Differentiation of c, d1 cytochrome and copper nitrite reductase production in denitrifiers

Abstract Gas chromatographic analyses revealed that rates of release of nitrous oxide from nitrite or nitric oxide in extracts of the c , d ₁ cytochrome nitrite reductase-producing denitrifiers, Paracoccus denitrificans and Pseudomonas perfectomarina , were unaffected by preincubation with the metal chelator, diethyldithiocarbamate (DDC). In contrast, preincubation with DDC completely inhibited generation of nitrous oxide from nitrite in extracts of copper protein nitrite reductase-producing denitrifiers, " Achromobacter cycloclastes " and Rhodopseudomonas sphaeroides forma species denitrificans . Pre-exposure to DDC lessened but did not completely inhibit nitric oxide reduction in extracts of the copper protein nitrite reductase-producing denitrifiers. Proton consumption values resulting from pulsing with nitrite were similarly completely inhibited by preincubation with DDC of extracts of the two copper protein-producing denitrifiers. Uptake values related to pulsing with nitric oxide were also lessened but not completely inhibited by prior exposure to DDC. As anticipated, proton consumption was not affected by preincubation with DDC in extracts of P. denitrificans pulsed with nitrite or nitric oxide. Differential sensitivity of copper protein nitrite reductase activity to DDC could provide the simple assay method needed for determination of the distribution of two types of nitrite reductase producers among populations of denitrifiers in nature.     

Evidence for the role of soluble cytochrome c in the dissimilatory reduction of nitrite and nitrous oxide by cells of Paracoccus denitrificans.

The role of periplasmic cytochrome c in the denitrification pathway has been investigated using a wild-type and/or a cytochrome c deficient strain of Paracoccus denitrificans. The reconstitution experiments with the isolated proteins showed that bacterial cytochrome c-550 restored the electron transport from the cytoplasmic membrane to soluble nitrite reductase (cytochrome cd1). In response to decreased aeration lasting 3 h, the HUUG25 strain synthesized nitrous-oxide reductase significantly starved of electrons from the respiratory chain and only very small amounts of soluble cytochrome c. The membrane-bound part of the respiratory chain catalyzing the reduction of soluble cytochrome c resembled an autologous region in wild-type cells kinetically and by its sensitivity to antimycin. In the periplasmic fraction obtained from anaerobically grown wild-type cells N2O caused the reoxidation of endogenous cytochrome(s) c previously reduced by N,N,N',N' tetramethyl-p-phenylenediamine plus ascorbate. All these results indicate the involvement of soluble cytochrome(s) c as the electron donor(s) for the reduction of NO2- and N2O in the periplasmic space of cells.     

Aerobic dissimilatory reduction of nitrite by cells of Paracoccus denitrificans: the role of nitric oxide

With anaerobically grown cells of Paracoccus denitrificans it was previously found (Kučera, I. and Dadák, V. (1983) Biochem. Biophys. Res. Commun. 117, 252–258) that, in the presence of an uncoupler, nitrite as terminal acceptor was preferred to oxygen, the consumption of which was simultaneously inhibited. In the present study it is shown that besides an increased inhibition of terminal oxidases brought about by NO₂⁻ anion another potent inhibitor originating in the course of nitrite reductase reaction affects the division of electron flow between oxygen and nitrite. The inhibitor, the creation of which is accompanied by the aerobic nitrite reduction, is formed, even in the absence of an uncoupler, only as a result of a slight inhibition of oxygen respiration exhibited by hydroxylamine addition. From the comparison of the inhibitory effect of the intermediate on aerobically grown cells and membrane vesicles derived from them, it was proved that at neutral pH this substance does not carry an electric charge. A complex absorbing at 563 nm was formed due to the interaction of the inhibitor (generated from nitrite in the presence of uncoupler) with ferricytochrome c from bovine heart. From these findings we were led to conclude that it was most probably nitric oxide (NO).     

