Reduction of superoxide production by mitochondria oxidizing NADH in the presence of organic acids

Effects of multiple substrates on oxygen uptake and superoxide production by mitochondria isolated from the pericarp tissue of green bell pepper (Capsicum annuum L.) were studied. Mitochondria isolated from peppers stored at 4 °C for 5 and 6 days had higher rates of oxygen uptake and were less sensitive to cyanide than mitochondria isolated from freshly harvested peppers. Succinate enhanced state 2 and state 4 rates of oxygen uptake with exogenous NADH in the absence of cytochrome path inhibitors, but not state 3 rates by mitochondria isolated from either freshly harvested or cold-stored bell peppers. The sensitivity of NADH oxidation to cyanide was reduced by both malate and succinate in mitochondria from cold-stored bell peppers, whereas only succinate was effective in mitochondria from freshly harvested peppers.Mitochondria isolated from both freshly harvested peppers and those stored at 4 °C for 5 and 6 days produced superoxide in the absence of exogenous substrates. Superoxide production by mitochondria from freshly harvested bell peppers increased when the mitochondria were supplied with malate, succinate or NADH, but only NADH enhanced superoxide production by mitochondria from cold-stored peppers. Both succinate and malate reduced the production of superoxide by mitochondria isolated from cold-stored bell peppers. Succinate and malate as second substrates also reduced the production of superoxide with NADH by mitochondria from both freshly harvested and cold-stored bell peppers. Malonate, a competitive inhibitor of succinate dehydrogenase, was inhibitory to oxygen uptake and to superoxide production.Mitochondria isolated from cold-stored bell peppers converted succinate to pyruvate at 25 °C at considerably higher rates than those of mitochondria from freshly harvested bell peppers. Since pyruvate has been shown to activate the alternative oxidase and the presence of pyruvate is essential for continued alternative oxidase activity, we suggest that pyruvate limits superoxide production by enhancing the flow of electrons through the alternative path. A direct scavenging of superoxide by succinate, malate and pyruvate, however, cannot be ruled out.     

An evaluation of mitochondrial heterosis and in vitro mitochondrial complementation in wheat,barley and maize

Summary Two families each of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and maize (Zea mays L.) were studied for mitochondrial heterosis and in vitro mitochondrial complementation. Inbred parents and their hybrids were compared for seedling heights and rate of oxygen uptake by the whole tissue to find out if the hybrids showed greater growth and respiratory activity at the seedling stage. Further comparisons were made by isolating mitochondria from the seedling tissues and measuring their ADP0 ratio, respiratory control ratio and cytochrome c oxidase activity for mitochondrial heterosis. Mixtures of parental mitochondria were similarly compared with parental and hybrid mitochondria for in vitro mitochondrial complementation. No evidence for mitochondrial heterosis or in vitro mitochondrial complementation was found, nor any correlation between the different mitochondrial parameters, seedling heights or rates of oxygen uptake by seedling tissue. The suggested use of mitochondrial heterosis and in vitro mitochondrial complementation for plant breeding is discussed.Data for this paper is taken from the author's dissertation written as a part of Ph.D. degree requirements at the Biology Department, Texas A & M University, College Station, Texas     

The carbon monoxide- and oxygen-reacting haemoproteins of Streptomyces clavuligerus: cytochrome aa 3 is the predominant terminal oxidase of the respiratory chain

The nature of the carbon monoxide- and oxygen-reacting haemoproteins in the respiratory chain of the filamentous antibiotic-producing bacterium Streptomyces clavuligerus has been investigated. CO-difference (i.e. CO+ reduced minus reduced) spectra of intact cells showed the presence of cytochrome aa ₃, a CO binding b-type cytochrome, and a pigment resembling cytochrome d. In addition, cells that were approaching the end of the growth phase showed the presence of cytochrome P450: this pigment was undetectable in cells harvested early in the growth cycle. High speed centrifugation of cell-free extracts prepared from cells broken by sonication showed that cytochrome aa ₃ was tightly membrane-bound and that cytochrome P450 was soluble. Inhibition of oxygen uptake rates of cells by cyanide indicated that one component, which showed 50% inhibition at 2–4 mM CN^–, was acting as major terminal oxidase: this was observed in cells harvested from all stages of growth. Photodissociation (i. e. photolysed, CO reduced minus CO reduced) spectra at-118°C, in the absence of oxygen, showed cytochrome aa ₃ to be the sole photolysable CO-reacting haemoprotein. At higher temperature (-87°C), in the presence of oxygen, cytochrome aa ₃ formed a complex with oxygen that could not be photolysed by similar intensities of light. By raising the temperature to-43°C, the oxidation of c-type cytochromes was observed. It is concluded that cytochrome aa ₃ is the predominant terminal oxidase in S. clavuligerus and that the other CO reacting haemoproteins, of unknown function, are unlikely to be oxidases.     

