Differential inactivation of Escherichia coli membrane dehydrogenases by a myeloperoxidase-mediated antimicrobial system.

Neutrophil myeloperoxidase, hydrogen peroxide, and chloride constitute a potent antimicrobial system with multiple effects on microbial cytoplasmic membranes. Among these is inhibition of succinate-dependent respiration mediated, principally, through inactivation of succinate dehydrogenase. Succinate-dependent respiration is inhibited at rates that correlate with loss of microbial viability, suggesting that loss of respiration might contribute to the microbicidal event. Because respiration in Escherichia coli can be mediated by dehydrogenases other than succinate dehydrogenase, the effects of the myeloperoxidase system on other membrane dehydrogenases were evaluated by histochemical activity stains of electrophoretically separated membrane proteins. Two bands of succinate dehydrogenase activity proved the most susceptible to inactivation with complete loss of staining activity within 20 min, under the conditions employed. A group with intermediate susceptibility, consisting of lactate, malate, glycerol-3-phosphate, and dihydroorotate dehydrogenases as well as three bands of glucose-6-phosphate dehydrogenase, was almost completely inactivated within 30 min. The relatively resistant group, including the dehydrogenases for glutamate, NADH, and NADPH and the remaining bands of glucose-6-phosphate dehydrogenase, retained substantial amounts of diaphorase activity for up to 60 min of incubation with the myeloperoxidase system. The differential effects of myeloperoxidase on dehydrogenase inactivation could not be correlated with published enzyme contents of flavin or iron-sulfur centers, potential targets of myeloperoxidase-derived oxidants. Despite the relative resistance of NADH dehydrogenase/diaphorase activity to myeloperoxidase-mediated inactivation, electron transport particles prepared from E. coli incubated for 20 min with the myeloperoxidase system lost 55% of their NADH oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competition of electrons to enter the respiratory chain: a new regulatory mechanism of oxidative metabolism in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are the external NADH dehydrogenases (Nde1p and Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle. In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p. Subsequently, glycerol 3-phosphate donates electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p). At saturating concentrations of NADH, the activation of external NADH dehydrogenases completely inhibits glycerol 3-phosphate oxidation. Studies on the functionally isolated enzymes demonstrated that neither Nde1p nor Nde2p directly inhibits Gut2p. Thus, the inhibition of glycerol 3-phosphate oxidation may be caused by competition for the entrance of electrons into the respiratory chain. Using single deletion mutants of Nde1p or Nde2p, we have shown that glycerol 3-phosphate oxidation via Gut2p is inhibited fully when NADH is oxidized via Nde1p, whereas only 50% of glycerol 3-phosphate oxidation is inhibited when Nde2p is functioning. By comparing respiratory rates with different respiratory substrates, we show that electrons from Nde1p are favored over electrons coming from Ndip (internal NADH dehydrogenase) and that when electrons come from either Nde1p or Nde2p and succinodehydrogenase, their use by the respiratory chain is shared to a comparable extent. This suggests a very specific competition for electron entrance into the respiratory chain, which may be caused by the supramolecular organization of the respiratory chain. The physiological consequences of such regulation are discussed.     

Idebenone-induced recovery of glycerol-3-phosphate and succinate oxidation inhibited by digitonin

Digitonin solubilizes mitochondrial membrane, breaks the integrity of the respiratory chain and releases two mobile redox-active components: coenzyme Q (CoQ) and cytochrome c (cyt c). In the present study we report the inhibition of glycerol-3-phosphate- and succinate-dependent oxygen consumption rates by digitonin treatment. Our results show that the inhibition of oxygen consumption rates is recovered by the addition of exogenous synthetic analog of CoQ idebenone (hydroxydecyl-ubiquinone; IDB) and cyt c. Glycerol-3-phosphate oxidation rate is recovered to 148 % of control values, whereas succinate-dependent oxidation rate only to 68 %. We find a similar effect on the activities of glycerol-3-phosphate and succinate cytochrome c oxidoreductase. Our results also indicate that succinate-dependent oxidation is less sensitive to digitonin treatment and less activated by IDB in comparison with glycerol-3-phosphate-dependent oxidation. These findings might indicate the different mechanism of the electron transfer from two flavoprotein-dependent dehydrogenases (glycerol-3-phosphate dehydrogenase and succinate dehydrogenase) localized on the outer and inner face of the inner mitochondrial membrane, respectively.     

