Characterization and phenotypic control of the cytochrome content of Escherichia coli

1. Electron-transport particles derived from Escherichia coli grown aerobically contain three b-type cytochromes with mid-point oxidation-reduction potentials at pH7 of +260mV, +80mV and -50mV, with n=1 for each. The variation of these values with pH was determined. 2. E. coli develops a different set of b-type cytochromes when grown anaerobically on glycerol with fumarate or nitrate as terminal electron acceptor. Electron-transport particles of fumarate-grown cells contain b-type cytochromes with mid-point potentials at pH7 of +140mV and +250mV (n=1). These two cytochromes are also present in cells grown with nitrate as terminal acceptor, where an additional cytochrome b with a mid-point potential of +10mV (n=1) is developed. 3. The wavelengths of the alpha-absorption-band maxima of the b-type cytochromes at 77K were: (a) for aerobically grown cells, cytochrome b (E(m7) +260mV), 556nm and 563nm, cytochrome b (E(m7) +80mV), 556nm and cytochrome b (E(m7)-50mV), 558nm; (b) for anaerobically grown cells, cytochrome b (E(m7) +250mV), 558nm, cytochrome b (E(m7) +40mV), 555nm and cytochrome b (E(m7) +10mV), 556nm. 4. Cytochrome d was found to have a mid-point potential at pH7 of +280mV (n=1). 5. Cytochrome a(1) was resolved as two components of equal magnitude with mid-point potentials of +260mV and +160mV (n=1). 6. Redox titrations performed in the presence of CO showed that one of the b-type cytochromes in the aerobically grown cultures was reduced, even at the upper limits of our range of electrode potentials (above +400mV). Cytochrome d was also not oxidizable in the presence of CO. Neither of the cytochromes a(1) was affected by the presence of CO.     

Cytochrome components of plant microsomes.

The electron transport components of the microsomal fraction of cauliflower buds and mung bean hypocotyls were investigated using split-beam and dual wavelength spectrophotometry under a variety of reducing conditions. Cauliflower microsomes were found to contain an ascorbate-reducible component, termed cytochrome b-559.5 [E'0 = +135 +/- 20 mV; lambdamax (reduced minus oxidised) = 559.5, 527 and 429 nm at 23 degrees C], cytochrome b5 [E'0 = -20 +/- 20 mV; lambdamax (reduced minus oxidised) = 556, 526 and 425 nm at 23 degrees C], cytochromes P-450 and P-420. On the basis of binding studies with ethyl isocyanide, degradation of cytochrome P-450 to P-420, redox potential, aniline binding, and relative rates of reduction by NADPH and NADH, it is suggested that the cytochrome P-450 system is analogous to that mammalian microsomes. Other components, reducible only by dithionite, may also be present. Mung bean microsomes were found to contain an ascorbate-reducible component, termed cytochrome b-562 [E'0 = +120 +/- 20 mV; lambdamax (reduced minus oxidised) = 562, 528 and 430 nm at 23 degrees C], cytochrome b5, and a low potential component which was reducible only by sodium dithionite. No cytochrome P-450 or P-420 could be detected. A general method of analysis of the cytochromes was developed and applied to the microsomes from a variety of plant sources. The results indicate that large variations, both in type and amount of components, occur between the microsomes from different plant materials.     

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 alpha-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 alpha-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 (lambda m (25 degrees C) = 553.5 nm; E'0 = 238 mV) and four cytochromes b (lambda m (25 degrees C) = 558.6, 561.2, 562.1, 566.1 nm and E'0 = -83, 26, 85, -60 mV).     

