The participation of cytochromes in the process of nitrate respiration in Klebsiella (Aerobacter) aerogenes

1. The participation of cytochromes in the membrane-bound, nitrate and oxygen respiratory systems of Klebsiella (Aerobacter) aerogenes has been investigated. The membrane preparations contained the NADH, succinate, lactate and formate oxidase systems, and in addition a high respiratory nitrate reductase activity.2. Difference spectra indicated the presence of cytochromes b, a₁, d, and o. Cytochromes of the c-type could not be detected in these membranes. Both cytochrome b content and respiratory nitrate reductase activity were the highest in bacteria grown anaerobically in the presence of nitrate.3. Cytochrome b was the only cytochrome which, after being reduced by NADH, could be partially reoxidized anaerobically in the presence of nitrate. Furthermore, nitrate caused a lower aerobic steady state reduction only of cytochrome b.4. NADH oxidase and NADH-linked respiratory nitrate reductase activities were both inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and KCN. NADH oxidase activity was selectively inhibited by CO, while azide was found to inhibit only the respiratory nitrate reductase. In the presence of azide, nitrate did not affect the level of reduction of cytochrome b.5. The evidence presented suggests that cytochrome b is a carrier in the electron transport systems to both nitrate and oxygen; from cytochrome b branching occurs, with one branch linked to the respiratory nitrate reductase and one branch linked to oxidase systems, containing the cytochromes a₁, d and o.     

Aging-related decrease in hepatic cytochrome oxidase of the Fischer 344 rat

The effect of aging on the hepatic mitochondrial population has been determined using a rigorously defined group of Fischer 344 rats with known survivorship data. The age groups studied included mature adult controls (8.5 months; 100% survivorship), an intermediate aged group (17.5 months; 90% survivorship), and an aged group (29 months; 20% survivorship). Cytochrome oxidase activity and content were determined in homogenates and mitochondrial fractions. The mitochondrial fractions were characterized by determination of respiratory activity, and monoamine oxidase activity as well as evaluation of the polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The yield of protein in the isolated mitochondrial fraction as well as the mitochondrial specific content decreased significantly as a function of aging. Mitochondrial specific content was determined from the specific activities of cytochrome oxidase in the homogenate (per gram liver) and in the isolated mitochondrial fraction (per mg protein). Specific activity of hepatic cytochrome oxidase decreased approximately 15% (P = 0.035) in homogenates from the 17.5-month animals with a further, highly significant (P = 0.0002) decrease (29%) in the 29-month animals. In contrast, there was no statistically significant difference among the age groups in the cytochrome oxidase specific activity in the isolated hepatic mitochondrial fractions. However, the percentage of the total homogenate cytochrome oxidase activity recovered in the isolated mitochondrial fraction decreased significantly in the 29-month animals (P = 0.0063 vs the 8.5-month controls; P = 0.022 vs the 17.5-month group). Cytochrome aa₃ content of total liver homogenates from aged animals decreased (P = 0.00064) which is in agreement with the decline in cytochrome oxidase specific activity in this age group. In the mitochondrial fraction from the aged animals, cytochrome aa₃ content was essentially unchanged which is consistent with the lack of aging-related change in mitochondrial cytochrome oxidase specific activity. In freshly isolated mitochondrial fractions, no aging-related alterations were observed in respiratory control and $\text{ADP}\text{O}$ ratios. The addition of exogenous NADH and cytochrome c did not change significantly the respiratory rate of hepatic mitochondria from control or aged animals. These results demonstrate the integrity of freshly isolated mitochondrial preparations from both control and aged Fischer 344 rats. In addition, there was no aging-related alteration in either monoamine oxidase specific activity or polypeptide composition. The similarities observed in the specific activities of cytochrome oxidase and monoamine oxidase, as well as in the cytochrome aa₃ content and polypeptide composition of the isolated mitochondrial fraction, suggest a generalized decrease in hepatic mitochondrial content as a function of aging rather than a selective loss of mitochondrial components.     