Respiratory Rate as a Regulatory Factor in the Biosynthesis of the Denitrification Pathway of the Bacterium Paracoccus denitrificans

The decrease in the electron flow of the aerobic respiratory chain of the bacterium Paracoccus denitrificans, owing to either the drop in the saturation of terminal oxidases by oxygen or to the inhibition of the rate of respiration by azide or nitrite, resulted in the synthesis of dissimilatory nitrate reductase and nitrite reductase. The dependence of the resulting activities of the two enzymes (after a three-hour adaptation) on the initial value of the parameter V_max/k_La (oxidase activity of the volume unit of the culture divided by the volumetric oxygen transfer coefficient) or on the concentrations of the inhibitors had a similar form, characterized by the appearance of a maximum. The increasing parts of the obtained curves reflect the synthesis of enzymes, probably initiated by the increase in the intracellular degree of reduction, the subsequent drop being evidently in connection with the lack of metabolic energy for biosynthesis. The possible mechanisms of the effect of nitrogenous terminal acceptors (NO^-₃ and NO^-₂) on the formation of the denitrification pathway are discussed.     

Anaerobic respiration and energy conservation in Paracoccus denitrificans. Functioning of iron-sulfur centers and the uncoupling effect of nitrite.

1. Electron paramagnetic resonance spectra at 8-60 K of NADH-reduced membrane particles prepared from Paracoccus denitrificans grown anaerobically with nitrate as terminal electron acceptor show the presence of iron-sulfur centers 1-4 in the NADH-ubiquinone segment of the respiratory chain. In addition resonance lines at g = 2.058, g = 1.953 and g = 1.88 are detectable in the spectra of succinate-reduced membranes at 15 K, which are attributed to the iron-sulfur-containing nitrate reductase. 2. Sulphate-limited growth under anaerobic conditions does not affect the iron-sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain-linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate-limited growth of P. denitrificans under aerobic conditions [Eur. J. Biochem. (1977) 81, 267-275]. 3. Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain-linked energy conservation observed during nitrate respiration [Arch. Microbiol. (1977) 112, 17-23] can be explained by the uncoupling action of nitrite.     

Nitrite reductase system involved in the terminal oxidation of the Streptomyces griseus respiratory particle.

A nitrite reductase system which was associated with the electron transfer system of the respiratory particle in Streptomyces griseus was studied. The electron transfer pathway consisted of the cytochrome oxidase and the nitrite reductase systems under aerobic and anaerobic conditions respectively, and these systems showed the exact opposite response to 2-n-heptyl-4-hydroxyquinoline-N-oxide and azide. Azide inhibited specifically the nitrite reductase system. It seems that cytochrome d works as the nitrite reductase and the reduced cytochrome b works as an intermediate electron donor for cytochrome d respectively. The respiratory particle also had a hydroxylamine reductase activity and ammonia was identified as the product of hydroxylamine reduction by the respiratory particle. A terminal electron transfer pathway in Streptomyces griseus was proposed.     

The energy-conserving nitric-oxide-reductase system in Paracoccus denitrificans. Distinction from the nitrite reductase that catalyses synthesis of nitric oxide and evidence from trapping experiments for nitric oxide as a free intermediate during denitrification

