitochondrial cytochrome c

Three lysine residues of horse heart cytochrome $\text{c}$ were modified by reaction with methyl-4-mercaptobutyrimidate hydrochloride and the free SH group of the latter was covalently linked to p-azidophenacyl bromide yielding a photoaffinity-labeled cytochrome $\text{c}$. The photoaffinity-labeled cytochrome $\text{c}$ was bound by irradiation into a covalent complex with cytochrome $\text{c}$ oxidase.     

Electron transfer in <Emphasis Type="Italic">Paracoccus denitrificans</Emphasis> with the modified <Emphasis Type="Italic">fbc</Emphasis> operon

Membrane fragments of two mutant strains of Paracoccus denitrificans genetically modified in the bc ₁ complex have been studied for comparison of enzymic activities of succinate-cytochrome-c reductase and its components, viz. succinate dehydrogenase (Complex II) and ubiquinol-cytochrome-c reductase (Complex III) and their response to changes in concentration of succinate, cytochrome c, ionic strength, pH, temperature and sensitivity to antimycin A. The mutants synthesized and assembled the b and c hemes in the ratio characteristic for the wild type strain. The mutant strain M 71 expressing the truncated copy of cytochrome c ₁ (devoid of a stretch of 150 mainly acidic amino acids) was less sensitive to increasing concentration of cytochrome c and changes in ionic strength of the medium, but maintained the original affinity to succinate and sensitivity to antimycin A. The mutant strain M 36 with an overexpressed bc ₁ content showed the highest response to changes in ionic strength and physical parameters, exhibited the lowest turnover number values with succinate-cytochrome-c reductase, but positively affected the succinate dehydrogenase. In view of the interaction of the redox components in native membranes the functional analyses of separated Complexes II and III should be regarded with caution.     

Reduced Pyridine Nucleotide Oxidases of Euglena gracilis var. bacillaris*

SYNOPSIS. Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (K_m) determined were as follows: NADH diaphorase, 350 μM; NADPH diaphorase, 200 μM; NADH cytochrome c reductase, 13 μM; NADPH cytochrome c reductase, 9 μM; NADH oxidase, 100 μM; NADPH oxidase 150 μM; NADH lipoyl dehydrogenase, 0.35 μM. Enzyme activities after storage at –5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles.     

Oxidative enzyme activities in rat brain subcellular fractions. The effect of deoxycholate, freeze-thaw, and water

(1) The distributions of four oxidative enzymes were studied in crude brain fractions. (2) Freeze-thaw cycle treatment and frozen storage of homogenate fractions gave apparent enhancement of cytochrome oxidase and NADH cytochrome c reductase activities. (3) Deoxycholate released cytochrome oxidase and NADH cytochrome c reductase activities from low-speed precipitates. The NADH diaphorase was enhanced to a small degree while NADPH cytochrome c reductase was not affected by deoxycholate. (4) Distilled water coupled with a single homogenization released trapped soluble enzymes and mitochondria and gave nearly maximal cytochrome oxidase activity as judged by deoxycholate treatment. The total distilled water activity of NADH cytochrome c reductase was much less than that of deoxycholate-stimulated fractions. The activities of other enzymes were not markedly affected by distilled water although their distribution was changed.     

Analysis of cytoplasmic membrane from wild type and mutant Paracoccus denitrificans containing excess nitrate reductase protein and cytochrome b

Cytoplasmic membranes were isolated from wild type and mutants strain M-1 of Paracoccus denitrificans grown with low aeration to promote synthesis of nitrate reductase protein and cytochrome b. The presence of 10-100-fold excess of nitrate reductase in the wild type or the corresponding enzymically inactive protein in the mutant did not significantly affect respiratory oxidase activities with NADH, succinate or TMPD-ascorbate as electron donor. A cytochrome b-nitrate reductase complex was resolved by isoelectric focussing of Triton X-100 solubilized membranes from the wild type grown with azide and from the mutant, whereas the enzyme complex from nitrate-grown wild type was not resolved from cytochrome c. Preparations from azideinduced wild type or from the mutant could be a suitable source of the cytochrome b associated with nitrate reductase for more detailed studies.Non standard abbreviations IEF isoelectric focussing - TMPD N, N, N, N-tetramethylphenylenediamine - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

The cytochrome c binding site on cytochrome c oxidase.

