Measuring and interpreting respiratory critical oxygen pressures in roots

BACKGROUND AND AIMS: Respiratory critical oxygen pressures (COPR) determined from O(2)-depletion rates in media bathing intact or excised roots are unreliable indicators of respiratory O(2)-dependency in O(2)-free media and wetlands. A mathematical model was used to help illustrate this, and more relevant polarographic methods for determining COPR in roots of intact plants are discussed. METHODS: Cortical [O(2)] near the root apex was monitored indirectly (pea seedlings) from radial oxygen losses (ROL) using sleeving Pt electrodes, or directly (maize) using microelectrodes; [O(2)] in the root was controlled by manipulating [O(2)] around the shoots. Mathematical modelling of radial diffusive and respiratory properties of roots used Michaelis-Menten enzyme kinetics. KEY RESULTS: Respiration declined only when the O(2) partial pressure (OPP) in the cortex of root tips fell below 0.5-4.5 kPa, values consistent with depressed respiration near the centre of the stele as confirmed by microelectrode measurements and mathematical modelling. Modelling predictions suggested that the OPP of a significant core at the centre of roots could be below the usual detection limits of O(2)-microelectrodes but still support some aerobic respiration. CONCLUSIONS: In O(2)-free media, as in wetlands, the COPR for roots is likely to be quite low, dependent upon the respiratory demands, dimensions and diffusion characteristics of the stele/stelar meristem and the enzyme kinetics of cytochrome oxidase. Roots of non-wetland plants may not differ greatly in their COPRs from those of wetland species. There is a possibility that trace amounts of O(2) may still be present in stelar 'anaerobic' cores where fermentation is induced at low cortical OPPs.     

Studies on the effects of copper deficiency on rat liver mitochondria. II. Effects on oxidative phosphorylation

Effects of dietary copper deficiency in rats on respiratory enzymes of isolated rat liver mitochondria have been studied. After 2 weeks of Cu-depletion, cytochrome c oxidase (EC 1.9.3.1) activity had declined by 42% and between 4 and 8 weeks exhibited between 20 and 25% of the activity of control mitochondria. Activities of NADH cytochrome c reductase (EC 1.6.99.3) and succinate cytochrome c reductase (EC 1.3.99.1), were unaffected initially but declined by 32 and 46%, respectively, after 8 weeks of Cu-depletion. After 4 weeks there was a significant (34%) decline in succinate supported state 3 respiration with only a modest (18%) decline in state 4 respiration. The ADP:O ratio was unaffected by Cu-depletion after 6 and 8 weeks of dietary Cu-restriction. State 3 respiration was significantly reduced after 6 weeks when glutamate/malate or beta-hydroxybutyrate were used as substrates, whereas state 4 respiration and ADP:O ratios were unaffected. The fall in state 3 respiration was of sufficient magnitude at 8 weeks to cause a significant decline in the respiratory control ratio with all substrates. Comparisons between the relative activities of cytochrome c oxidase and reductase activities in Cu-deficient preparations, the relatively specific effect of the deficiency on state 3 respiration with all substrates tested and the ability to increase significantly oxygen consumption in excess of maximal state 3 respiration by the uncoupler 2,4-dinitrophenol suggest that the defect in Cu-deficient mitochondria cannot be attributed solely to the decreased activity of cytochrome c oxidase.     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

The role of oxidized cytochrome c in regulating mitochondrial reactive oxygen species production and its perturbation in ischaemia