Tissue-specific expression of the alternative oxidase in soybean and siratro

Alternative oxidase activity (cyanide-insensitive respiration) was measured in mitochondria from the shoots, roots, and nodules of soybean (Glycine max L.) and siratro (Macroptilium atropurpureum) plants. Activity was highest in the shoots and lowest in the nodules. Alternative oxidase activity was associated with one (roots) or two (shoots) proteins between 30 and 35 kilodaltons that were detected by western blotting with a monoclonal antibody against Sauromatum guttatum alternative oxidase. No such protein was detected in nodule mitochondria. Measurements of oxygen uptake by isolated soybean root and nodule cells in the presence of cyanide and salicylhydroxamic acid indicated that alternative oxidase activity was confined to the uninfected cortex cells of the nodule. Immunoprecipitation of translation products of mRNA isolated from soybean shoots revealed a major band at 43 kilodaltons that is assumed to be the precursor of an alternative oxidase protein. This band was not seen when mRNA from nodules was treated in the same fashion. The results indicate that tissue-specific expression of the alternative oxidase occurs in soybean and siratro.     

Binding of Butyl Gallate to Plant Mitochondria : II. Relationship to the Presence or Absence of the Alternative Pathway

[¹⁴C]butyl gallate was used in binding studies to investigate the cyanide-resistant respiratory pathway in mitochondria isolated from a variety of sources displaying varying levels of cyanide resistance. Highly cyanide-resistant mitochondria were isolated from aroid spadices, while moderately cyanide-resistant mitochondria were isolated from either mung bean (Vigna radiata L.) hypocotyls or carbon dioxide/oxygen/ethylene-treated tubers. Totally cyanide-sensitive mitochondria were isolated from untreated tubers and rat liver. With one exception, all the plant mitochondria showed a reversible butyl gallate binding site which saturated at a level of 1.0 to 2.0 nanomoles per milligram protein. The exception, freshly harvested white potato tubers (<1 month from harvest), showed little specific butyl gallate binding, and also showed no appreciable induction of the cyanide-resistant pathway following carbon dioxide/oxygen/ethylene treatment. Only a low level, linear binding, well below that seen with plant mitochondria, was observed with rat liver mitochondria. Taken together, these results suggest a model for the interaction of the alternative pathway with the cytochrome pathway. In this model, the butyl gallate binding site (alternative oxidase) is a constitutive component in those mitochondria that are capable of developing the alternative pathway, and the binding sites associated with a second, inducible component that functions to couple the oxidase to the cytochrome pathway.     

Biochemical and cytological studies of mitochondrial development in chloramphenicol-induced rice coleoptiles

The ultrastructure and respiratory activity of mitochondria in rice coleoptile grown in the presence and in the absence of an inhibitor of mitochondrial protein synthesis (chloramphenicol) have been studied. It is shown that during the first 48 h of germination a rapid development of mitochondrial cristae takes place without notable influence of chloramphenicol on biogenesis of mitochondria. But the presence of the inhibitor has a significant effect in the subsequent period (48–144 h): a gradual and almost complete reduction of mitochondrial cristae is observed. These unusual “noncristate” mitochondria, although greatly lacking cytochrome oxidase, have a high respiratory activity. The respiration of “noncristate” mitochondria is resistant to KCN. It is supposed that chloramphenicol-induced rice coleoptile can be used as a new convenient object for studies of the nature of alternative oxidase as well as the biogenesis of mitochondria with cyanide-insensitive respiration.     

Energy conservation by the plant mitochondrial cyanide-insensitive oxidase. Some additional evidence.