Redox and acid-base characterization of cytochrome b-559 in photosystem II particles

The redox and acid/base states and midpoint potentials of cytochrome b-559 have been determined in oxygen-evolving photosystem II (PS II) particles at room temperature in the pH range from 6.5 to 8.5. At pH 7.5 the fresh PS II particles present about 2/3 of their cytochrome b-559 in its reduced and protonated (non-auto-oxidizable) high-potential form and about 1/3 in its oxidized and non-protonated low-potential form. Potentiometric reductive titration shows that the protonated high-potential couple is pH-independent (E'0, + 380 mV), whereas the low-potential couple is non-protonated and pH-independent above pH 7.6 (E'0, pH greater than 7.6, + 140 mV), but becomes pH-dependent below this pH, with a slope of -72 mV/pH unit. Moreover, evidence is presented that in PS II particles cytochrome b-559 can cycle, according to its established redox and acid/base properties, as an energy transducer at two alternate midpoint potentials and at two alternate pKa values. Red light absorbed by PS II induces reduction of cytochrome b-559 in these particles at room temperature, the reaction being completely blocked by dichlorophenyldimethylurea.     

Energy tranduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the co-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata.

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E'0 at pH 7.0 +413+/-5, +270+/-5, +148+/-5, +56+/-5 and -32+/-5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b148, b56, and b-32) vary as a function of pH with a slope of 30 mV per pH unit. 2. In the presence of a CO/N2 mixture, the apparent E'0 of cytochrome b270 shifts markedly towards higher potentials (+355mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain. 3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c2 and of virtually all cytochrome cc'. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 nor the CO-binding cytochrome cc' are involved in this pathway. 4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in C0-difference spectra and with an alpha band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome "0" and considered the alternative CO-sensitive oxidase.     

Spectral and potentiometric analysis of cytochromes from Bacillus subtilis

Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain. At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion-exchange chromatography. They include two terminal oxidases with CO-binding properties and cyanide sensitivity. One of these is an aa3-type cytochrome c oxidase which has characteristic absorption maxima in the reduced-oxidized difference spectrum at 601 nm in the alpha-band and at 443 nm in the Soret band regions. In the alpha-band two separate electron transitions with Em = +205 mV and Em = +335 mV can be discriminated by redox potentiometric titration. The other CO-binding cytochrome c oxidase contains two cytochrome b components with alpha-band maxima at 556 nm and 559 nm. Cytochrome b556 can be reduced by ascorbate and has an Em + +215 mV, whereas cytochrome b559 has an Em = +140 mV. Furthermore a complex consisting of a cytochrome b564 (Em = +140 mV) associated with a cytochrome c554 (Em = +250 mV) was found. This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical alpha-peak and stains for heme-catalyzed peroxidase activity on SDS-containing polyacrylamide gels. A protein with a molecular mass of about 30 kDa is responsible for this activity. A cytochrome b559 (Em = +65 mV) appears to be an essential part of succinate dehydrogenase. Finally a cytochrome c550 component with an apparent mid-point potential of Em = +195 mV has been detected.     

Hepatic uroporphyrin accumulation and uroporphyrinogen decarboxylase activity in cultured chick-embryo hepatocytes and in Japanese quail (Coturnix coturnix japonica) and mice treated with polyhalogenated aromatic compounds.

A soluble cytochrome b was purified from Acinetobacter calcoaceticus L.M.D. 79.41. On the basis of the alpha-band maximum of a reduced preparation, measured at 25 degrees C, it is designated as cytochrome b-562. This cytochrome is a basic monomeric protein (pI 10.2; Mr 18,000), containing one protohaem group per molecule. The reduced form, at 25 degrees C, showed absorption bands at 428, 532 and 562 nm. At 77 K the alpha-band shifted to 560 nm (with a shoulder at 558 nm). The reduced cytochrome did not react with CO. Cytochrome b-562 is most probably (loosely) attached to the outside of the cytoplasmic membrane, since substantial amounts of it, equimolar to quinoprotein glucose dehydrogenase (GDH), were present in the culture medium when cells were grown in the presence of low concentrations of Triton X-100. The midpoint potential at pH 7.0 was found to be +170 mV, a value that was lowered to +145 mV by the presence of GDH. Since the GDH was shown to have a midpoint potential of +50 mV, cytochrome b-562 could function as the natural primary electron acceptor. Arguments to substantiate this view and to propose a role of ubiquinone-9 as electron acceptor for cytochrome b-562 are presented.     

Fumarate reduction in Proteus mirabilis.