Oxidized cytochrome c is a powerful superoxide scavenger within the mitochondrial IMS (intermembrane space), but the importance of this role in situ has not been well explored. In the present study, we investigated this with particular emphasis on whether loss of cytochrome c from mitochondria during heart ischaemia may mediate the increased production of ROS (reactive oxygen species) during subsequent reperfusion that induces mPTP (mitochondrial permeability transition pore) opening. Mitochondrial cytochrome c depletion was induced in vitro with digitonin or by 30 min ischaemia of the perfused rat heart. Control and cytochrome c-deficient mitochondria were incubated with mixed respiratory substrates and an ADP-regenerating system (State 3.5) to mimic physiological conditions. This contrasts with most published studies performed with a single substrate and without significant ATP turnover. Cytochrome c-deficient mitochondria produced more H?O? than control mitochondria, and exogenous cytochrome c addition reversed this increase. In the presence of increasing [KCN] rates of H?O? production by both pre-ischaemic and end-ischaemic mitochondria correlated with the oxidized cytochrome c content, but not with rates of respiration or NAD(P)H autofluorescence. Cytochrome c loss during ischaemia was not mediated by mPTP opening (cyclosporine-A insensitive), neither was it associated with changes in mitochondrial Bax, Bad, Bak or Bid. However, bound HK2 (hexokinase 2) and Bcl-xL were decreased in end-ischaemic mitochondria. We conclude that cytochrome c loss during ischaemia, caused by outer membrane permeabilization, is a major determinant of H?O? production by mitochondria under pathophysiological conditions. We further suggest that in hypoxia, production of H?O? to activate signalling pathways may be also mediated by decreased oxidized cytochrome c and less superoxide scavenging.     

A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum.

It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.     

Oxyconformity in the intertidal worm Sipunculus nudus: the mitochondrial background and energetic consequences

The energetic consequences of strict oxyconformity in the intertidal worm S. nudus were studied by characterizing the Po2 dependence of respiration in mitochondria isolated from the body wall tissue. Mitochondrial respiration rose in a Po2 range between 2.8 and 31.3 kPa from a mean of 56.5 to 223.9 nmol O mg protein(-1) h(-1). Respiration was sensitive to both salicylhydroxamic acid (SHAM) and KCN. Po2 dependence remained unchanged with saturating and non-saturating substrate levels (malate, glutamate and ADP). A concomitant decrease of the ATP/O ratio revealed a lower ATP yield of aerobic metabolism at elevated Po2. Obviously, oxyconforming respiration implies progressive uncoupling of mitochondria. The decrease in ATP/O ratios at higher Po2 was completely reversible. Addition of 90.9 micromol H2O2 l(-1) did not inhibit ATP synthesis. Both observations suggest that oxidative injury did not contribute to oxyconformity. The contribution of the rates of mitochondrial ROS production and proton leakiness to mitochondrial oxygen consumption and uncoupling was investigated by using oligomycin as a specific inhibitor of the ATP synthase. The maximum contribution of oligomycin independent respiration to state 3 respiration remained below 6% and showed a minor, insignificant increase at elevated Po2, at a slope significantly lower than the increment of state 3 respiration. Therefore, Po2 dependent mitochondrial proton leakage or ROS production cannot explain oxyconformity. In conclusion Po2 dependent state 3 respiration likely relates to the progressive contribution of an alternative oxidase (cytochrome o), which is characterized by a low affinity to oxygen and an ATP/O ratio similar to the branched respiratory system of bacteria. The molecular nature of the alternative oxidase in lower invertebrates is still obscure.     

Two free radical pathways mediate chemical hypoxia-induced glutamate release in synaptosomes from the prefrontal cortex

It has been known that the inhibition of mitochondrial cytochrome c oxidase is one of the earliest events occurring under hypoxia and this inhibition can lead to neuronal damages. Thus, the cytochrome c oxidase inhibitor sodium cyanide (NaCN) is widely used to produce a model of chemical hypoxia by inhibiting this enzyme. However, the downstream signaling pathways of the inhibition of the cytochrome c oxidase remain to be studied. In the present paper, we used sodium cyanide to mimic the inhibition of the mitochondrial cytochrome c oxidase and studied its effect on glutamate release in synaptosomes from the prefrontal cortex using on-line fluorimetry. We also further investigated the mechanisms underlying the enhancing effect of sodium cyanide on glutamate release using pharmacological approaches combined with other techniques. The results showed that sodium cyanide significantly increased glutamate release from synaptosomes of prefrontal cortex; the broad-spectrum free radical scavenger MnTBAP and melatonin completely abolished the effect of sodium cyanide on glutamate release; the H2O2-NMDA receptor pathway mediated one part, whereas the lipid peroxyl radicals-ATP synthase pathway mediated another part of the sodium cyanide-induced glutamate release; scavenging H2O2 and enhancing ATP synthase activity could completely abolish the sodium cyanide-induced glutamate release.     

