D-lactate oxidation and generation of the proton electrochemical gradient in membrane vesicles from Escherichia coli GR19N and in proteoliposomes reconstituted with purified D-lactate dehydrogenase and cytochrome o oxidase

The respiratory chain in the cytochrome d deficient mutant Escherichia coli GR19N is a relatively simple, linear system consisting of primary dehydrogenases, ubiquinone 8, cytochrome b-556, and cytochrome o oxidase. By use of right-side-out and inside-out membrane vesicles from this strain, various oxidase activities and the generation of the H+ electrochemical gradient were studied. Oxidation of ubiquinol 1 or N,N,-N',N'-tetramethyl-p-phenylenediamine, which donate electrons directly to the terminal oxidase, generates a H+ electrochemical gradient comparable to that observed during D-lactate oxidation. In contrast, D-lactate/ubiquinone 1 or D-lactate/ferricyanide oxidoreductase activity does not appear to generate a membrane potential, suggesting that electron flow from D-lactate dehydrogenase to ubiquinone is not electrogenic. Moreover, proteoliposomes reconstituted with purified D-lactate dehydrogenase, ubiquinone 8, and purified cytochrome o catalyze D-lactate and ubiquinol 1 oxidation and generate a H+ electrochemical gradient similar to that observed in membrane vesicles. Strikingly, in inside-out vesicles, NADH oxidation generates a H+ electrochemical gradient that is very significantly greater than that produced by either D-lactate or ubiquinol 1; furthermore, NADH/ubiquinone 1 and NADH/ferricyanide oxidoreductase activities are electrogenic. It is suggested that the only component between D-lactate dehydrogenase or ubiquinol and oxygen in GR19N membranes that is directly involved in the generation of the H+ electrochemical gradient is cytochrome o, which functions as a "half-loop" (i.e., the oxidase catalyzes the scalar release of 2 H+ from ubiquinol on the outer surface of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biophysical properties of a major membrane phospholipid, dielaidoylphosphatidylethanolamine, found in an Escherichia coli fatty acid auxotroph.

Dielaidoylphosphatidylethanolamine, a principal lipid component of membranes of Escherichia coli fatty acid auxotrophs enriched in elaidic acid, has been studied by paramagnetic resonance, fluorescence, and calorimetric methods. EPR measurements with perdeutero-di-tert-butylnitroxide and 2,2,6,6-tetramethyl piperidine-1-oxyl indicate that, when dispersed in aqueous media, this phospholipid undergoes an abrupt order leads to disorder transition at 37.5 degrees C and 36.5 C, respectively. A similar transition temperature is suggested by experiments with 9-doxyl-dimyristoylphosphatidylethanolamine (DEPE). cis- and trans-Parinaric-acid fluorescence polarization measurements indicate that the midpoint of this transition occurs at 34.0 degrees C and 35.5 degrees C, respectively. Differential scanning calorimetry of DEPE revealed a single, sharp endotherm at 38.5 degrees C with increasing temperature; two exotherms of similar magnitude were observed at 36.5 degrees C and 34.5 degrees C upon cooling. This double transition was not observed by any of the other methods. From these results we conclude that the major structural transition at 30-31 degrees C observed previously with 5-, 12-, and 16-doxyl stearate in intact E. coli membranes is due to the DEPE present (Morrisett, J.D., Pownall, H.J., Plumlee, R.T., Smith, L.C., Zehner, Z.E., Esfahani, M., and Wakil, S.J. (1975) J. Biol. Chem. 250, 6969-6976).     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes. A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This 'stiffening' effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35 degrees C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This correspond to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10 degrees C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

Correlation between lipid fluidity and tryptic susceptibility of Ca2+-ATPase in sarcoplasmic reticulum membranes