1. Proteus mirabilis formed fumarate reductase under anaerobic growth conditions. The formation of this reductase was repressed under conditions of growth during which electron transport to oxygen or to nitrate is possible. In two of three tested chlorate-resistant mutant strains of the wild type, fumarate reductase appeared to be affected. 2. Cytoplasmic membrane suspensions isolated from anaerobically grown P. mirabilis oxidized formate and NADH with oxygen and with fumarate, too. 3. Spectral investigation of the cytoplasmic membrane preparation revealed the presence of (probably at least two types of) cytochrome b, cytochrome a1 and cytochrome d. Cytochrome b was reduced by NADH as well as by formate to approximately 80%. 4. 2-n-Heptyl-4-hydroxyquinilone-N-oxide and antimycin A inhibited oxidation of both formate and NADH by oxygen and fumarate. Both inhibitors increased the level of the formate/oxygen steady state and the formate/fumarate steady state. 5. The site of inhibition of the respiratory activity by both HQNO and antimycin A was located at the oxidation side of cytochrome b. 6. The effect of ultraviolet-irradiation of cytoplasmic membrane suspensions on oxidation/reduction phenomena suggested that the role of menaquinone is more exclusive in the formate/fumarate pathway than in the electron transport route to oxygen. 7. Finally, the conclusion has been drawn that the preferential route for electron transport from formate and from NADH to fumarate (and to oxygen) includes cytochrome b as a directly involved carrier. A hypothetical scheme for the electron transport in anaerobically grown P. mirabilis is presented.     

Purification and properties of cytochrome b556 in the respiratory chain of aerobically grown Escherichia coli K12.

Cytochrome b556, a major component of type b cytochromes in the respiratory chain of aerobically grown Escherichia coli, was purified to near homogeneity. It was solubilized from cytoplasmic membranes by treatment with Sarkosyl/cholate mixture and purified by gel filtration on Sephadex G-200. The purified cytochrome b556 is an oligomer composed of identical polypeptides, with a molecular weight of 17,500, determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains equimolar amounts of heme and polypeptide but no detectable non-heme iron, phospholipid, or dehydrogenase. Its isoelectric point was determined to be 8.5. The cytochrome b556 is highly hydrophobic in its amino acid composition and does not contain any half-cystine residues. The purified cytochrome b556 is spectrophotometrically pure and the alpha absorption peak in its difference spectrum at 77 K is at 556 nm. The molar extinction coefficient of cytochrome b556 was determined as 22.8 cm-1 mM-1. Its oxidation-reduction potential was found to be -45 mV. It could be reduced by D-lactate dehydrogenase of E. coli in the presence of menadione.     

A new membrane-bound b-type cytochrome, cytochrome b-558, from photosynthetically grown Rhodopseudomonas sphaeroides

A new membrane-bound b-type cytochrome, cytochrome b-558, was removed from chromatophore membranes of photosynthetically grown Rhodopseudomonas sphaeroides strain R-26 by deoxycholate-cholate extraction. The cytochrome was purified by ammonium sulfate fractionation and ion-exchange chromatography. Cytochrome b-558 had absorption maxima at 280 and 405 nm in the oxidized form, and at 558, 528, and 420 nm in the reduced form. It had a midpoint potential of--130 mV at pH 7.0. The minimal molecular weight of this protein was 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it contained one mole heme per mole of protein. The isoelectric point was 8.5. The electrophoretic pattern of heme-carrying proteins and the redox potentiometry showed that cytochrome b-558 was present in membranes from wild type, strain R-26, and strain GA grown photosynthetically, but not from any strain grown aerobically.     

Energy transduction in photosynthetic bacteria. X. Composition and function of the branched oxidase system in wild type and respiration deficient mutants of Rhodopseudomonas capsulata.

