A potentiometric and kinetic study on the respiratory chain of ferrous-iron-grown Thiobacillus ferrooxidans

The type and number of respiratory chain components present in membranes of Thiobacillus ferrooxidans have been investigated. These redox components were resolved potentiometrically and kinetically. Using optical techniques two cytochromes a₁, multiple cytochromes c and two cytochromes b were detected. By using electron paramagnetic resonance, two copper-containing centres, high and low spin ferric haems and a ferredoxin centre were detected. Based on the combination of a potentiometric resolution and a kinetic study a model for the sequence of the respiratory chain components in the Fe²⁺ to O₂ branch of the T. ferrooxidans respiratory chain is proposed.     

Midpoint potentials of the mitochondrial cytochromes of Crithidia fasciculata.

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

Role of quinones in the branch of the Escherichia coli respiratory chain that terminates in cytochrome o.

The role of quinones in the cytochrome o branch of the Escherichia coli respiratory chain was investigated by using mutant strains lacking the cytochrome d terminal oxidase complex. The only cytochromes present were cytochrome b556 and the cytochrome o complex, consisting of cytochrome b555-b562. Mutant strains missing ubiquinone, menaquinone, or both were constructed in the cytochrome d-minus (cyd) background. The steady-state levels of cytochrome b reduction were examined and compared in these strains to assess the effects of the quinone deficiencies. The data clearly show that a ubiquinone deficiency results in a lower level of cytochrome b reduction in the steady state. The data are consistent with a simple model in which ubiquinone is placed on the dehydrogenase side of all the cytochromes in this branch of the respiratory chain. There is no evidence from these experiments for a role of quinones in the respiratory chain at any site besides this one.     

Investigation of Endomyces magnusii respiratory system. Characteristics of mitochondria from cells grown in the presence of antimycin A]

Mitochondria from AA-cells are a unique model with separately finctioning 1st and 3rd points of energy coupling and with completely blocked 2nd coupling point. In vivo the terminal segment of the respiratory chain does not operate, cytochromes c and a+a3 remain oxidized, and dominating terminal oxidase appears to be a component inhibiting by salicylhydroxamate and bound with the respiratory chain via cytochromes b or ubiquinone pool. The blocking of the 2nd coupling point is due to firm and, probably, quantitative binding of AA with cytochromes b, especially with b559, and also to a partial denaturation of the complex III.     

Redox state of respiratory chain enzymes and potassium transport in silkworm mid-gut.

The midgut of Hyalophora cecropia actively transports potassium from hemolymph to lumen and the energy for this process appears to be intimately linked to oxidative metabolism. In the present investigation, we monitored concurrently the rate of active transport and the redox levels of the components of the respiratory chain in the intact tissue under a variety of experimental conditions. Approximately equal concentrations of cytochromes a3, a, c and b-557 were found. Other investigators (Pappenheimer, Jr, A.M. and Williams, C.M. (1954) J. Biol. Chem. 209, 915, Shappirio, D.G. and Williams, C.M. (1957) Proc. R. Soc. Lond. Ser. B 147, 233 and Chance, B. and Pappenheimer, Jr, A.M. (1957) J. Biol, Chem, 209, 931) have indentified cytochrome b-557 with b5 and found that it exists primarily in an extramitochondrial location. Steady-state experiments demonstrated that all these cytochromes were approximately 50% reduced while active transport proceeded at a high rate in regular cecropia Ringer containing 32 mM KCl. When the potassium concentration was reduced, the active transport decreased and all the cytochromes became more oxidized. Addition of 1 mM cyanide inhibited active transport by 90% and caused a 100% reduction of all cytochromes. Redox state and short circuit current (Isc) kinetics measured as the tissue was made anoxic showed that all the respiratory enzymes, except cytochrome b-557, became fully reduced at a faster rate than the rate of inhibition of the Isc. The rate of cytochrome b-557 reduction followed kinetically the Isc. These observations are interpreted in a scheme where cytochrome b-557 (possibly b5) branches off cytochrome c from the conventional resporatory chain, utilizing cytochrome a3 as the terminal oxidase for both branches. Cytochrome b-557 may be involved in providing a direct link between oxidative metabolism and active transport in the midgut of the silkworm.     