Cytochrome $\text{c}$ was chemically coupled to cytochrome $\text{c}$ oxidase using the reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) which couples amine groups to carboxyl residues. The products of this reaction were analyzed on 2.5–27% polyacrylamide gradient gels electrophoretically. Since cytochrome $\text{c}$ binds to cytochrome oxidase electrostatically in an attraction between certain of its lysine residues and carboxyl residues on the oxidase surface, EDC is an especially appropriate reagent probe for binding-subunit studies. Coupling of polylysine to cytochrome oxidase using EDC was also performed, and the products of this reaction indicate that polylysine, an inhibitor of the cytochrome $\text{c}$ reaction with oxidase, binds to the same oxidase subunit as does cytochrome $\text{c}$, subunit IV in the gel system used.     

Effect of pyridoxal phosphate on the formation of cytochrome c1-c and cytochrome c-cytochrome oxidase complexes.

The cytochrome c-cytochrome oxidase complex is formed when c reacts with cytochrome oxidase (Kuboyama et al. (1962) Biochem. Biophys. Res. Commun. 9, 534) and the cytochrome c1-cytochrome c complex is formed when c reacts with cytochrome c1 in the presence of the hinge protein (Kim, C.H. and King, T.E. (1981) Biochem. Biophys. Res. Commun. 101, 607). Both complexes are considered to be possible intermediates in electron transfer reaction between these cytochromes. Triply substituted modified cytochrome c by pyridoxal phosphate at lysine residues (Lys-79, 86 and one to be identified) abolishes both complex formations and electron transfer activity with succinate cytochrome c reductase or cytochrome oxidase.     

The use of a water-soluble carbodiimide to cross-link cytochrome c to plastocyanin

A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, has been used to cross-link horse heart cytochrome c to spinach chloroplast plastocyanin. The complex was formed in yields up to 90%, and was found to have a stoichiometry of 1 mol plastocyanin per mol cytochrome c. The cytochrome c in the complex was fully reducible by ascorbate and potassium ferrocyanide, and had a redox potential only 25 mV less than that of native cytochrome c. The complex was nearly completely inactive towards succinate-cytochrome c reductase and cytochrome c oxidase, suggesting that the heme crevice region of cytochrome c was blocked. We propose that the carbodiimide promoted the formation of amide cross-links between lysine amino groups surrounding the heme crevice of cytochrome c and complementary carboxyl groups on plastocyanin. It is of interest that the high-affinity site for cytochrome c binding on bovine heart cytochrome c oxidase has recently been found to involve a sequence of subunit II with some homology to the copper-binding sequence of plastocyanin.     

Nitrobacter agilis Cytochrome c-550: Isolation, Physicochemical and Enzymatic Properties, and Primary Structure

Nitrobacter agilis cytochrome c-550 was purified to an electrophoreticallyhomogeneous state, and some of its properties were determined.The cytochrome showed an absorption peak at 410 nm in the oxidizedform, and peaks at 416, 521 and 550 nm in the reduced form.Its isoelectric point was 8.1 at 5?C. Analysis of the aminoacid composition showed that the cytochrome molecule was composedof 108 amino acid residues, 16 of which were lysine residues. The cytochrome reacted rapidly with N. agilis cytochrome c oxidaseand yeast cytochrome c peroxidase and more slowly with Pseudomonasaeruginosa nitrite reductase and bovine cytochrome c oxidase.The reactivities with these redox enzymes suggested that thecytochrome might be an evolutionary stage between bacterialand eukaryotic cytochromes c. The primary structure of the cytochrome from the N-terminusto the 85th residue was determined. The N-terminal sequencewas homologous to the corresponding portion of the primary structureof horse cytochrome c. ¹ Present adress: Department of Chemistry, Faculty of Science,Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo,152, Japan. (Received December 3, 1981; Accepted January 28, 1982)     

The terminal oxidases of Paracoccus denitrificans

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding sub unit I of the aa₃-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa₃-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem-containing oxidase, called cytochrome bb₃, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::Km^R) an alternative cyto-chrome c oxidase has been characterized; this cbb₃-type oxidase has been partially purified. Both cytochrome aa₃ and cytochrome bb₃ are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb₃ has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.