Modes of coenzyme Q function in electron transfer

Summary In the mitochondrial respiratory chain, coenzyme Q acts in different ways. A diffusable coenzyme Q pool as a common substrate-like intermediate links the low-potential complexes with complex III. Its diffusion in the lipids is not rate-limiting for electron transfer, but its content is not saturating for maximal rate of NADH oxidation. Protein-bound coenzyme Q is involved in energy conservation, and may be part of enzyme supercomplexes, as in succinate cytochromec reductase. The reason for lack of kinetic saturation of the respiratory chain by quinone concentration is in the low extent of solubility of monomeric coenzyme Q in the membrane lipids. Assays of respiratory enzymes are performed using water soluble coenzyme Q homologs and analogs; several problems exist in using oxidized quinones as acceptors of coenzyme Q reductases. In particular, for complex I no acceptor appears to favorably substitute the endogenous quinone. In addition, quinone reduction sites in complex III compete with the sites in the dehydrogenases, particularly when using duroquinone. The different extent by which these sites operate when different donor substrates (NADH, succinate, glycerol-3-phosphate) are used is best explained by different exposure of the quinone acceptor sites in the dehydrogenases.     

In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria

During respiratory glucose dissimilation, eukaryotes produce cytosolic NADH via glycolysis. This NADH has to be reoxidized outside the mitochondria, because the mitochondrial inner membrane is impermeable to NADH. In Saccharomyces cerevisiae, this may involve external NADH dehydrogenases (Nde1p or Nde2p) and/or a glycerol-3-phosphate shuttle consisting of soluble (Gpd1p or Gpd2p) and membrane-bound (Gut2p) glycerol-3-phosphate dehydrogenases. This study addresses the physiological relevance of these mechanisms and the possible involvement of alternative routes for mitochondrial oxidation of cytosolic NADH. Aerobic, glucose-limited chemostat cultures of a gut2Delta mutant exhibited fully respiratory growth at low specific growth rates. Alcoholic fermentation set in at the same specific growth rate as in wild-type cultures (0.3 h(-1)). Apparently, the glycerol-3-phosphate shuttle is not essential for respiratory glucose dissimilation. An nde1Delta nde2Delta mutant already produced glycerol at specific growth rates of 0.10 h(-1) and above, indicating a requirement for external NADH dehydrogenase to sustain fully respiratory growth. An nde1Delta nde2Delta gut2Delta mutant produced even larger amounts of glycerol at specific growth rates ranging from 0.05 to 0.15 h(-1). Apparently, even at a low glycolytic flux, alternative mechanisms could not fully replace the external NADH dehydrogenases and glycerol-3-phosphate shuttle. However, at low dilution rates, the nde1Delta nde2Delta gut2Delta mutant did not produce ethanol. Since glycerol production could not account for all glycolytic NADH, another NADH-oxidizing system has to be present. Two alternative mechanisms for reoxidizing cytosolic NADH are discussed: (i) cytosolic production of ethanol followed by its intramitochondrial oxidation and (ii) a redox shuttle linking cytosolic NADH oxidation to the internal NADH dehydrogenase.     

Kinetic regulation of the mitochondrial glycerol-3-phosphate dehydrogenase by the external NADH dehydrogenase in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the two most important systems for conveying excess cytosolic NADH to the mitochondrial respiratory chain are external NADH dehydrogenase (Nde1p/Nde2p) and the glycerol-3-phosphate dehydrogenase shuttle. In the latter system, NADH is oxidized to NAD+ and dihydroxyacetone phosphate is reduced to glycerol 3-phosphate by the cytosolic Gpd1p; glycerol 3-phosphate gives two electrons to the respiratory chain via mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p)-regenerating dihydroxyacetone phosphate. Both Nde1p/Nde2p and Gut2p are located in the inner mitochondrial membrane with catalytic sites facing the intermembranal space. In this study, we showed kinetic interactions between these two enzymes. First, deletion of either one of the external dehydrogenases caused an increase in the efficiency of the remaining enzyme. Second, the activation of NADH dehydrogenase inhibited the Gut2p in such a manner that, at a saturating concentration of NADH, glycerol 3-phosphate is not used as respiratory substrate. This effect was not a consequence of a direct action of NADH on Gut2p activity because both NADH dehydrogenase and its substrate were needed for Gut2p inhibition. This kinetic regulation of the activity of an enzyme as a function of the rate of another having a similar physiological function may be allowed by their association into the same supramolecular complex in the inner membrane. The physiological consequences of this regulation are discussed.     