Lipid fluidity in native and denatured sarcoplasmic reticulum membranes and extracted lipids was monitored between -30 and 30 degrees C using trans-parinaric acid as a fluorescent probe. In addition to a large increase in fluidity between -30 and 0 degree C in each system, a phase change centered near 10 degrees C was observed in the extracted lipids but not in either the native or denatured membranes. A significant change in fluorescence intensity near 15 degrees C was observed in native sarcoplasmic reticulum membranes, however, when trans-parinaric acid was excited by energy transfer from tryptophan residues of the membrane protein. When Ca2+-ATPase was subjected to proteolytic cleavage by trypsin as a function of temperature, a change in susceptibility was detected at about 15-20 degrees C in the native membranes but not in a solubilized preparation. It is proposed that one or more structural changes in the microenvironment of Ca2+-ATPase in the native membrane occur between 15 and 20 degrees C which may be related to the change in apparent activation energy which is observed for this enzyme.     

Four different b-type cytochromes in the halophilic archaebacterium, Halobacterium halobium

The halophilic archaebacterium, Halobacterium halobium has been found to contain four different b-type cytochromes. The four components were recognized by their potentiometric characteristics in situ in their functional environment in the membrane of H. halobium. Oxidation-reduction midpoint potentials of these four b-type cytochromes were determined to be +261, +160, +30, and -153 mV, respectively. We also demonstrate that the pathway involved in the transport of reducing equivalents from succinate to oxygen proceeds through the b-type cytochromes with oxidation-reduction midpoint potentials of +261 and +161 mV. The cytochrome with oxidation-reduction midpoint potential of -153 mV was not substrate reducible by NADH but was chemically reducible by dithionite. Antimycin inhibits reduction of b-type cytochrome in the succinate pathway, but has no effect on b-type cytochrome reduction when reducing equivalents are provided by NADH. The carbon monoxide difference spectrum of H. halobium membranes shows at least one carbon monoxide-binding b-type cytochrome, indicating a terminal oxidase. A scheme for electron transport in H.halobium involving the b-type cytochromes and terminal oxidase is suggested.     

A biochemical study of the reconstitution of D-lactate dehydrogenase-deficient membrane vesicles using fluorine-labeled components

Fluorine-19 labeled compounds have been incorporated into lipids and proteins of Escherichia coli. 19F-Labeled membrane vesicles, prepared by growing a fatty acid auxotroph of a D-lactate dehydrogenase-deficient strain on 8,8-difluoromyristic acid, can be reconstituted for oxidase and transport activities by binding exogenous D-lactate dehydrogenase. 19F-Labeled D-lactate dehydrogenases prepared by addition of fluorotryptophans to a tryptophan-requiring strain are able to reconstitute D-lactate dehydrogenase-deficient membrane vesicles. Thus, lipid and protein can be labeled independently and used to investigate protein-lipid interactions in membranes.     

Lipid ordering and enzymic activities in chromaffin granule membranes.

1. The interaction of a variety of fluorescent probes with the membranes of adrenal medullary chromaffin granules is described. 2. Changes in the motional properties of the bound probes with temperature were investigated and evidence is presented which indicates that ordering of the membrane lipids occurs below 33 degrees C. 3. The ordering is characteristics of the membrane lipids and is retained by sonicated aqueous dispersions of the total lipid extracted from chromaffin granule membranes. 4. The ATPase and NADH:acceptor oxidoreductase activities of the chromaffin granule membrane have discontinuous Arrhenius temperature versus activity relationships with 'transitions' at 33 degrees C. 5. The ATPase has a second transition at 36.5 degrees C. 6. The 33 degrees C 'transition' for the NADH:acceptor oxidoreductase is removed by treatment with the detergent Triton X-100. 7. The correlation between the onset of lipid ordering and the change in activation energy of the membrane-bound enzyme activities is discussed in terms of the co-operative interactions of the different membrane components. The possible role of lipid ordering in exocytosis is discussed.     