Derepression of mitochondria and their enzymes in yeast: regulatory aspects

We have performed a detailed analysis of the properties of glucose-repressed cells of a commercial strain of Saccharomyces cerevisiae. They contain measurable amounts of the respiratory enzymes NADH oxidase, cytochrome c oxidase, succinate dehydrogenase, succinate:cytochrome c reductase and NADH:cytochrome c reductase (antimycin A-sensitive) as well as the dehydrogenases for l-malate, l-glutamate, and l₈-isocitrate. Cytochromes b, c₁, and aa₃ are present in amounts that may be in excess of those required for cytochrome-linked enzyme activities. Enzymes and cytochromes are localized in large, presumably mitochondrial organelles among which no compositional or functional heterogeneity could be detected.We have also analyzed the kinetics of synthesis of respiratory enzymes and cytochromes during the release from catabolite(glucose) repression. All activities assayed except for cytochrome c oxidase begin their derepression before the external glucose concentration falls below 0.4%; derepression of cytochrome oxidase occurs only after the glucose concentration falls below 0.1%. The earlier events comprise the “fermentative” phase of derepression while the later events comprise the “oxidative” phase. The two phases can be distinguished operationally by their sensitivity to antimycin A. Only the oxidative phase is blocked by the inhibitor. Respiratory enzymes and cytochromes appear to fall into two classes distinguishable by their increase during derepression. An apparently constitutive one consists of cytochrome c oxidase, ATPase, and cytochromes aa₃, b, and c₁; these entities increase in amount per cell but not in amount per unit of mitochondrial mass and are of the order of 5-fold or less. The second class consists of those activities that increase by more than 6-fold and may be considered derepressible in the strict sense. Thus, proliferation and differentiation of mitochondria both contribute to the cellular changes associated with derepression.The fermentative phase of derepression does not require mitochondrial function, mitochondrial protein, or RNA synthesis, or the gradual accumulation of regulatory elements for either its initiation or persistence. This phase of derepression also occurs in cytoplasmic petites. In contrast, the oxidative phase of derepression requires mitochondrial function. Mitochondrial gene expression is required for the biogenesis of fully functional mitochondria but, except for cytochrome c, it plays little or no role in regulating the expression of nuclear genes the products of which are localized in mitochondria.     

Respiratory Activities, Cytochrome Composition and Cytochrome c Oxidase Subunit II Levels of Mitochondria from Alloplasmic Wheats Having Aegilops Cytoplasms

Respiratory activities, cytochrome composition and cytochromec oxidase (EC 1.9.3.1 [EC] ) subunit II (COXII) levels of isolatedmitochondria in one euplasmic and four alloplasmic lines ofwheat (Triticum aestivum), having cytoplasms of Aegilops columnaris,Ae.crassa (4x),Ae. mutica and Ae. triuncialis, which show variousgrowth abnormalities, were studied. Cytoplasm-specific variationon the respiratory activities has been revealed among the cytoplasmsof five Triticum and Aegilops species. The cyanide-resistantrespiratory pathway does not seem to contribute to the variationin activity. The low level of state 3 and cytochrome oxidaseactivities were evident in lines having Ae. columnaris and Ae.triuncialis cytoplasms, which also show growth inhibition. Thelow cytochrome oxidase activity in both cytoplasms is consistentwith the low amount of cytochrome aa₃ and the severe reductionin COXII levels. The relatively low amount of cytochrome aa₃in Ae. mutica is also consistent with the reduced level of theCOXII. On the other hand, relatively low amounts of cytochromeb (b-557) are found in the Ae. crassa cytoplasmic line. Theseresults suggest that the growth abnormalities found in the alloplasmicwheat lines are related to the variation in respiratory activitiesand cytochrome composition. In particular, growth inhibitionfound in the alloplasmic wheats having Ae. columnaris and Ae.triuncialis may be caused by the severe depression of respiratoryactivity due to the impaired cytochrome oxidase activity oftheir mitochondria. ³Present address: Department of Biochemistry, Dalhousie University,Halifax, NS, Canada B3H 4H7     

Effect of carbon source and oxidative metabolism on secondary metabolism inStreptomyces thermoviolaceus

The effect of carbon source on a number of cultural parameters was investigated inStreptomyces thermoviolaceus. Glucose-grown cultures produced the antibiotic granaticin during pH-controlled growth in a fermenter. Biphasic growth occurred for all the carbon sources tested at 45°C, the inflexion of which occurred at a biomass density of between 0.5 and 0.6 gL^–1 and which coincided with the onset of appearance of secondary metabolites. Maximum antibiotic production occurred in proline-grown cultures, which also had the slowest growth rates during the secondary phase of growth. Respiratory chain activity was probed by measuring NADH oxidation in membrane preparations exposed to a range of cyanide concentrations. Modulation of the terminal oxidase activity was apparent so that membranes prepared from cultures in the antibiotic-producing phase were less sensitive to KCN than those prepared from the early exponential phase of growth. The probable reason for this difference was the synthesis of cytochrome oxidased during later stages of growth. These changes in respiratory activity are discussed in relation to patterns of growth and timing of the appearance of secondary metabolites synthesised byS. thermoviolaceus.     

Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803

The cytochrome b₆f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b₆f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane.Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase.     

Biochemistry and physiological role of methionine sulfoxide residues in proteins

The cytochrome system in eggs and embryos of the sea urchin, Hemicentrotus pulcherrimus, was investigated. Difference spectra of the mitochondrial fraction demonstrated the presence of a complete cytochrome system in unfertilized eggs. Cytochrome levels and the activities of respiratory enzymes were measured in crude extracts of eggs both before and after fertilization. Unfertilized eggs contained cytochromes aa₃, b, and c + c₁ in a ratio of 1.0:1.8:0.7. Gastrulae contained almost the same amount of cytochromes aa₃and b as unfertilized eggs. However, the amount of cytochrome c + c₁ in gastrulae was 1.5 times greater than that in unfertilized eggs. The activity of cytochrome oxidase remained unchanged during development. No cytochrome oxidase inhibitor was found in unfertilized eggs. Both antimycin A-sensitive and insensitive NADH-cytochrome c reductase activities increased during development. The activity of succinate-cytochrome c reductase increased during early development, reached a temporary plateau, and then declined at the pluteus stage. These results are discussed in relation to the increase of respiration during early development.     

The Respiratory Chain of Plant Mitochondria: VIII. Reduction Kinetics of the Respiratory Chain Carriers of Mung Bean Mitochondria with Reduced Nicotinamide Adenine Dinucleotide

Addition of 90 micromolar reduced nicotinamide adenine dinucleotide (NADH) in the presence of cyanide to a suspension of aerobic mung bean (Phaseolus aureus) mitochondria depleted with ADP and uncoupler gives a cycle of reduction of electron transport carriers followed by reoxidation, as NADH is oxidized to NAD⁺ through the cyanide-insensitive, alternate oxidase by excess oxygen in the reaction medium. Under these conditions, cytochrome b₅₅₃ and the nonfluorescent, high potential flavoprotein Fp_ha of the plant respiratory chain become completely reduced with half-times of 2.5 to 2.8 seconds for both components. Reoxidation of flavoprotein Fp_ha on exhaustion of NADH is more rapid than that of cytochrome b₅₅₃. There is a lag of 1.5 seconds after NADH addition before any reduction of ubiquinone can be observed, whereas there is no lag perceptible in the reduction of flavoprotein Fp_ha and cytochrome b₅₅₃. The half-time for ubiquinone reduction is 4.5 seconds, and the extent of reduction is 90% or greater. About 30% of cytochrome b₅₅₇ is reduced under these conditions with a half-time of 10 seconds; both cytochrome b₅₆₂ and the fluorescent, high potential flavoprotein Fp_hf show little, if any, reduction. The two cytochromes c in these mitochondria, c₅₄₇ and c₅₄₉, are reduced in synchrony with a half-time of 0.8 second. These two components are already 60% reduced in the presence of cyanide but absence of substrate, and they become completely reduced on addition of NADH. These results indicated that reducing equivalents enter the respiratory chain from exogenous NADH at flavoprotein Fp_ha and are rapidly transported through cytochrome b₅₅₃ to the cytochromes c; once the latter are completely reduced, reduction of ubiquinone begins. Ubiquinone appears to act as a storage pool for reducing equivalents entering the respiratory chain on the substrate side of coupling site 2. It is suggested that flavoprotein Fp_ha and cytochrome b₅₅₃ together may act as the branching point in the plant respiratory chain from which forward electron transport can take place to oxygen through the cytochrome chain via cytochrome oxidase, or to oxygen through the alternate, cyanide-insensitive oxidase via the fluorescent, high potential flavoprotein Fp_hf.     