An Escherichia coli mutant conditionally altered in respiratory chain components

Nitrosoguanidine mutagenesis was employed to isolate an Escherichia coli mutant conditionally altered in respiratory chain components. Mutant R25 was able to grow on glucose, fructose, and glycerol but failed to grow on succinate and acetate (suc-). Also, R25 exhibited leaky growth on DL-lactate, fumarate, and malate (lct*). The lct* mutation pleiotropically affected a number of respiratory chain components and its expression was conditional with the growth substrate. Glucose-grown R25 resting cell suspensions oxidized DL-lactate and formate; however, these two substrates were not oxidized by fructose- or glycerol-grown cell suspensions. The same conditional pattern was observed for the concentration of cytochrome components, the membrane-associated oxidation of NADH and formate, and formate phenazine methosulfate (PMS) reductase activity; succinate oxidase and PMS reductase activities were not exhibited by membranes under any growth condition due to the suc- mutation. R25 membrane-associated H(+)-translocating ATPase activity was not conditional with the growth substrate. R25PC, a spontaneous lct+ suc- partial revertant of R25, did not exhibit the conditional pattern of R25. The lct* mutation was found to map in the 27-30-min region and the suc- mutation in the 15-17-min region of the E. coli genome. Two distinct classes of R25 P1kc transductants were isolated that differed in both their growth response on succinate and DL-lactate and their oxidase activities.     

The mitochondrial respiratory chain of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill

Rhizopus stolonifer (Ehrenb.:Fr.) Vuill mitochondria contain the complete system for oxidative phosphorylation, formed by the classical components of the electron transport chain (complexes I, II, III, and IV) and the F₁F₀-ATP synthase (complex V). Using the native gel electrophoresis, we have shown the existence of supramolecular associations of the respiratory complexes. The composition and stoichiometry of the oxidative phosphorylation complexes were similar to those found in other organisms. Additionally, two alternative routes for the oxidation of cytosolic NADH were identified: the alternative NADH dehydrogenase and the glycerol-3-phosphate shuttles. Residual respiratory activity after inhibition of complex IV by cyanide was inhibited by low concentrations of n-octyl gallate, indicating the presence of an alternative oxidase. The K_0.5 for the respiratory substrates NADH, succinate, and glycerol-3-phosphate in permeabilized cells was higher than in isolated mitochondria, suggesting that interactions of mitochondria with other cellular elements might be important for the function of this organelle.     

The stereospecificity of oxidation of alpha-[4R-2H]NADH by dehydrogenases

The stereospecificity of the enzyme-dependent oxidation of alpha-[4R-2H]NADH has been determined for four dehydrogenases: two pro-R specific enzymes, pig heart malate dehydrogenase and yeast alcohol dehydrogenase; and two pro-S specific enzymes, rabbit muscle glycerol-3-phosphate dehydrogenase and Rhodopseudomonas spheroides 3-hydroxybutyrate dehydrogenase. In all cases, an enzyme-dependent and substrate-specific oxidation to alpha-NAD+ is observed with the stereochemistry of oxidation identical with that found for the oxidation of the correspondingly labeled beta-NADH. The ability of dehydrogenases from diverse sources to utilize alpha-NADH in a stereochemically competent fashion is discussed in relation to proposed interactions between the nicotinamide sugar moiety and active site residues or obligatory alignments of the pyridine and sugar moieties.     

Enzyme activity and content of glycerophosphate shuttle metabolites in the cortex and medulla oblongata during hyperbaric oxygenation therapy of acute hemorrhage

The experiments on cats have demonstrated that hyperbaric oxygenation (303.9 kPa, 60 min) in acute blood loss (24.0 +/- 0.8 ml/kg) has an essential effect on glycerophosphate shuttle mechanism: it stimulates mitochondrial glycerol-3-phosphate dehydrogenase in the sensorimotor cortex and medulla oblongata, providing glycerol-3-phosphate dehydrogenation, activates cytoplasm hydrogen delivery to mitochondrial respiratory chain, prevents glycerol-3-phosphate and NADH level increase and cytoplasm glycerol-3-phosphate dehydrogenase inhibition, limiting lactate production.     

Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.     

Effects of short-term ozone exposure on lung mitochondrial oxidative and energy metabolism