1. A Clark-type electrode that responds to nitric oxide has been used to show that cytoplasmic membrane vesicles of Paracoccus denitrificans have a nitric-oxide reductase activity. Nitrous oxide is the reaction product. NADH, succinate or isoascorbate plus 2,3,5,6-tetramethyl-1,4-phenylene diamine can act as reductants. The NADH-dependent activity is resistant to freezing of the vesicles and thus the NADH:nitric-oxide oxidoreductase activity of stored frozen vesicles provides a method for calibrating the electrode by titration of dissolved nitric oxide with NADH. The periplasmic nitrite reductase and nitrous-oxide reductase enzymes are absent from the vesicles which indicates that nitric-oxide reductase is a discrete enzyme associated with the denitrification process. This conclusion was supported by the finding that nitric-oxide reductase activity was absent from both membranes prepared from aerobically grown P. denitrificans and bovine heart submitochondrial particles. 2. The NADH: nitric-oxide oxidoreductase activity was inhibited by concentrations of antimycin or myxothiazol that were just sufficient to inhibit the cytochrome bc1 complex of the ubiquinol--cytochrome-c oxidoreductase. The activity was deduced to be proton translocating by the observations of: (a) up to 3.5-fold stimulation upon addition of an uncoupler; and (b) ATP synthesis with a P:2e ratio of 0.75. 3. Nitrite reductase of cytochrome cd1 type was highly purified from P. denitrificans in a new, high-yield, rapid two- or three-step procedure. This enzyme catalysed stoichiometric synthesis of nitric oxide. This observation, taken together with the finding that the maximum rate of NADH:nitric-oxide oxidoreductase activity catalysed by the vesicles was comparable with that of NADH:nitrate-oxidoreductase, is consistent with a role for nitric-oxide reductase in the physiological conversion of nitrate or nitrite to dinitrogen gas. 4. Intact cells of P. denitrificans also reduced nitric oxide in an antimycin- or myxothiazol-sensitive manner. However, nitric oxide was not detected by the electrode during the reduction of nitrate. Nitric-oxide synthesis from nitrate could be detected with cells in the presence of very low concentrations of Triton X-100 which selectively inhibits nitric-oxide reductase activity. 5. Nitric oxide was detected as an intermediate in denitrification by including haemoglobin with an anaerobic suspension of cells that was reducing nitrate. The characteristic spectrum of the nitric oxide derivative of haemoglobin was observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nitric Oxide-Mediated Inhibition of the Mitochondrial Respiratory Chain in Cultured Astrocytes

Abstract: The Ca²⁺-independent form of nitric oxide synthase was induced in rat neonatal astrocytes in primary culture by incubation with lipopolysaccharide (1 µg/ml) plus interferon-γ (100 U/ml), and the activities of the mitochondrial respiratory chain components were assessed. Incubation for 18 h produced 25% inhibition of cytochrome c oxidase activity. NADH-ubiquinone-1 reductase (complex I) and succinate-cytochrome c reductase (complex II–III) activities were not affected. Prolonged incubation for 36 h gave rise to a 56% reduction of cytochrome c oxidase activity and a 35% reduction in succinate-cytochrome c reductase activity, but NADH-ubiquinone-1 reductase activity was unchanged. Citrate synthase activity was not affected by any of these conditions. The inhibition of the activities of these mitochondrial respiratory chain complexes was prevented by incubation in the presence of the specific nitric oxide synthase inhibitor N ^G-monomethyl- l -arginine. The lipopolysaccharide/interferon-γ treatment of the astrocytes produced an increase in glycolysis and lactate formation. These results suggest that inhibition of the mitochondrial respiratory chain after induction of astrocytic nitric oxide synthase may represent a mechanism for nitric oxide-mediated neurotoxicity.     

Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms

The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain. Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH. By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and Bacillus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD. In the bovine and P. denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase. In the bovine NADH-ubiquinone reductase complex (complex I), [14C]DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000. The time course of labeling of the 29,000 molecular weight subunit with [14C]DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

The nitric oxide reductase of Paracoccus denitrificans.

The nitric oxide (NO) reductase activity of the cytoplasmic membrane of Paracoccus denitrificans can be solubilized in dodecyl maltoside with good retention of activity. The solubilized enzyme lacks NADH-dependent activity, but can be assayed with isoascorbate plus 2,3,5,6-tetramethylphenylene-1,4-diamine as electron donor and with horse heart cytochrome c as mediator. Reduction of NO was measured with an amperomeric electrode. The solubilized enzyme could be separated from other electron-transport components, including the cytochrome bc1 complex and nitrite reductase, by several steps of chromatography. The purified enzyme had a specific activity of 11 mumols.min-1.mg of protein-1 and the Km(NO) was estimated as less than 10 microM. The enzyme formed N2O from NO with the expected stoichiometry. These observations support the view that NO reductase is a discrete enzyme that participates in the denitrification process. The enzyme contained both b- and c-type haems. The former was associated with a polypeptide of apparent molecular mass 37 kDa and the latter with a polypeptide of 18 kDa. Polypeptides of 29 and 45 kDa were also identified in the purified protein which showed variable behaviour on electrophoresis in polyacrylamide gels.     