The reduction of cytochrome b558 and the activity of the respiratory burst oxidase from human neutrophils.

It is known that in respiratory burst oxidase preparations engaged in O2- production, cytochrome b558, a characteristic oxidase component, is partly reduced. This result has been interpreted in terms of a mechanism in which cytochrome b558 functions as an electron-carrying component of the respiratory burst oxidase, its level of reduction reflecting a steady-state partitioning of the cytochrome between reduced and oxidized forms as it ferries electrons from NADPH to oxygen. Kinetic arguments based on this interpretation have supported the proposal that the cytochrome is reduced at a rate sufficient to account for the rate of O2- production by activated neutrophils. We have confirmed the partial reduction of cytochrome b558 in neutrophil cytoplasts and in oxidase preparations exposed to NADPH, but have found that the reduction of the cytochrome bears no apparent relation to the activity of the oxidase, and can occur when NADPH is added to neutrophil membrane preparations that are unable to manufacture O2-. We therefore conclude that the NADPH-dependent reduction of cytochrome b558 seen in these preparations is unlikely to be a reflection of a catalysis-related steady state and that inferences drawn from such observations regarding the kinetic competence of the cytochrome may need to be reconsidered.     

Endogenous and endobiotic induced reactive oxygen species formation by isolated hepatocytes.

The rat hepatocyte catalyzed oxidation of 2',7'-dichlorofluorescin to form the fluorescent 2,7'-dichlorofluorescein was used to measure endogenous and xenobiotic-induced reactive oxygen species (ROS) formation by intact isolated rat hepatocytes. Various oxidase substrates and inhibitors were then used to identify the intracellular oxidases responsible. Endogenous ROS formation was markedly increased in catalase-inhibited or GSH-depleted hepatocytes, and was inhibited by ROS scavengers or desferoxamine. Endogenous ROS formation was also inhibited by cytochrome P450 inhibitors, but was not affected by oxypurinol, a xanthine oxidase inhibitor, or phenelzine, a monoamine oxidase inhibitor. Mitochondrial respiratory chain inhibitors or hypoxia, on the other hand, markedly increased ROS formation before cytotoxicity ensued. Furthermore, uncouplers of oxidative phosphorylation inhibited endogenous ROS formation. This suggests endogenous ROS formation can largely be attributed to oxygen reduction by reduced mitochondrial electron transport components and reduced cytochrome P450 isozymes. Addition of monoamine oxidase substrates increased antimycin A-resistant respiration and ROS formation before cytotoxicity ensued. Addition of peroxisomal substrates also increased antimycin A-resistant respiration but they were less effective at inducing ROS formation and were not cytotoxic. However, peroxisomal substrates readily induced ROS formation and were cytotoxic towards catalase-inhibited hepatocytes, which suggests that peroxisomal catalase removes endogenous H(2)O(2) formed in the peroxisomes. Hepatocyte catalyzed dichlorofluorescin oxidation induced by oxidase substrates, e.g., benzylamine, was correlated with the cytotoxicity induced in catalase-inhibited hepatocytes.     

Azorhizobium caulinodans uses both cytochrome bd (quinol) and cytochrome cbb3 (cytochrome c) terminal oxidases for symbiotic N2 fixation.