Cyanide-insensitive Respiration in Plant Mitochondria

Pathways of electron transport have been studied in mitochondria isolated from hypocotyls of etiolated mung bean seedlings and skunk cabbage spadices that show cyanide-resistant respiratory activity. The residual flux through cytochrome c oxidase is shown to be small in comparison with the flux through an unidentified alternative oxidase that is known to have a high affinity for oxygen. This alternative oxidase is not a cytochrome. Skunk cabbage and mung bean mitochondria contain cytochromes a and a₃ that have absorption peaks differing slightly from those of animal preparations. A slow oxidation-reduction of cytochrome a₃-CN has been demonstrated. Cytochromes b undergo oxidation and reduction in the presence of cyanide but play no essential role in the cyanide-resistant pathway. Antimycin inhibits to an extent similar to that of cyanide; the respiratory chain bifurcates on the substrate side of the antimycin-sensitive site. Evidence is presented for the selective inhibition by thiocyanate, α, α′-dipyridyl, and 8-hydroxyquinoline of the alternative oxidase pathway, which may therefore contain a non-heme iron protein.     

The Respiratory Chain of Plant Mitochondria: VII. Kinetics of Flavoprotein Oxidation in Skunk Cabbage Mitochondria

The oxidation kinetics of the two high potential flavo-proteins, one (Fp_hf) fluorescent and the other (Fp_ha) nonfluorescent, in mitochondria from skunk cabbage (Symplocarpus foetidus) spadices have been measured by combined spectrophotometry and fluorimetry. In the absence of respiratory inhibitors, both flavoproteins are oxidized at nearly the same rate with half-times between 120 and 160 milliseconds at 24 C. When slight differences in rate are observed, it is Fp_ha which consistently has the shorter half-time. The presence of 0.3 millimolar KCN has no perceptible effect on the oxidation rate of either component. Antimycin A (2 nanomoles per milligram of protein) increases the oxidation half-time of Fp_ha about 3-fold, but it has no effect on the oxidation half-time of Fp_hf. In contrast to these two inhibitors, m-chlorobenzhydroxamic acid—an inhibitor specific to the cyanide insensitive, alternate oxidase pathway in these mitochondria—increases the oxidation half-time of Fp_hf 10-fold to about 2 seconds, while increasing that of Fp_ha only some 20%. This result implies that the flavoprotein Fp_hf mediates electron transport to the alternate oxidase from the region of the mitochondrial respiratory chain encompassing Fp_ha, ubiquinone, and the cytochromes b. The oxidation rate of cytochrome b₅₅₇ is unaffected by either m-chlorobenzhydroxamic acid or cyanide but is strongly inhibited by antimycin A. This result implies that cytochrome b₅₅₇ plays no direct role in the respiratory pathway to the alternate oxidase and is different from cytochrome b₇ found in mitochondria from the spadices of Arum maculatum.     

Isolation of Sinorhizobium meliloti Tn5 mutants with altered cytochrome terminal oxidase expression and improved symbiotic performance

The relationship between whole-cell redox potential, cytochrome composition in free-living culture and symbiotic activity of Sinorhizobium meliloti was studied. Three Tn5-induced mutants with increased cellular redox potential were generated. Stationary cultures of mutants Tb9 and Tb16 in contrast to the parental strain produced the b-type terminal oxidase that may be similar to the symbiotically essential cytochrome oxidase cbb₃ of Bradyrhizobium japonicum. Increase in the symbiotic effectiveness of all three mutants and in O₂ consumption rate in free-living cultures was observed. Mutants Tb1 and Tb16 were also characterized by an increase in fixNOQP gene expression. Consequently, the mutations probably affect at least two different steps of rhizobial respiratory metabolism operating both in free-living cells and endosymbiotic forms.     

Different effects of 2-n-heptyl-4-hydroxyquinoline-N-oxide on oxygen and nitrate respiration in Klebsiella aerogenes

The inhibition of the oxidase and respiratory nitrate reductase activity in membrane preparations from Klebsiella aerogenes by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) has been investigated. Addition of HQNO only slightly affected the aerobic steady-state reduction of cytochrome b₅₅₉ with NADH, but caused a significantly lower nitrate reducing steady-state of this cytochrome. The changes in the redox states of the cytochromes during a slow transition from anaerobic to aerobic conditions in the presence and absence of HQNO showed that the inhibition site of HQNO is located before cytochrome d. Inhibition patterns obtained upon titration of the NADH oxidase and NADH nitrate reductase activity with HQNO indicated one site of inhibitor interaction in the NADH nitrate reductase pathway and suggested a multilocated inhibition of the NADH oxidase pathway. Difference spectra with ascorbate-dichlorophenolindophenol as electron donor indicated the presence of a cytochrome b₅₆₃ component which was not oxidized by nitrate, but was rapidly oxidized by oxygen. The latter oxidation was prevented by HQNO. A scheme for the electron transport to oxygen and nitrate is presented. In the pathway to oxygen, HQNO inhibits both at the electron-accepting side of cytochrome b₅₅₉ and at the electron-donating side of cytochrome b₅₆₃, whereas in the pathway to nitrate, inhibition occurs only at the electron-accepting side of cytochrome b₅₅₉.     