Denitrification and nitrite reduction: Pseudomonas aeruginosa nitrite-reductase

Present knowledge of the different enzymatic steps of the denitrification chains in various bacteria, particularly Paracoccus denitrificans and Pseudomonas aeruginosa has been briefly reviewed. The question whether nitric oxide (NO), nitrous oxide (N2O) and other nitrogen derivatives are obligatory intermediates has been discussed. The second part is an extensive review of the structure and the function of a key enzyme in denitrification, cytochrome c551-nitrite-oxidoreductase from P. aeruginosa. Recent results on the stoichiometry of nitrite reduction have been discussed.     

Trapping of nitric oxide produced during denitrification by extracellular hemoglobin

A spectrophotometric method has been developed that uses extracellular hemoglobin (Hb) to trap nitric oxide (NO) released during denitrification as nitrosyl hemoglobin (HbNO). The rate of complexation of NO with Hb is about at the diffusion controlled limit for protein molecules and the product, HbNO, is essentially stable. Hb was added to an anaerobic bacterial suspension and denitrification was initiated with either KNO2 or KNO3. HbNO formation was observed for six species of denitrifying bacteria and showed isosbestic points at 544, 568, and 586 nm. Cellular NO production, presumably by nitrite reductase, was kinetically distinct from the much slower chemical reaction of Hb with KNO2 to form methemoglobin and HbNO. The rate of HbNO formation was proportional to cell density, essentially independent of pH from 6.8 to 7.4, nearly zero order in [Hb] and, at least with Paracoccus denitrificans, strongly inhibited by rotenone and antimycin A. The Cu chelator, diethyldithiocarbamate, had no effect on HbNO formation by Pa. denitrificans, but abolished that by Achromobacter cycloclastes which uses a Cu-containing nitrite reductase known to be inactivated by the chelator. HbNO formation did not occur with non-denitrifying bacteria. The stoichiometry at high [Hb] for conversion of Hb to HbNO was 1.3-1.8 KNO2 per Hb for Pa. denitrificans, Pseudomonas aeruginosa, and A. cycloclastes and about 3.4 for Pseudomonas stutzeri. The former range of values corresponds to a partition of about 2 N atoms in 3 toward trapping and 1 in 3 toward reduction on the pathway to N2. Nitrogen not trapped appeared largely as N2O in presence of acetylene. The results are consistent with a model in which NO is a freely diffusible intermediate between nitrite and N2O, providing that nitric oxide reductase is or nearly is a diffusion controlled enzyme.     

New pathway of amine oxidation respiratory chain of Paracoccus denitrificans IFO 12442.

The physiological electron acceptor of quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans IFO 12442 was identified by biochemical and electrochemical methods. Of three types of heme c-containing proteins purified together with QH-AmDH from the periplasm of n-butylamine-grown cells, only constitutive cytochrome c-550 was reduced by the addition of QH-AmDH and n-butylamine. Reconstitution of the respiratory chain revealed that cytochrome c-550 mediates the electron transfer from QH-AmDH to the terminal oxidase. This is a new pathway of the amine oxidation respiratory chain of P. denitrificans.     

Solubilization and resolution of the membrane-bound nitrite reductase from Paracoccus halodenitrificans into nitrite and nitric oxide reductases

Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-cholamidopropyldimethylammonio)-1-(2-hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.Abbreviations PMS phenazine methosulfate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - CHAPSO 3-(3-cholamidopropyl-dimethylammonio)-1-(2-hydroxy-1-propanesulfonate) - NH buffer 150 mM NaCl-50 mM - HEPES pH 7.5; octylglucoside, octyl--d glucopyranoside - NIR intrite reductase (nitrite to nitric oxide) - NOR nitric oxide reductase (nitric oxide to nitrous oxide)     