Menaquinone function in the respiratory chain of Micrococcus lysodeikticus]

Menaquinone-9 which is destructed under long-wave UV-irradiation is isolated from Micrococcus lysodeikticus membranes. NAD-H, malate and lactate oxidases are observed to be inhibited under irradiation, dehydrogenases of these substrates being almost intact. Photoinactivation of menaquinone results in the reduction of only one from two cytochromes b, presented in the membrane, thus testifying the location of menaquinone-9 between cytochromes b in the respiratory chain. Reconstruction of malate, NAD-H and lactate oxidases after irradiation took place when natural menaquinone (MQ-9) or menadione (MQ-0) were added. Detailed scheme of M. lysodeikticus respiratory chain is given.     

Effects of iron limitation on the respiratory chain and the membrane cytochrome pattern of the Euryarchaeon Halobacterium salinarum

The effects of iron limitation on the electron transport chain of the extremely halophilic Euryarchaeon Halobacterium salinarum were analyzed. When iron was growth-limiting, the respiratory rates as well as the inhibition pattern of the membranes were significantly different from membranes of iron replete cells. Changes in the availability of iron cause the formation of different respiratory pathways including different entry sites for electrons, different terminal oxidases of the respiratory chain, and drastic changes of the cytochrome composition and of the relative amounts of cytochromes. Under iron-limiting conditions, mainly low-potential cytochromes were measured. EPR spectroscopic studies revealed that the amount of proteins containing iron-sulfur clusters is reduced in membranes under iron-limiting growth conditions. Taken together, our results strongly suggest for the first time an important role of iron supply for the bioenergetics of an Archaeon.     

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.     

The development of the respiratory chain of Saccharomyces carlsbergensis during respiratory adaptation

1. Subcellular fractionation of sphaeroplasts produced at different stages during the first 4h of respiratory adaptation of anaerobically grown glucose-de-repressed Saccharomyces carlsbergensis gave mitochondrial fractions that contained all the detectable c- and a-type cytochromes. 2. The rates of cytochrome formation were studied; individual cytochromes were produced at different rates so as to give respiratory chains having widely differing cytochrome ratios. A CO-reacting haemoprotein other than cytochrome a(3) also increased throughout 8h of respiratory adaptation. 3. Even after short periods of aeration, organisms contained mitochondria in which cytochrome-cytochrome interactions and the reaction of cytochrome a(3) with O(2) proceeded at rates almost as fast as in organelles from aerobically grown cells. 4. The technique of flow-flash photolysis enabled kinetic resolution of the reoxidation of cytochromes a(3) and a to be achieved and their individual contributions to extinction changes in the Soret region were assessed. The ratio cytochrome a(3)/cytochrome a increased over the early stages of adaptation.     

Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration

An extension of the respiratory chain to the cell surface is necessary to reduce extracellular electron acceptors like ferric iron or manganese oxides. In the past few years, more and more compounds were revealed to be reduced at the surface of the outer membrane of Gram-negative bacteria, and the list does not seem to have an end so far. Shewanella as well as Geobacter strains are model organisms to discover the biochemistry that enables the dissimilatory reduction of extracellular electron acceptors. In both cases, c-type cytochromes are essential electron-transferring proteins. They make the journey of respiratory electrons from the cytoplasmic membrane through periplasm and over the outer membrane possible. Outer membrane cytochromes have the ability to catalyze the last step of the respiratory chains. Still, recent discoveries provided evidence that they are accompanied by further factors that allow or at least facilitate extracellular reduction. This review gives a condensed overview of our current knowledge of extracellular respiration, highlights recent discoveries, and discusses critically the influence of different strategies for terminal electron transfer reactions.     

Structural homology of cytochromes c.