Azorhizobium caulinodans employs both cytochrome bd (cytbd; quinol oxidase) and cytcbb3 (cytc oxidase) as terminal oxidases in environments with very low O2 concentrations. To investigate physiological roles of these two terminal oxidases both in microaerobic culture and in symbiosis, knockout mutants were constructed. As evidenced by visible absorbance spectra taken from mutant bacteria carrying perfect gene replacements, both the cytbd- and cytcbb3- mutations were null alleles. In aerobic culture under 2% O2 atmosphere, Azorhizobium cytbd- and cytcbb3- single mutants both fixed N2 at 70 to 90% of wild-type rates; in root nodule symbiosis, both single mutants fixed N2 at 50% of wild-type rates. In contrast, Azorhizobium cytbd- cytcbb3-double mutants, which carry both null alleles, completely lacked symbiotic N2 fixation activity. Therefore, both Azorhizobium cytbd and cytcbb3 oxidases drive respiration in environments with nanomolar O2 concentrations during symbiotic N2 fixation. In culture under a 2% O2 atmosphere, Azorhizobium cytbd- cytcbb3- double mutants fixed N2 at 70% of wild-type rates, presumably reflecting cytaa3 and cytbo (and other) terminal oxidase activities. In microaerobic continuous cultures in rich medium, Azorhizobium cytbd- and cytcbb3- single mutants were compared for their ability to deplete a limiting-O2 sparge; cytbd oxidase activity maintained dissolved O2 at 3.6 microM steady state, whereas cytcbb3 oxidase activity depleted O2 to submicromolar levels. Growth rates reflected this difference; cytcbb3 oxidase activity disproportionately supported microaerobic growth. Paradoxically, in O2 limited continuous culture, Azorhizobium cytbd oxidase is inactive below 3.6 microM dissolved O2 whereas in Sesbania rostrata symbiotic nodules, in which physiological, dissolved O2 is maintained at 10 to 20 nM, both Azorhizobium cytbd and cytcbb3 seem to contribute equally as respiratory terminal oxidases.     

Changes in mitochondrial electron partitioning in response to herbicides inhibiting branched-chain amino acid biosynthesis in soybean

The adaptation of the respiratory metabolism in roots of soybean (Glycine max L. Merr. cv Ransom) treated with herbicides that inhibit the enzyme acetolactate synthase (ALS) was analyzed. A new gas phase dual-inlet mass spectrometry system for simultaneous measurement of 34O2 to 32O2 and O2 to N2 ratios has been developed. This system is more accurate than previously described systems, allows measurements of much smaller oxygen gradients, and, as a consequence, works with tissues that have lower respiration rates. ALS inhibition caused an increase of the alternative oxidase (AOX) protein and an accumulation of pyruvate. The combination of these two effects is likely to induce the activation of the alternative pathway and its participation in the total respiration. Moreover, the start of the alternative pathway activation and the increase of AOX protein were before the decline in the activity of cytochrome pathway. The possible role of AOX under ALS inhibition is discussed.     

Respiratory pathways in aged soybean seeds

Respiratory activities in soybean seeds (Glvcine max (L.) Merr. cv. Chippewa 64) have been examined in the first minutes after water imbibition and after three hours of imbibition, using either particles or intact axes. Cyanide and azide were utilized as inhibitors of the cytochrome oxidase pathway of respiration, and SHAM inhibitions were interpreted as effects on the alternative pathway, since in unaged axes inhibitions by SHAM were obtained only when respiratory activity had either been restricted with inhibitors of the cytochrome oxidase pathway or expanded by an uncoupler (CCCP). From the experiments with these inhibitors, it is suggested that unaged seeds utilize both respiration pathways in the cotyledon but only the cytochrome pathway in the axis. Accelerated aging causes a marked deterioration of respiration, especially that through the cytochrome pathway, and there is an associated engagement of the alternative pathway in the seed axis. It is suggested that the lowering of respiratory activity and the shift in respiratory pathways may play a major role in the decline of germinability and vigor.     

Role of MicroRNA Processing in Adipose Tissue in Stress Defense and Longevity

The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner?mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S.?cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of?a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.     