Several measures of energy conservation, namely ADP/O ratio, P/O ratio, ATP/O ratio and phosphorylation detected by continuous assay with purified firefly luciferase and luciferin, all show phosphorylation can occur with mung-bean mitochondria at cyanide concentrations sufficient to inhibit the cytochrome oxidase system. Phosphorylation in the presence of cyanide is uncoupler- oligomycin- and salicylhydroxamate-sensitive. The participation of phosphorylation site 1 is excluded, phosphorylation being attributable to a single phosphorylation site associated with the cyanide-insensitive oxidase. The cyanide-insensitive oxidase has also been shown to support a variety of other energy-linked functions, namely, Ca2+ uptake, reversed electron transport and the maintenance of a membrane potential detected by the dye probes 8-anilinonaphthalene-1-sulphonate and safranine. High concentrations of cyanide have uncoupler-like activity, decreasing the ADP/O ratio and the t 1/2 for the decay of a pH pulse through the the mitochondrial membrane. This uncoupler-like effect is most marked with aged mitochondria. The observations of energy conservation attributable to the cyanide-insensitive oxidase are compared with other reports where it is concluded that the alternative oxidase is uncoupled.     

Heat-induced changes in respiratory pathways and mitochondrial structure during microcycle conidiation of Neurospora crassa

Changes in both respiratory pathways and mitochondrial structure of Neurospora crassa occurred under conditions of microcycle conidiation. Upon heat-treatment at 46°C, conidia developed a highly cyanide-insensitive, hydroxamate-sensitive respiration associated with morphological alterations in mitochondrial membranes; such changes were time-dependent. When heat-treated conidia were shifted down to 25°C, the alternate, hydroxamate-sensitive respiration decreased significantly, paralleling the recovery of well-cristated mitochondria with an electron-dense matrix in the germ tubes. The decrease in hydroxamate-sensitivity was associated with two periods of increase in cyanide sensitivity corresponding to the events of germination and precocious proconidial budding.     

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.     

The impact of the thermal sensitivity of cytochrome c oxidase on the respiration rate of Arctic charr red muscle mitochondria

To assess if cytochrome c oxidase could determine the response of mitochondrial respiration to changes in environmental temperature in ectotherms, we performed KCN titration of the respiration rate and cytochrome c oxidase activity in mitochondria from Arctic charr (Salvelinusfontinalis) muscle at four different temperatures (1 degrees C, 6 degrees C, 12 degrees C, and 18 degrees C). Our data showed an excess of cytochrome c oxidase activity over the mitochondrial state 3 respiration rate. Mitochondrial oxygen consumption rates reached approximately 12% of the cytochrome c oxidase maximal capacity at every temperature. Also, following titration, the mitochondrial respiration rate significantly decreased when KCN reached concentrations that inhibit almost 90% of the cytochrome c oxidase activity. This strongly supports the idea that the thermal sensitivity of the maximal mitochondrial respiration rate cannot be dictated by the effect of temperature on cytochrome c oxidase catalytic capacity. Furthermore, the strong similarity of the Q10s of mitochondrial respiration and cytochrome c oxidase activity suggests a functional or structural link between the two. The functional link could be coevolution of parts of the mitochondrial system to maintain optimal functions in most of the temperature range encountered by organisms.     

Interrelation of active oxygen species,membrane damage and altered calcium functions

Incubation of freshly isolated rat liver mitochondria in the presence of oxygen free radical generating hypoxanthine —xanthine oxidase system led to swelling of mitochondria as measured by the change in optical density, which was reversed by the addition of superoxide dismutase. O₂ ^– in the presence of CaCl₂ enhanced the peroxidative decomposition of mitochondrial membrane lipids along with swelling of the organelle. Free radical generation led to enhancement of monoamine oxidase activity while glutathione peroxidase and cytochrome c oxidase were inhibited. Tertbutyl hydroperoxide (t-BHP) caused mitochondrial swelling through oxidative stress. Incorporation of ruthenium red, which is a Ca²⁺ transport blocker, during assay abolished peroxidative membrane damage and swelling. Dithiothreitol (DTT) accorded protection against t-BHP induced mitochondrial swelling. The above in vitro data suggest a possible interrelationship of active oxygen species, membrane damage and calcium dynamics.     

Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin

Reactive oxygen species (ROS) are considered a key factor in mitochondrial dysfunction associated with brain aging process. Mitochondrial respiration is an important source of ROS and hence a potential contributor to brain functional changes with aging. In this study, we examined the effect of aging on cytochrome c oxidase activity and other bioenergetic processes such as oxygen consumption, membrane potential and ROS production in rat brain mitochondria. We found a significant age-dependent decline in the cytochrome c oxidase activity which was associated with parallel changes in state 3 respiration, membrane potential and with an increase in H₂O₂ generation. The cytochrome aa₃ content was practically unchanged in mitochondria from young and aged animals. The age-dependent decline of cytochrome c oxidase activity could be restored, in situ, to the level of young animals, by exogenously added cardiolipin. In addition, exposure of brain mitochondria to peroxidized cardiolipin resulted in an inactivation of this enzyme complex. It is suggested that oxidation/depletion of cardiolipin could be responsible, at least in part, for the decline of cytochrome c oxidase and mitochondrial dysfunction in brain aging. Melatonin treatment of old animals largely prevented the age-associated alterations of mitochondrial bioenergetic parameters. These results may prove useful in elucidating the molecular mechanisms underlying mitochondrial dysfunction associated with brain aging process, and may have implications in etiopathology of age-associated neurodegenerative disorders and in the development of potential treatment strategies.     