Cytochrome b type oxidases in the respiratory chains of the phytopathogenic fluorescent bacteria Pseudomonas cichorii and Pseudomonas aptata

Membrane fragments from the phytopathogenic bacteria Pseudomonas cichorii and Pseudomonas aptata have been examined. A branched respiratory chain is operative in P. cichorii whereas a linear electron transport system characterizes the related bacterium P. aptata. Both species contain several b type cytochromes resolved by redox titration analysis, but no a type components may be detected. In contrast, only P. cichorii is endowed with c type cytochromes and hence with cytochrome c oxidase activity. Among the b type cytochromes, two high-potential components, with Em_7.0 at +250 mV and +380 mV, have been kinetically characterized and tentatively associated with cyanideresistant and cytochrome c oxidase activities, respectively. Cytochrome b-250 should correspond to the spectrally detectable cytochrome o whereas cytochrome b-380 is functionally similar to cytochrome b-410 described in Rhodopseudomonas capsulata. This conclusion seems to blur previous reported data on other obligate aerobes in which cytochrome o has been generally associated with cytochrome c oxidase and also suggests that a more accurate reconsideration of the actual physiological role of cyt. o in bacterial respiration is necessary. Furthermore the question arises whether cyt. b-410 like oxidases, i. e. high-potential b's similar to cyt. b-410 of R. capsulata, may be widely distributed among aerobes rather than restricted to facultative photosynthetic prokaryotes.     

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.     

The peculiarities of the structural and functional state of the cytochrome component of the liver mitochondrial respiratory chain under conditions of acetaminophen-induced hepatitis on the background of alimentary protein deprivation

The activity of a key enzyme of the cytochrome component of the respiratory chain (cytochrome oxidase), the quantitative redistribution of mitochondrial cytochromes b, c ₁, c, and aa ₃, as well as the activities of the key enzymes of cytochrome heme metabolism (δ-aminolevulinate synthase and heme oxygenase) under conditions of acetaminophen-induced liver injury were studied on the background of dietary protein deprivation. Under conditions of acetaminophen-induced hepatitis that developed on the background of alimentary protein deprivation, an inhibition of cytochrome oxidase activity and a decrease in the contents of mitochondrial cytochromes on the background of an increase in the δ-aminolevulinate synthase and heme oxygenase activity were observed. In animals with a toxic liver injury that were kept under conditions of dietary protein deprivation, the contents of mitochondrial cytochromes b, c ₁, c, and aa ₃ progressively decreased, which was accompanied by an increase in heme oxygenase activity, whereas δ-aminolevulinate synthase activity remained at the control level. It was concluded that dietary protein deprivation is a critical factor for the development of disturbances in the structural-functional integrity of the cytochrome component of the respiratory chain. The identified changes can be considered as a possible mechanism that underlies the disturbance in the function of the energy biotransformation system under conditions of dietary protein deprivation.     

Assembly of a chimeric respiratory chain from bovine heart submitochondrial particles and cytochrome bd terminal oxidase of Escherichia coli

Cytochrome bd is a terminal quinol oxidase in Escherichia coli. Mitochondrial respiration is inhibited at cytochrome bc₁ (complex III) by myxothiazol. Mixing purified cytochrome bd oxidase with myxothiazol-inhibited bovine heart submitochondrial particles (SMP) restores up to 50% of the original rotenone-sensitive NADH oxidase and succinate oxidase activities in the absence of exogenous ubiquinone analogs. Complex III bypassed respiration and is saturated at amounts of added cytochrome bd similar to that of other natural respiratory components in SMP. The cytochrome bd tightly binds to the mitochondrial membrane and operates as an intrinsic component of the chimeric respiratory chain.     

A four-subunit cytochrome bc1 complex complements the respiratory chain of Thermus thermophilus

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc₁ complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc₁ subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc₁ complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c₁ carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c₅₅₂, mediating electron transfer to the ba₃ oxidase. Identification of this cytochrome bc₁ complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.     