The respiratory chain of Rhodopseudomonas capsulata, strain St. Louis and of two respiration deficient mutants (M6 and M7) has been investigated by examining the redox and spectral characteristics of the cytochromes and their response to substrates and to specific respiratory inhibitors. Since the specific lesions of M6 and M7 have been localized on two different branches of the multiple oxidase system of the wild type strain, the capability for aerobic growth of these mutants can be considered as a proof of the physiological significance of both branched systems "in vivo". Using M6 and M7 mutants the response of the branched chain to respiratory inhibitors could be established. Cytochrome oxidase activity, a specific function of an high potential cytochrome b (E'0 = +413 mV) is sensitive to low concentrations of KCN (5-10(-5) M); CO is a specific inhibitor of an alternative oxidase, which is also inhibited by high concentrations of KCN (10(-3) M). Antimycin A inhibits preferentially the branch of the chain affected by low concentrations of cyanide. Redox titrations and spectral data indicate the presence in the membrane of three cytochromes of b type (E'0 = +413, +260, +47 vM) and two cytochromes of c type (E'0 = +342, +94 mV). A clear indication of the involvement in respiration of cytochrome b413, cytochrome c342 and cytochrome b47 has been obtained. Only 50% of the dithionite reducible cytochrome b can be reduced by respiratory substrates also in the presence of high concentrations of KCN or in anaerobiosis. The presence and function of quinones in the respiratory electron transport system has been clearly demonstrated. Quinones, which are reducible by NADH and succinate to about the same extent can be reoxidized through both branches of the respiratory chain, as shown by the response of their redox state to KCN. The possible site of the branching of the electron transport chain has been investigated comparing the per cent level of reduction of quinones and of cytochromes b and c as a function of KCN concentrations in membranes from wild type and M6 mutants cells. The site of the branching has been localized at the level of quinones-cytochrome b47. A tentative scheme of the respiratory chains operating in Rhodopseudomonas capsulata, St. Louis and in the two respiration deficient mutants, M6 and M7 is presented.     

Do photosynthetic bacteria contain cytochrome c1?

A method is described for characterizing, c-type cytochromes in bacterial membrane preparations according to molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Applied to the photosynthetic bacterium Rhodopseudomonas sphaeroides this technique is used, together with spectroscopic measurements, to demonstrate that a membrane-bound cytochrome c of mol.wt. 30000 is active in photosynthetic electron transport in addition to the well-known soluble cytochrome, cytochrome c2. The membrane cytochrome has a midpoint potential (E'0) at pH 7 of +290 mV, as compared with +360 mV for purified cytochrome c2. Its alpha-band has a peak near 552 nm, as compared with 550 nm for cytochrome c2. Evidence is presented that chromatophores contain roughly equal amounts of the two cytochromes.     

Effect of oxygen supply during growth on the production of cytochromes, enzymes, and acid end products by Haemophilus parasuis.

Haemophilus parasuis, grown under conditions of high aeration, was found to lack a tricarboxylic acid cycle but to possess phosphoenolpyruvate carboxylase and a reductive pathway leading to the production of succinate. Such organisms contained approximately equal quantities of b-, c-, and d-type cytochromes and excreted acetate. When the oxygen supply for growth was either reduced or eliminated, the specific activities of phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, and NADH: fumarate oxidoreductase were increased substantially, and the acid products were succinate, acetate, and formate. Organisms grown under the latter conditions also contained increased quantities of b- and c-type cytochromes, some of which were low-potential cytochromes. These low-potential cytochromes were reduced by NADH and oxidized by fumarate, and hence, appeared to be components of NADH: furmarate oxidoreductase. Our results indicate that in H. parasuis, growing aerobically in medium containing glucose, the sole function of the reductive pathway is to provide intermediates for biosynthetic processes, and oxygen is the preferred electron acceptor. As the supply of oxygen is reduced or eliminated, the reductive pathway becomes more involved in NAD+ recycling and fumarate becomes the acceptor. In effect, irrespective of the oxygen supply, the growth of H. parasuis is absolutely dependent upon the presence of an electron transport system.     