Analysis of the respiratory chain in Ethanologenic Zymomonas mobilis with a cyanide-resistant bd-type ubiquinol oxidase as the only terminal oxidase and its possible physiological roles

The respiratory chain of the ethanologenic bacterium Zymomonas mobilis was investigated, in which the pyruvate-to-ethanol pathway has been demonstrated to be mainly responsible for NADH oxidation and the tricarboxylic acid cycle is incomplete. Membranes from cells cultivated under aerobic or anaerobic growth conditions showed dehydrogenase and oxidase activities for NADH, D-lactate and D-glucose and ubiquinol oxidase activity. Intriguingly, the NADH oxidase activity level of membrane fractions from cells grown aerobically was found to be higher than that of membrane fractions from Escherichia coli or Pseudomonas putida grown aerobically, indicating a crucial role of the respiratory chain in NADH oxidation in the organism. Cyanide-resistant terminal oxidase activity was observed and appeared to be due to a bd-type ubiquinol oxidase as the only terminal oxidase encoded by the entire genome. The terminal oxidase with a relatively strong ubiquinol oxidase activity exhibited remarkably weak signals of cytochrome d. Considering these findings and the presence of a type-II NADH dehydrogenase but not a type-I, a simple respiratory chain that generates less energymay have evolved in Z. mobilis.     

Thermal transitions in erythrocyte membranes revealed by their permeability to ANS

A number of breaks were recorded on the curve of Arrhenius relationship of the rate constant of the dye 1-anilino-8-naphthalenesulphonate sodium salt (ANS) input into human erythrocytes of 20, 28, 36, 42 and 46 degrees C. Variations in the values of activation energies within the temperature range of 28-36 degrees and 42-46 degrees C obtained in various blood samples allow to consider these temperatures as those at which structural changes of the membranes take place. The values of activation energy of the process for temperature "conformers" of the erythrocyte membrane are 12(10-20 degrees C), 26.5 (20-28 degrees C), 34.2(36-42 degrees C) and 47 kcal/mol (t is greater than 46 degrees C). Within the temperature range of 28-36 degrees and 42-46 degrees C an irreversible decrease of permeability to ANS of the erythrocyte ghost after their incubation for 10 min at increased temperatures were observed. Thus the temperature regions of the change in erythrocyte permeability correspond to those at which the resealing of ghost takes place. The break in Arrhenius graph at 20 degrees C seems to characterize a highly cooperative "point" transition. The lipid nature of the initiator of structural transition within 28-36 degrees C is proved by a sharp increase of the permeability of liposomes prepared from erythrocyte membrane lipids to ANS at 28 degrees C. The nature of the initiators of two other thermal transitions is discussed.     

Study of a time lag in the assay of Escherichia coli membrane-bound dehydrogenases based on tetrazolium salt reduction

The Escherichia coli membrane-bound D-lactate dehydrogenase and succinate dehydrogenase were assayed on the basis of the phenazine methosulfate- (PMS-) mediated reduction of the tetrazolium salt, MTT. An initial slower phase (lag) in the time-course of the reaction was observed and analyzed. The results were as follows. (1) The time lag in the assay of the D-lactate dehydrogenase was eliminated by preincubating the membranes with PMS plus D-lactate, with PMS plus succinate, or with PMS plus NADH (conditions which implicated PMS reduction). (2) When the D-lactate dehydrogenase was assayed by another method based on the measurement of the pyruvate formed, neither was a time lag observed nor was the enzyme activity affected by membrane preincubation with PMS plus D-lactate. (3) Although the superoxide radical was involved in MTT reduction, this radical seemed not to participate in the generation of the time lag. (4) Membranes whose D-lactate dehydrogenase activity had previously been destroyed by heating at 80 degrees C for 1 min, were able to prolong the time lag in MTT reduction when added to the assay medium for the D-lactate dehydrogenase from untreated membranes, whereas membranes previously heated at 100 degrees C instead of 80 degrees C did not have this effect. It was concluded that the E. coli membranes interfered in the dehydrogenase assay based on the PMS-mediated reduction of MTT. The time lag was interpreted as a period during which the interfering substance reacted with reduced PMS inhibiting the reduction of MTT.     