The bioenergetics of denitrification

In anaerobically grown Paracoccus denitrificans the dissimilatory nitrate reductase is linked to the respiratory chain at the level of cytochromes b. Electron transport to nitrite and nitrous oxide involves c-type cytochromes. During electron transport from NADH to nitrate one phosphorylation site is passed, whereas two sites are passed during electron transport from NADH to oxygen, nitrite and nitrous oxide. The presentation of a respiratory chain as a linear array of electron carriers gives a misleading picture of the efficiency of energy conservation since the location of the reductases is not taken into account. For the reduction of nitrite and nitrous oxide, protons are utilized from the periplasmic space, whereas for the reduction of oxygen and nitrate, protons are utilized from the cytoplasmic side of the inner membrane. Evidence for two transport systems for nitrate was obtained. One is driven by the proton motive force; this system is used to initiate nitrate reduction. The second system is a nitrate-nitrite antiport system. A scheme for proton translocation and electron transport to nitrate, nitrite, nitrous oxide and oxygen is presented. The number of charges translocated across the membrane during flow of two electrons from NADH is the same for all nitrogenous oxides and is 67-71% of that during electron transfer to oxygen via cytochrome o. These findings are in accordance with growth yield studies. YMAX electron values determined in chemostat cultures for growth with various substrates and hydrogen acceptors are proportional to the number of charges translocated to these hydrogen acceptors during electron transport.     

Respiratory detoxification of nitric oxide by the cytochrome c nitrite reductase of Escherichia coli

Nitric oxide is a key element in host defense against invasive pathogens. The periplasmic cytochrome c nitrite reductase (NrfA) of Escherichia coli catalyzes the respiratory reduction of nitrite, but in vitro studies have shown that it can also reduce nitric oxide. The physiological significance of the latter reaction in vivo has never been assessed. In this study the reduction of nitric oxide by Escherichia coli was measured in strains active or deficient in periplasmic nitrite reduction. Nrf(+) cells, harvested from cultures grown anaerobically, possessed a nitric-oxide reductase activity with physiological electron donation of 60 nmol min(-1) x mg dry wt(-1), and an in vivo turnover number of NrfA of 390 NO* s(-1) was calculated. Nitric-oxide reductase activity could not be detected in Nrf(-) strains. Comparison of the anaerobic growth of Nrf(+) and Nrf(-) strains revealed a higher sensitivity to nitric oxide in the NrfA(-) strains. A higher sensitivity to the nitrosating agent S-nitroso-N-acetyl penicillamine (SNAP) was also observed in agar plate disk-diffusion assays. Oxygen respiration by E. coli was also more sensitive to nitric oxide in the Nrf(-) strains compared with the Nrf(+) parent strain. The results demonstrate that active periplasmic cytochrome c nitrite reductase can confer the capacity for nitric oxide reduction and detoxification on E. coli. Genomic analysis of many pathogenic enteric bacteria reveals the presence of nrf genes. The present study raises the possibility that this reflects an important role for the cytochrome c nitrite reductase in nitric oxide management in oxygen-limited environments.     

Inhibition by capsaicin of NADH-quinone oxidoreductases is correlated with the presence of energy-coupling site 1 in various organisms

The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by capsaicin and dihydrocapsaicin, which are the pungent principles of red pepper. This inhibition was correlated with the presence of an energy transducing site in this segment of the respiratory chain. Where the NADH-quinone oxidoreductase segment involved an energy coupling site (e.g., in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes and bovine heart mitochondria), capsaicin acted as an inhibitor of ubiquinone reduction by NADH. In contrast, where this energy coupling site was absent (e.g., in Saccharomyces cerevisiae mitochondria and Bacillus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by capsaicin. The capsaicin inhibition of Paracoccus membranes was reversed by washing the membranes with medium containing bovine serum albumin. In the E. coli and Paracoccus membranes and bovine submitochondrial particles, capsaicin acted as a noncompetitive inhibitor for ubiquinone-1 at lower concentrations of ubiquinone-1 (less than 20 microM) and as a competitive inhibitor at higher concentrations of ubiquinone-1 (greater than 50 microM). In addition, the concentrations of capsaicin required for 50% inhibition of NADH oxidase activity of bovine submitochondrial particles were increased when ubiquinone-10 was added to the particles. The mechanism by which capsaicin inhibits the energy-transducing NADH-quinone oxidoreductase is discussed.