Action de l'antimycine a sur l'activité succinoxydase des mitochondries du tubercule de Pomme de Terre,et comportement des cytochromes de type b

A kinetic study of the effect of antimycin A on succinate oxidase from plant mitochondria produced sigmoidal curves for the reduction of cytochromes b₅₆₀ and b₅₈₅ and for the inhibition of succinate oxidase. In the stationary state the interaction of the various components of the respiratory chain (flavins, ubiquinone, cytochromes…) occurs in a sequential mode which allows the application of simple equations utilizing rate constants and cytochrome concentrations. In these equations, it is assumed that there exists an excess of ubiquinone relative to other components of the respiratory chain as suggested by Kröger & Klingenberg (1970) and that the reoxidation of b cytochromes is fast. The inhibition by antimycin A, characterized by non-linear inhibition curves for succin-oxidase and inhibitor fixation in complex III on a component other than cytochrome c₁, is interpreted in terms of this model. This hypothesis presupposes the existence of an F factor between cytochrome b₅₆₀ and cytochrome c₁ as suggested by other authors. Utilizing these equations, theoretical curves for the inhibition of the reduction of cytochrome b₅₆₀ have been constructed and the results agree with the experimental data. The kinetic behavior of cytochrome b₅₆₆ during the induction of anaerobiosis suggests that it is not directly involved in the electron transfer chain but rather is either in thermodynamic equilibrium with cytochrome b₅₆₀ or in a shunt between cytochrome b₅₆₀ and factor F. From the experimental data, an equation is derived for the inhibition of the reduction of cytochrome b₅₆₆ by antimycin A. The actual effects of ATP and mClCCP on succinoxidase agree well with those predicted by the model.     

Particulate cytochromes of mung bean seedlings

Efforts have been made to solubilize cytochrome components from particulate fractions of etiolated mung bean seedlings. Low temperature spectrophotometry reveals that the cytochrome composition of mitochondria isolated from whole seedlings is the same as that reported by Bonner for mung bean hypocotyls. On the basis of the identity in position of the α-bands in low temperature difference spectra for mitochondria, for a partially purified haemoprotein from mitochondria, and for purified cytochrome b-555, it is suggested that cytochrome b-555 is an intrinsic component of mung bean mitochondria. Difference spectra show that both the mitochondrial and microsomal fractions contain at least 2 b-type cytochromes. Cytochrome b-555 is almost certainly present in the microsomes, since the low temperature difference spectrum for the cytochrome is identical with the spectrum for this particulate fraction.

By freezing and thawing mung bean mitochondria in 4% cholate and centrifuging, cytochrome oxidase activity can be concentrated in the supernatant fraction, although it is not completely solubilized. The oxidase is inhibited by high concentrations of cytochrome c. A particle-bound cytochrome c can be obtained from mitochondria by digestion with snake venom. However, the autoxidizability of the preparation indicates that the cytochrome has been solubilized in a modified form. A CO-binding pigment can be obtained from mung bean microsomes by digestion with snake venom.

     

Partial purification of the superoxide-generating system of macrophages. Possible association of the NADPH oxidase activity with a low-potential (−247 mV) cytochrome b

NADPH oxidase activity was solubilized by detergent treatment of subcellular particles obtained from guinea-pig peritoneal macrophages stimulated with phorbol myristate acetate. Gel filtration of the material containing the NADPH oxidase activity gave two peaks of proteins, one of which eluted with the void and the other with the included volume of an AcA 22 column. The material eluted in the void volume contained more than 50% of the NADPH oxidase activity and less than 10% of the NAD(P)H cytochrome c reductase activity. A b-type cytochrome with peaks of absorption at 558, 528 and 426 nm was also enriched in the fraction which contained the NADPH oxidase activity. The distribution of flavoproteins as revealed by the measurement of FAD was different from that of NADPH oxidase and cytochrome b, and followed the elution profile of NADH cytochrome c reductase. Studies in subcellular particles showed that the b cytochromes of mitochondria and endoplasmic reticulum reduced by selective biochemical means accounted for only a minor part of the total b-type cytochromes and that the new cytochrome b previously described in neutrophils is the major chromophore also in macrophages. Oxidation-reduction midpoint potential of the partially purified cytochrome b was shown to be −247 mV. Association of cytochrome b with the NADPH oxidase activity and its very low E_m7.0 makes it a suitable candidate to be part of the superoxide-generating system also in macrophages.