Evidence that lipid lateral phase separation induces functionally significant structural changes in the Ca+2ATPase of the sarcoplasmic reticulum.

We have studied lipid lateral phase separation (LPS) in the intact sarcoplasmic reticulum (SR) membrane and in bilayers of isolated SR membrane lipids as a function of temperature, [Mg+2], and degree of hydration. Lipid LPS was observed in both the intact membrane and in the bilayers of isolated SR lipids, and the LPS behavior of both systems was found to be qualitatively similar. Namely, lipid LPS occurs only at relatively low temperature and water content, independently of the [Mg+2], and the upper characteristic temperature (th) for lipid LPS for both the membrane and bilayers of its isolated lipids coincide to within a few degrees. However, at similar temperatures, isolated lipids show more LPS than the lipids in the intact membrane. Lipid LPS in the intact membrane and in bilayers of the isolated lipids is fully reversible, and more extensive for samples partially dehydrated at temperatures below th. Our previous x-ray diffraction studies established the existence of a temperature-induced transition in the profile structure of the sarcoplasmic reticulum Ca+2ATPase which occurs at a temperature corresponding to the [Mg+2]-dependent upper characteristic temperature for lipid LPS in the SR membrane. Furthermore, the functionality of the ATPase, and in particular the lifetime of the first phosphorylated enzyme conformation (E1 approximately P) in the Ca+2 transport cycle, were also found to be linked to the occurrence of this structural transition. The hysterisis observed in lipid LPS behavior as a function of temperature and water content provides a possible explanation for the more efficient transient trapping of the enzyme in the E1 approximately P conformation observed in SR membranes partially dehydrated at temperatures below th. The observation that LPS behavior for the intact SR membrane and bilayers of isolated SR lipids (no protein present) are qualitatively similar strongly suggests that the LPS behavior of the SR membrane lipids is responsible for the observed structural change in the Ca+2ATPase and the resulting significant increase in E1 approximately P lifetime for temperatures below th.     

Studies on Mitochondrial Proteins. II. Localization of Components in the inner membrane labelling with diazobenzenesulfonate,a non-penetrating probe

The topography of the inner membrane of rat liver mitochondria was studied using a probe, diazobenzenesulfonate, which interacts preferentially with surface components. Inner membranes were examined both in a native orientation as found in the intact mitochondrion or in an inverted state as found in isolated inner membranes prepared by sonication.Enzyme inactivation as a consequence of diazobenzenesulfonate labeling was employed to determine the localization of a number of inner membrane activities. In inner membranes labeled on the outer surface, NADH and succinate oxidation were strongly inhibited while ATPase and $\text{ascorbate}\text{-N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenylene-diamine}$ (TMPD) oxidase activities were unaffected. In inner membranes labeled on the inner surface. ATPase and succinate oxidation were inactivated while NADH oxidation and ascorbate-TMPD oxidase were unaffected. Succinate dehydrogenase was inhibited only by labeling the inner surface while NADH dehydrogenase was inhibited to a similar extent by treatment of either surface.Sodium dodecylsulfate-polypeptides (66 000 and 26 000) on the outer surface of the inner membrane and five polypeptides (80 000, 66 000, 51 000-48 000, and 26 000) on the inner surface. These results indicate a highly asymmetric localization of inner membrane components.     

Thermally induced changes in reconstituted and membranous cytochrome c oxidase.