Redox properties of membrane-bound b-type cytochromes and a soluble c-type cytochrome of nitrate reductase in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

In order to identify the b-type cytochrome involved in the nitrate reduction in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, the b-type cytochromes in the spheroplast membranes were characterized. Difference spectra at 77K of spheroplast membranes indicated the presence of two b-type cytochromes with a bands at 556.5 and 562 nm. Three components considered to be of the b-type cytochrome were resolved by anaerobic potentiometric titration at 560-572 nm. Their midpoint potentials at pH 7, Em,7, were - 135 mV, +40 mV and +175 nm and their approximate reduced minus oxidized maxima were determined to be at 565 nm (562 nm at 77K), 560 nm (556.5 nm) and 560 nm (556.5 nm), respectively. These values are almost the same as those reported for R. sphaeroides. The Em,7 value of the cytochrome c involved in the nitrate reductase of this denitrifier was determined to be 250 mV. A b-type cytochrome reduced with NADH and FMN was oxidized by nitrate in chromatophore membranes. The possibility that cytochrome b (Em,7 = 175 mV) is involved in the nitrate reduction is discussed.     

Inhibition of electron transfer from ferrocytochrome b to ubiquinone, cytochrome c1 and duroquinone by antimycin.

The effect of antimycin on (i) the respiratory activity of the KCN-insensitive pathway of mitochondria of Neurospora grown on chloramphenicol (chloramphenicol-grown) with durohydroquinone and succinate or NADH as substrate, (ii) the electron transfer from the b-type cytochromes to ubiquinone with durohydroquinone as electron donor as well as (iii) the electron transfer from the b-type cytochromes to duroquinone with succinate as electron donor in chloramphenicol-grown Neurospora and beef heart submitochondrial particles was studied. All experiments were performed in the uncoupled state. 1. The respiratory chain of chloramphenicol-grown Neurospora mitochondria branches at ubiquinone into two pathways. Besides the cytochrome oxidase-dependent pathway, a KCN-insensitive branch equiped with a salicylhydroxamate-sensitive oxidase exists. Durohydroquinone, succinate or NADH are oxidized via both pathways. The durohydroquinone oxidation via the KCN-insensitive pathway is inhibited by antimycin, wheras the succinate or NADH oxidation is not. The titer for ful inhibition is one mol antimycin per mol cytochrome b-563 or cytochrome b-557. 2. The electron transfer from durohydroquinone to ubiquinone, which takes place in the KCN-inhibited state, does not occur in the antimycin-inhibited state. 3. The reduction of duroquinone by succinate in the presence of KCN is inhibited by antimycin. The titer for full inhibition is one mol antimycin per mol cytochrome b-566 or cytochrome b-562 for beef heart (or cytochrome b-563 or cytochrome b-557 for Neurospora). 4. When electron transfer from the b-type cytochromes to cytochrome C1, ubiquinone and duroquinone is inhibited by antimycin, the hemes of cytochrome b-566 and cytochrome b-562 (or cytochrome b-563 and cytochrome b-557) are in the reduced state. 5. The experimental results suggest that the two b-type cytochromes form a binary complex the electron transferring activity of which is inhibited by antimycin, the titer for full inhibition being one mol of antimycin per mol of complex. The electron transfer from the b-type cytochromes to ubiquinone is inhibited in a non-linear fashion.     

Terminal oxidases of Escherichia coli aerobic respiratory chain. I. Purification and properties of cytochrome b562-o complex from cells in the early exponential phase of aerobic growth

Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli K12, was isolated in a highly purified form. The purified oxidase is composed of equimolar amounts of two polypeptides, with Mr = 33,000 and 55,000, determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains 19.5 nmol of heme and 16.8 nmol of copper/mg of protein, but no detectable nonheme iron, phospholipid, ubiquinone, or menaquinone. In the difference spectrum at room temperature, the oxidase shows a single alpha absorption peak at 560 nm and at 77 K it shows two alpha absorption peaks at 555 and 562 nm. This oxidase combines with CO and the CO difference spectrum at room temperature has a peak at 416 nm and a trough at 430 nm in the Soret region. Its oxidation-reduction potential is estimated to be 125 mV (pH 7.4) and it is pH-dependent (-60 mV/pH) in medium of pH 6.0 to 7.4. It catalyzes electron transport to oxygen via ubiquinol and ascorbate in the presence of phenazine methosulfate or N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. This oxidase activity depends on phospholipids and is sensitive to respiratory inhibitors, such as 2-heptyl-4-hydroxyquinoline N-oxide, piericidin A, KCN and NaN3. The divalent cations Zn2+, Cd2+, and Co2+ inhibit the oxidase activity extensively. The oxidase activity of the cytochrome b562-o complex was inhibited by photoinactivation with rose bengal, suggesting that the inhibition by zinc ion results from modification of a histidine residue of cytochrome o.     