Ozone effects on lung mitochondrial oxidative metabolism were examined after short-term exposure of rats and monkeys to O₃. Exposure of animals to 2 ppm O₃ for 8 hr or to 4 ppm O₃ for 4 hr caused a 15–27% (P < 0.05) depression of lung mitochondrial O₂ consumption, using 2-oxoglutarate, succinate, and glycerol-1-phosphate. but not ascorbate plus Wurster's blue as substrates. Under these exposure conditions (4 ppm 4 hr) the ADP:O ratios dropped 25–36% (P < 0.05) and the respiratory control indices decreased 27–33% (P < 0.02) for oxidation of all substrates examined. Lung mitochondria from control animals were relatively impermeable to added NADH, but those from O₃-exposed animals showed an increased permeability as judged from NADH oxidation at a rate 3-fold higher than the control. Likewise, added cytochrome c caused a 22% (P < 0.01) stimulation of succinate oxidation in exposed lung mitochondria as against 5% (nonsignificant) in controls. Ozone exposure also caused a 20% (P < 0.01) oxidation of thiol groups in lung mitochondria, but no lipid peroxidation products were detectable in O₃-exposed lung tissue. The depression of substrate utilization, coupled phosphorylation and respiratory control observed in lung mitochondria of O₃-exposed animals might be related to alteration of membrane permeability, and inhibition of respiratory enzymes (dehydrogenases) due to oxidation of functional thiol groups.     

Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae

Keeping a cytosolic redox balance is a prerequisite for living cells in order to maintain a metabolic activity and enable growth. During growth of Saccharomyces cerevisiae, an excess of NADH is generated in the cytosol. Aerobically, it has been shown that the external NADH dehydrogenase, Nde1p and Nde2p, as well as the glycerol-3-phosphate dehydrogenase shuttle, comprising the cytoplasmic glycerol-3-phosphate dehydrogenase, Gpdlp, and the mitochondrial glycerol-3-phosphate dehydrogenase, Gut2p, are the most important mechanisms for mitochondrial oxidation of cytosolic NADH. In this review we summarize the recent results showing (i) the contribution of each of the mechanisms involved in mitochondrial oxidation of the cytosolic NADH, under different physiological situations; (ii) the kinetic and structural properties of these metabolic pathways in order to channel NADH from cytosolic dehydrogenases to the inner mitochondrial membrane and (iii) the organization in supramolecular complexes and, the peculiar ensuing kinetic regulation of some of the enzymes (i.e. Gut2p inhibition by external NADH dehydrogenase activity) leading to a highly integrated functioning of enzymes having a similar physiological function. The cell physiological consequences of such an organized and regulated network are discussed.     

Cooperative Substrate Oxidation by Mitochondria from Castor Bean Hypocotyls

Cooperative oxidation of succinate and exogenous NADH was followed in the mitochondria from five- to six-day-old castor bean (Ricinus communisL.) seedlings. Although succinate was oxidized at a much higher rate than NADH, the former inconsiderably (less than 15%) inhibited the oxidation of the latter substrate in state 4, while, in state 3 (in the presence of ATP), the two substrates did not compete and were jointly oxidized. When two substrates were oxidized by the mitochondria with the alternative CN-resistant oxidase (AO) inhibited with salicylhydroxamic acid, the rate of NADH oxidation in state 4 dropped by over 40% as compared to the initial rate. Meanwhile, the rate of succinate oxidation was not considerably affected by AO inhibition. We believe that one of the AO functions in the mitochondria is to provide for noncompeting oxidation of two (or more) substrates by employing two (or several) dehydrogenases of the respiratory chain.     

Effect of inhibitors on the substrate-dependent quenching of 9-aminoacridine fluorescence in inside-out membrane vesicles of Escherichia coli.

The effect of various inhibitors on the substrate-dependent quenching of the fluorescence of 9-aminoacridine was measured in inside-out membrane vesicles of Escherichia coli. The rate of fluorescence quenching in the presence of inhibitors was dependent on the rate of electron transfer through the respiratory chain with NADH, succinate, D-lactate or DL-glycerol 3-phosphate as substrates. Several patterns of response were given by the inhibitors. Inhibitors competitive with substrate, or those acting only on the dehydrogenases, gave a direct relationship between the extent of inhibition of oxidase activity and the rate of quenching. A biphasic relationship was given by 2-heptyl-4-hydroxyquinoline N-oxide and piericidin A which was due to these compounds acting both as inhibitors of the respiratory chain and, at higher concentrations, as uncoupling agents. Uncouplers inhibited fluorescence quenching with minimal inhibition of oxidase activity. The transmembrane pH difference was calculated from the extent of fluorescence quenching and the intravesicular volume. The maximum pH difference of 3.3--3.7 units was generated by each of the substrates tested.     