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. I. Isolation and preliminary characterization.

Summary Three nuclear mutants of Neurospora crassa, temperature-sensitive for the synthesis of cytochrome aa ₃ have been isolated. When grown at 41°C the mutants have large amounts of KCN-insensitive respiration, reduced amounts of cytochrome aa ₃ and cytochrome c oxidase activity, and grow more slowly than wild-type cultures grown at the same temperature. When the mutants are grown at 23°C, they are virtually indistinguishable from wild-type strains.The mutants were selected on the basis of their slow growth at 41°C in medium containing salicylhydroxamic acid, and by their inability to reduce 2,3,5-triphenyltetrazolium chloride at 41°C. The selection technique was designed to eliminate mutants that did not carry thermolabile electron transport chain components. However, studies on the thermolability of the cytochrome oxidase activity in isolated mitochondria indicate that the enzyme of the mutants is no more susceptible to heat denaturation than is the enzyme in wild-type mitochondria. This suggests that the synthesis or assembly of cytochrome aa ₃ may be altered in the mutants at the restrictive temperature.Supported by National Research Council of Canada Grant Number A-6351Recipient of a National Research Council of Canada Postgraduate Scholarship     

Regulation of Alternative Oxidase Activity by Pyruvate in Soybean Mitochondria

The regulation of alternative oxidase activity by the effector pyruvate was investigated in soybean (Glycine max L.) mitochondria using developmental changes in roots and cotyledons to vary the respiratory capacity of the mitochondria. Rates of cyanide-insensitive oxygen uptake by soybean root mitochondria declined with seedling age. Immunologically detectable protein levels increased slightly with age, and mitochondria from younger, more active roots had less of the protein in the reduced form. Addition of pyruvate stimulated cyanide-insensitive respiration in root mitochondria, up to the same rate, regardless of seedling age. This stimulation was reversed rapidly upon removal of pyruvate, either by pelleting mitochondria (with succinate as substrate) or by adding lactate dehydrogenase with NADH as substrate. In mitochondria from cotyledons of the same seedlings, cyanide-insensitive NADH oxidation was less dependent on added pyruvate, partly due to intramitochondrial generation of pyruvate from endogenous substrates. Cyanide-insensitive oxygen uptake with succinate as substrate was greater than that with NADH, in both root and cotyledon mitochondria, but this difference became much less when an increase in external pH was used to inhibit intramitochondrial pyruvate production via malic enzyme. Malic enzyme activity in root mitochondria declined with seedling age. The results indicate that the activity of the alternative oxidase in soybean mitochondria is very dependent on the presence of pyruvate: differences in the generation of intramitochondrial pyruvate can explain differences in alternative oxidase activity between tissues and substrates, and some of the changes that occur during seedling development.     

Alternative pathway respiration and lipoxygenase activity in aged potato slice mitochondria

Mitochondrial preparations isolated from aged white potato (Solanum tuberosum L.) slices exhibited classical cyanide-insensitive O(2) uptake which was inhibited by salicylhydroxamic acid and tetraethylthiuram disulfide (disulfiram). These mitochondria also possessed lipoxygenase activity, as determined by O(2) uptake in the presence of 4 millimolar linoleic acid. Purification of the mitochondrial preparation on a continuous Percoll gradient resulted in a large decrease in lipoxygenase activity whereas cyanide-insensitive (disulfiram sensitive) O(2) consumption was still observed. These data indicate that cyanide-insensitive O(2) consumption in mitochondrial preparations isolated from aged white potato slices is of mitochondrial origin and not due to lipoxygenase contamination.     