Localization and characterization of cytochromes from membrane vesicles of Escherichia coli K-12 grown in anaerobiosis with nitrate.

Cytochromes b of anaerobically nitrate-grown Escherichia coli cells are analysed. Ascorbate phenazine methosulfate distinguishes low and high potential cytochromes b. Reduction kinetics performed at 559 nm presents a very complex pattern which can be analysed assuming that at least four b-type cytochromes are present. The electron transport chain from formate to oxygen would contain a low potential cytochrome b-556, a cytochrome b-558 associated to the oxidase, and a cytochrome d as the principle oxidase. Cytochrome o is also present, but seems to be functional only at low oxygen concentrations. A cytochrome b-556 associated to nitrate reductase is shown to belong to a branch of the formate-oxidase chain. 2-N-Heptyl-4-hydroxyquinoline-N-oxide affects the reduction kinetics in a very complex way. One inhibition site is in evidence between cytochrome b-558 and cytochrome d; another between the cytochrome associated to nitrate reductase and the nitrate reductase. A third inhibition site is located in the common part of the formate-nitrate and the formate-oxidase systems. Ascorbate phenazine methosulfate is shown to donate electrons near cytochrome b-558.     

Assignment of ESR signals of Escherichia coli terminal oxidase complexes

The ESR signals of all the major components of the aerobic respiratory chain of Escherichia coli were measured and assigned at liquid helium temperature. Cytochrome b-556 gives a weak high-spin signal at g = 6.0. The terminal oxidase cytochrome b-562 . o complex gives signals at g = 6.0, 3.0 and 2.26, and the terminal oxidase cytochrome b-558 . d complex gives signals at g = 6.0, 2.5 and 2.3. A signal derived from cupric ions in the purified cytochrome b-562 . o complex was observed near g = 2.0. It was shown by the effects of KCN or NaN3 on cytochromes under the air-oxidized conditions that cytochrome o has a high-spin heme and cytochrome d has a low-spin heme. The E'm values for cytochromes b-558 and d, respectively, determined by potentiometric titration of the ESR signals were 140 and 240 mV in the membrane preparation, and 30 and 240 mV in the purified preparation. The oxidized cytochrome d gave intense low-spin signals at g = 2.5 and 2.3, while cytochrome d under the air-oxidized conditions gave corresponding signals of only very low intensity. These results suggested that most of the cytochrome d under the air-oxidized conditions contains a diamagnetic iron atom with a bound dioxygen.     

Potentiometric analysis of the cytochromes of an Escherichia coli mutant strain lacking the cytochrome d terminal oxidase complex.

A combination of potentiometric analysis and electrochemically poised low-temperature difference spectroscopy was used to examine a mutant strain of Escherichia coli that was previously shown by immunological criteria to be lacking the cytochrome d terminal oxidase. It was shown that this strain is missing cytochromes d, a1, and b558 and that the cytochrome composition of the mutant is similar to that of the wild-type strain grown under conditions of high aeration. The data indicate that the high-aeration branch of the respiratory chain contains two cytochrome components, b556 (midpoint potential [Em] = +35 mV) and cytochrome o (Em = +165 mV). The latter component binds to CO and apparently has a reduced-minus-oxidized split-alpha band with peaks at 555 and 562 nm. When the wild-type strain was grown under conditions of low aeration, the components of the cytochrome d terminal oxidase complex were observed: cytochrome d (Em = +260 mV), cytochrome a1 (Em = +150 mV) and cytochrome b558 (Em = +180 mV). All cytochromes appeared to undergo simple one-electron oxidation-reduction reactions. In the absence of CO, cytochromes b558 and o have nearly the same Em values. In the presence of CO, the Em of cytochrome o is raised, thus allowing cytochromes b558 and o to be individually quantitated by potentiometric analysis when they are both present.     

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.