Adapting High-Resolution Respirometry to Glucose-Limited Steady State Mycelium of the Filamentous Fungus Penicillium ochrochloron: Method Development and Standardisation

Fungal electron transport systems (ETS) are branched, involving alternative NADH dehydrogenases and an alternative terminal oxidase. These alternative respiratory enzymes were reported to play a role in pathogenesis, production of antibiotics and excretion of organic acids. The activity of these alternative respiratory enzymes strongly depends on environmental conditions. Functional analysis of fungal ETS under highly standardised conditions for cultivation, sample processing and respirometric assay are still lacking. We developed a highly standardised protocol to explore in vivo the ETS—and in particular the alternative oxidase—in Penicillium ochrochloron. This included cultivation in glucose-limited chemostat (to achieve a defined and reproducible physiological state), direct transfer without any manipulation of a broth sample to the respirometer (to maintain the physiological state in the respirometer as close as possible to that in the chemostat), and high-resolution respirometry (small sample volume and high measuring accuracy). This protocol was aimed at avoiding any changes in the physiological phenotype due to the high phenotypic plasticity of filamentous fungi. A stable oxygen consumption (< 5% change in 20 minutes) was only possible with glucose limited chemostat mycelium and a direct transfer of a broth sample into the respirometer. Steady state respiration was 29% below its maximum respiratory capacity. Additionally to a rotenone-sensitive complex I and most probably a functioning complex III, the ETS of P. ochrochloron also contained a cyanide-sensitive terminal oxidase (complex IV). Activity of alternative oxidase was present constitutively. The degree of inhibition strongly depended on the sequence of inhibitor addition. This suggested, as postulated for plants, that the alternative terminal oxidase was in dynamic equilibrium with complex IV—independent of the rate of electron flux. This means that the onset of activity does not depend on a complete saturation or inhibition of the cytochrome pathway.     

Control of mitochondrial respiration by NO*, effects of low oxygen and respiratory state

Nitric oxide (NO.) inhibits mitochondrial respiration by binding to the binuclear heme a3/CuB center in cytochrome c oxidase. However, the significance of this reaction at physiological O2 levels (5-10 microM) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O2], and [NO.] were measured simultaneously at physiological O2 levels with constant O2 delivery, to model in vivo respiratory dynamics. Under these conditions NO. inhibited mitochondrial respiration with an IC50 of 0.14 +/- 0.01 microm in state 3 versus 0.31 +/- 0.04 microM in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO. is due to greater control over respiration by an NO.-dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO. in vivo and its implications for the control of vessel-parenchymal O2 gradients.     

The anesthetic nitrous oxide affects dioxygen utilization by bovine heart and bean seed mitochondrial particles

Nitrous oxide affects dioxygen utilization by both bean seed and bovine heart submitochondrial particles when either succinate or reduced cytochrome c are used as substrates. Bovine heart particles exhibit reversible, dose-dependent partial inhibition of respiratory activity when exposed to N2O. Bean seed particle respiration is stimulated by low levels of N2O, but higher concentrations are inhibitory. These findings can be explained in terms of one locus of anesthetic action: cytochrome c oxidase, the terminal component of the mitochondrial respiratory chain. Alterations in respiration rates are expected to make important contributions to anesthesia in animals and to control of germination in plants.     

Involvement of the alternative oxidase in respiration of Yarrowia lipolytica mitochondria is controlled by the activity of the cytochrome pathway

The degree of involvement of cyanide-resistant alternative oxidase in the respiration of Yarrowia lipolytica mitochondria was evaluated by comparing the rate of oxygen consumption in the presence of cyanide, which shows the activity of the cyanide-resistant alternative oxidase, and the oxidation rate of cytochrome c by ferricyanide, which shows the activity of the main cytochrome pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was associated with oxygen consumption due to the functioning of the alternative oxidase. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation) completely inhibited oxygen consumption by the mitochondria. Under these conditions, the inhibition of the alternative oxidase by benzohydroxamic acid (BHA) failed to affect the reduction of ferricyanide at the level of cytochrome c. BHA did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These data indicate that the alternative system is unable to compete with the cytochrome respiratory chain for electrons. The alternative oxidase only transfers the electrons that are superfluous for the cytochrome respiratory chain.