Cytochromes c from many eukaryotic and diverse prokaryotic organisms have been investigated and compared using high-resolution nuclear magnetic resonance spectroscopy. Resonances have been assigned to a large number of specific groups, mostly in the immediate environment of the heme. This information, together with sequence data, has allowed a comparison of the heme environment and protein conformation for these cytochromes. All mitochondrial cytochromes c are found to be very similar to the cytochromes c2 from Rhodospirillaceae. In the smaller bacterial cytochromes, Pseudomonas aeruginosa cytochrome c551 and Euglena gracilis cytochrome c552, the orientation of groups near the heme is very similar, but the folding of the polypeptide chain is different. The heme environment of these two proteins is similar to that of the larger bacterial and mitochondrial cytochromes. Two low-potential cytochromes, Desulfovibrio vulgaris cytochrome c553 and cytochrome c554 from a halotolerant micrococcus have heme environments which are not very similar to those of the other proteins reported here.     

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 growth 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 beta-types cytochromes b561, b560 and b558, and at least two c-type cytochromes c556 and c2 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 b560, 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 b561 with associated beta and gamma 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 c2 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.     

Effects of growth substrate and respiratory chain composition on bioenergetics in Acinetobacter sp. strain HO1-N.

The relationship between respiratory chain composition and efficiency of coupling phosphorylation to electron transport was examined in Acinetobacter sp. strain HO1-N. Cells containing only cytochrome o as a terminal oxidase displayed the same stoichiometries of adenosine 5'-triphosphate synthesis and proton extrusion as cells which contained both cytochromes o and d as terminal oxidases. In addition, CO inhibition and photo-relief of cytochromes o or d did not alter the efficiency of energy coupling. These findings indicate that adenosine 5'-triphosphate synthesis is coupled to electron transport through both cytochromes o and d in Acinetobacter.     

Carbon monoxide-insensitive respiratory chain of Pseudomonas carboxydovorans.

Experiments employing electron transport inhibitors, room- and low-temperature spectroscopy, and photochemical action spectra have led to a model for the respiratory chain of Pseudomonas carboxydovorans. The chain is branched at the level of b-type cytochromes or ubiquinone. One branch (heterotrophic branch) contained cytochromes b558, c, and a1; the second branch (autotrophic branch) allowed growth in the presence of CO and contained cytochromes b561 and o (b563). Electrons from the oxidation of organic substrates were predominantly channelled into the heterotrophic branch, whereas electrons derived from the oxidation of CO or H2 could use both branches. Tetramethyl-p-phenylenediamine was oxidized via cytochromes c and a exclusively. The heterotrophic branch was sensitive to antimycin A, CO, and micromolar concentrations of cyanide. The autotrophic branch was sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide, insensitive to CO, and inhibited only by millimolar concentrations of cyanide. The functioning of cytochrome a1 as a terminal oxidase was established by photochemical action spectra. Reoxidation experiments established the functioning of cytochrome o as an alternative CO-insensitive terminal oxidase of the autotrophic branch.     

Membrane-bound respiratory of Spirillum itersonii.

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.     

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

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

Cytochrome c biogenesis in mitochondria--Systems III and V

In c-type cytochromes, heme becomes covalently attached to the polypeptide chain by a reaction between the vinyl groups of the heme and cysteine thiols from the protein. There are two such cytochromes in mitochondria: cytochrome c and cytochrome c(1). The heme attachment is a post-translational modification that is catalysed by different biogenesis proteins in different organisms. Three types of biogenesis system are found or predicted in mitochondria: System I (the cytochrome c maturation system); System III (termed holocytochrome c synthase (HCCS) or heme lyase); and System V. This review focuses primarily on cytochrome c maturation in mitochondria containing HCCS (System III). It describes what is known about the enzymology and substrate specificity of HCCS; the role of HCCS in human disease; import of HCCS into mitochondria; import of apocytochromes c and c(1) into mitochondria and the close relationships with HCCS-dependent heme attachment; and the role of the fungal cytochrome c biogenesis accessory protein Cyc2. System V is also discussed; this is the postulated mitochondrial cytochrome c biogenesis system of trypanosomes and related organisms. No cytochrome c biogenesis proteins have been identified in the genomes of these organisms whose c-type cytochromes also have a unique mode of heme attachment.     