Ca2+-binding and Ca2+-independent respiratory NADH and NADPH dehydrogenases of Arabidopsis thaliana

Type II NAD(P)H:quinone oxidoreductases are single polypeptide proteins widespread in the living world. They bypass the first site of respiratory energy conservation, constituted by the type I NADH dehydrogenases. To investigate substrate specificities and Ca(2+) binding properties of seven predicted type II NAD(P)H dehydrogenases of Arabidopsis thaliana we have produced them as T7-tagged fusion proteins in Escherichia coli. The NDB1 and NDB2 enzymes were found to bind Ca(2+), and a single amino acid substitution in the EF hand motif of NDB1 abolished the Ca(2+) binding. NDB2 and NDB4 functionally complemented an E. coli mutant deficient in endogenous type I and type II NADH dehydrogenases. This demonstrates that these two plant enzymes can substitute for the NADH dehydrogenases in the bacterial respiratory chain. Three NDB-type enzymes displayed distinct catalytic profiles with substrate specificities and Ca(2+) stimulation being considerably affected by changes in pH and substrate concentrations. Under physiologically relevant conditions, the NDB1 fusion protein acted as a Ca(2+)-dependent NADPH dehydrogenase. NDB2 and NDB4 fusion proteins were NADH-specific, and NDB2 was stimulated by Ca(2+). The observed activity profiles of the NDB-type enzymes provide a fundament for understanding the mitochondrial system for direct oxidation of cytosolic NAD(P)H in plants. Our findings also suggest different modes of regulation and metabolic roles for the analyzed A. thaliana enzymes.     

Agrobacterium tumefaciens type II NADH dehydrogenase. Characterization and interactions with bacterial and thylakoid membranes

Type II NADH dehydrogenases (NDH-2) are monomeric enzymes that catalyse quinone reduction and allow electrons to enter the respiratory chain in different organisms including higher plant mitochondria, bacteria and yeasts. In this study, an Agrobacterium tumefaciens gene encoding a putative alternative NADH dehydrogenase (AtuNDH-2) was isolated and expressed in Escherichia coli as a (His)6-tagged protein. The purified 46 kDa protein contains FAD as a prosthetic group and oxidizes both NADH and NADPH with similar Vmax values, but with a much higher affinity for NADH than for NADPH. AtuNDH-2 complements the growth (on a minimal medium) of an E. coli mutant strain deficient in both NDH-1 and NDH-2, and is shown to supply electrons to the respiratory chain when incubated with bacterial membranes prepared from this mutant. By measuring photosystem II chlorophyll fluorescence on thylakoid membranes prepared from the green alga Chlamydomonas reinhardtii, we show that AtuNDH-2 is able to stimulate NADH-dependent reduction of the plastoquinone pool. We discuss the possibility of using heterologous expression of NDH-2 enzymes to improve nonphotochemical reduction of plastoquinones and H2 production in C. reinhardtii.     

Identification and characterization of a bacterial glycerol-1-phosphate dehydrogenase: Ni(2+)-dependent AraM from Bacillus subtilis

The exclusive presence of glycerol-1-phosphate dehydrogenases (G1PDH) has been postulated to be a key feature that distinguishes archaea from bacteria. However, homologues of G1PDH genes can be found in several bacterial species, among them the hitherto uncharacterized open reading frame araM from Bacillus subtilis. We produced recombinant AraM in Escherichia coli and demonstrate that the purified protein forms a homodimer that reversibly reduces dihydroxyacetone phosphate (DHAP) to glycerol-1-phosphate (G1P) in a NADH-dependent manner. AraM, which constitutes the first identified G1PDH from bacteria, has a similar catalytic efficiency as its archaeal homologues, but its activity is dependent on the presence of Ni (2+) instead of Zn (2+). On the basis of these findings and the analysis of an araM knockout mutant, we propose that AraM generates G1P for the synthesis of phosphoglycerolipids in Gram-positive bacterial species.     

Characterization of an Escherichia coli mutant pleiotropically altered in membrane-bound oxidoreductase activities

An Escherichia coli mutant pleiotropically altered in membrane-bound oxidoreductase activities was isolated following nitrosoguanidine treatment. Mutant R23 was able to grow on glucose, but was unable to grow on succinate or other oxidizable substrates as a sole energy source. Isolated membranes prepared from R23 failed to oxidize succinate and formate; while NADH was oxidized at a reduced rate by membranes. The mutant also exhibited markedly reduced cytochrome content, but normal DL-lactate PMS reductase and H(+)-translocating ATPase activities relative to the parent strain. Bacteriophage Plkc was used to transduce R23 to growth on glycerol, DL-lactate or succinate; regardless of the selection procedure, each of the 179 transductants had gained the ability to grow on all three substrates. The suc- mutation in R23 appeared to be responsible for the loss of growth on oxidizable substrates, altered membrane-bound oxidoreductase activities, resistance to neomycin, and reduced levels of cytochrome components. The suc- mutation was localized in the 6 to 6.5 min region of the E. coli chromosome map utilizing episomal transfers.     

Membrane-bound respiratory of Spirillum itersonii.

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