Isolating the segment of the mitochondrial electron transport chain responsible for mitochondrial damage during cardiac ischemia

Ischemia damages the mitochondrial electron transport chain (ETC), mediated in part by damage generated by the mitochondria themselves. Mitochondrial damage resulting from ischemia, in turn, leads to cardiac injury during reperfusion. The goal of the present study was to localize the segment of the ETC that produces the ischemic mitochondrial damage. We tested if blockade of the proximal ETC at complex I differed from blockade distal in the chain at cytochrome oxidase. Isolated rabbit hearts were perfused for 15 min followed by 30 min stop-flow ischemia at 37 °C. Amobarbital (2.5 mM) or azide (5 mM) was used to block proximal (complex I) or distal (cytochrome oxidase) sites in the ETC. Time control hearts were buffer-perfused for 45 min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated. Ischemia decreased cytochrome c content in SSM but not in IFM compared to time control. Blockade of electron transport at complex I preserved the cytochrome c content in SSM. In contrast, blockade of electron transport at cytochrome oxidase with azide did not retain cytochrome c in SSM during ischemia. Since blockade of electron transport at complex III also prevented cytochrome c loss during ischemia, the specific site that elicits mitochondrial damage during ischemia is likely located in the segment between complex III and cytochrome oxidase.     

Non-phosphorylating bypass of the plant mitochondrial respiratory chain by stress protein CSP 310

Recently, it has been reported that the cold-stress protein CSP 310, discovered in the cytoplasm of cold-resistant winter cereals, causes uncoupling of oxidative phosphorylation during cold stress. To understand how the uncoupling mechanism of CSP differs from that of cyanide-insensitive alternative oxidase and plant mitochondrial uncoupling protein, we determined the effect of respiratory-chain inhibition on winter wheat (Triticum aestivum L. cv. Zalarinka) mitochondria. Our data show a possible involvement of stress protein CSP 310 in mitochondrial electron transport in winter wheat. CSP 310 shunts electrons around the main cytochrome pathway of the mitochondrial respiratory chain, i.e. electron flow bypasses ubiquinone and complex III via CSP 310 to complex IV.     

Biochemical and cytological studies of mitochondrial development in chloramphenicol-induced rice coleoptiles.

The ultrastructure and respiratory activity of mitochondria in rice coleoptile grown in the presence and in the absence of an inibitor of mitochondrial protein synthesis (chloramphenicol) have been studied. It is shown that during the first 48 h of germination a rapid development of mitochondrial cristae takes place without notable influence of chloramphenicol on biogenesis of mitochondria. But the presence of the inhibitor has a significant effect in the subsequent period (48-144 h): a gradual and almost complete reduction of mitochondrial cristae is observed. These unusual "noncristate" mitochondria, although greatly lacking cytochrome oxidase, have a high respiratory activity. The respiration of "noncristate" mitochondria is resistant to KCN. It is supposed that chloramphenicol-induced rice coleoptile can be used as a new convenient object for studies of the nature of alternative oxidase as well as the biogenesis of mitochondria with cyanide-insensitive respiration.     

Early septic shock induces loss of oxidative phosphorylation yield plasticity in liver mitochondria

We aimed to study the change in mitochondrial oxidative phosphorylation efficiency occurring at the early stage of septic shock in an experimental model. Thirty-six male Wistar rats were divided into two groups. In the first group, a cecal ligation and puncture (CLP) was carried out to induce septic shock for 5 h. The second group includes sham-operated rats and constitutes the control group. Blood gas analysis, alanine amino transferase, and lactic acid dosages were assayed 5 h after surgery. Liver mitochondria were isolated for in vitro functional characterization, including mitochondrial respiratory parameters, oxidative phosphorylation efficiency, oxi-radical production, membrane potential, and cytochrome c oxidase activity and content. Liver interleukin 1β (IL-1β) and tumor necrosis α mRNA levels were determined. Septic shock induced a severe hypotension occurring 180 min after CLP in association with a metabolic acidosis, an increase in plasma alanine amino transferase, liver IL-1β gene expression, and mitochondrial reactive oxygen species production. The rates of mitochondrial oxygen consumption and the activity and content of cytochrome c oxidase were significantly decreased while no alterations in the oxidative phosphorylation efficiency and inner membrane integrity were found. These results show that contrary to what was expected, liver mitochondria felt to adjust their oxidative phosphorylation efficiency in response to the decrease in the mitochondrial oxidative activity induced by CLP. This loss of mitochondrial bioenergetics plasticity might be related to mitochondrial oxidative stress and liver cytokines production.     

The respiratory chain of the facultative thermophile,Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa ₃ and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa ₃ was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (_max 558 and _max 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.