Exploration of the 'cytochromome' of Desulfuromonas acetoxidans, a marine bacterium capable of powering microbial fuel cells

Recent progress in bacterial genomic analysis has revealed a vast number of genes that encode c-type cytochromes that contain multiple heme cofactors. This high number of multiheme cytochromes in several bacteria has been correlated with their great respiratory flexibility, and in what concerns biotechnological applications, has been correlated with electricity production in Microbial Fuel Cells. Desulfuromonas acetoxidans, a member of the Geobactereaceae family, is one of these organisms for which the genome was recently made available, coding for 47 putative multiheme cytochromes. The growth of D. acetoxidans in different media allowed the identification of the cytochromes dominant in each condition. The triheme cytochrome c(7) is always present suggesting a key role in the bioenergetic metabolism of this organism, and a dodecaheme cytochrome of low homology with other proteins in the databases was also isolated. Different cytochromes are found for different growth conditions showing that their roles can be assigned to specific bioenergetic electron transfer routes.     

Characterization of alternative respiratory pathways in the yeast Schwanniomyces castellii by the study of mutants deficient in cytochromes a+a3 and/or b.

After a general review of the proposed mechanisms and physiological roles of the alternative respiratory pathways found in various organisms, the studies are focussed on the amylolytic yeast Schwaniomyces castellii. In addition to the cytochrome chain, the wild type presents two alternative pathways insensitive to antimycin A. One is salicylhydroxamic acid (SHAM)-sensitive and azide-insensitive; the other is SHAM-insensitive and sensitive to high azide concentration. Conditions for mutagenesis and screening are described, which allow isolation of mutants deficient in cytochromes a+a3 and/or b in this yeast previously classified as petite negative. The relative proportions of the alternative respiratory pathways are compared in the wild type and mutant strains following inhibition by SHAM and azide at optimal concentration as determined by iso-inhibition curves. The growth of the cytochrome deficient mutants on citrate, a non-fermentable carbon source, and the ability of the wild type to grow on citrate+antimycin A, after a lag of about 10 h, indicate an involvement of the alternative pathway(s) in energy production. Rotenone sensitivity of respiration and ATP level confirm the presence of a functional phosphorylation site 1. The role of each alternative respiratory pathway in energy production is discussed.     

The Respiratory Chain of Plant Mitochondria: XII. Some Aspects of the Energy-linked Reverse Electron Transport from the Cytochromes c to the Cytochromes b in Mung Bean Mitochondria

The cytochromes c of mung bean (Phaseolus aureus) mitochondria become reduced when sulfide, a cytochrome oxidase inhibitor free from uncoupling side effects, is added to the aerobic mitochondrial suspension in the absence of added substrate. The cytochromes b remain largely oxidized. Subsequent addition of ATP results in partial oxidation of the cytochromes c and partial reduction of the cytochromes b due to ATP-driven reverse electron transport through the second site of energy conservation, or coupling site, of the respiratory chain. Cytochrome a is also oxidized under these conditions, but there is no concomitant reduction of the flavoprotein components, of ubiquinone, or of endogenous pyridine nucleotide. The reaction is abolished by oligomycin. The reducing equivalents transported from the cytochromes c and a in ATP-driven reverse electron transport are about 2-fold greater than those which appear in the cytochromes b. It is suggested that the equivalents not accounted for are present in a coupling site enzyme at the second site of energy conservation which interacts with the respiratory chain carriers by means of the dithiol-disulfide couple; this couple would not show absorbance changes with redox state over the wavelength range examined. With succinate present, reverse electron transport can be demonstrated at both coupling sites in both the aerobic steady state and in anaerobiosis. ATP-driven reverse electron transport in anaerobiosis maintains cytochrome a 30% oxidized while endogenous pyridine nucleotide is 50% reduced.