The Arrhenius plots of electron transport activity in cytochrome c oxidase reconstituted with well-defined phospholipids have been shown to display a change in slope at 20--25 degrees C regardless of the chemical nature of the incorporated lipid. In native membranous cytochrome c oxidase, the discontinuity in Arrhenius activity plot occurred at 16--18 degrees C. These temperature breaks were found to correlate with changes in spin-label mobilities but not with the bulk lipid transition observed by differential scanning calorimetry. Temperature-dependent reciprocal equilibrium between the immobilized and fluid pools is demonstrated. It is suggested that the changes in kinetic and spin-label spectral characteristics in cytochrome c oxidase membranes are related very likely to a lipid-protein interaction prompted by a thermally induced change in the physical state of the lipids that does not involve a gel to liquid crystalline transition.     

Characterization of the mycoplasma membrane proteins. V. Release and localization of membrane-bound enzymes in Acholeplasma laidlawii.

The peripheral membrane protein fraction released by washing Acholeplasma laidlawii membranes with low-ionic strength buffers contained about 50% of the total membrane-bound ribonuclease and deoxyribonuclease activities. The ATPase, NADH oxidase and p-nitrophenylphosphatase activities remained bound to the membrane even when EDTA was added to the wash fluids, and thus appear to belong to the integral membrane protein group. Serving as a marker for peripheral membrane proteins, the membrane-bound ribonuclease activity was solubilized by bile salts much more effectively than the integral membrane-bound enzymes. On the other hand, the solubilized ribonuclease showed a much lower capacity to reaggregate with other solubilized membrane components to membranous structures. Yet, most of the ribonuclease molecules which were bound to the reaggregated membranes could not be released by low-ionic strength buffer. The reaggregated membranes differed from the native membranes in the absence of particles on their fracture faces obtained by freeze cleaving, and by their much higher labeling by the [125-I]lactoperoxidase iodination system. These results suggest that most of the proteins are exposed on the reaggregated membrane surfaces, with very little, if any, protein embedded in its lipid bilayer core. Enzyme disposition in the A. laidlawii membrane was studied by comparing the activity of isolated membranes with that of membranes of intact cells after treatment with pronase or with an antiserum to membranes. The data indicate the asymmetrical disposition of these activities, the ATPase and NADH oxidase being localized on the inner membrane surface, while the nucleases are exposed on the external membrane surface.     

Evaluation of structuro-functional heterogeneity of isolated mitochondria from the normal and the ischemic myocardium

The structural and functional heterogeneity of mitochondria isolated from intact and ischemic (after 60 min exposure at 37 degrees C) rabbit myocardium was evaluated. In the presence of cytochrome c. a relatively high (260 +/- 26 ng at O/min . mg of protein) rate of rotenone-sensitive NADH oxidation was observed, which was increased in ischemia. Cytochrome c stimulated the increase of NADH oxidation in mitochondria of normal and ischemic myocardium by the factors of 3.5 and 3.4, respectively. Succinate oxidation in the presence of bromthymol blue in normal and ischemic myocardium mitochondria was activated by cytochrome c 3.3- and 2.9-fold, respectively. The percentage of mitochondria with both structurally damaged membranes was 15% and 25% in normal and ischemic myocardium preparations, respectively. In the absence of ADP, cytochrome c contributed to the increase of the succinate oxidase activity in ischemic mitochondria; that in the 3rd state was inhibited in ischemia and normalized by cytochrome c. A principle was proposed for estimating the percentage of mitochondria with damaged outer membranes, the indices being equal to 34% in control and to 56% in ischemic myocardium. Evidence was obtained suggesting that this mitochondrial fraction was characterized by lowered coupling and absence of rotenone-sensitive NADH: oxidase activity. The percentage of intact mitochondria, in which succinate oxidation is inhibited by bromthymol blue and does not need exogenous cytochrome c, is 51% in control and 19% in ischemic myocardium mitochondria.     

Subzero temperature study of the inner mitochondrial membrane and related phospholipid membrane systems with the fluorescent probe, trans-parinaric acid.

The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0 degrees C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospho lipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at -25 degrees C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.     

A spin-label study of the chromaffin granule membrane.

The structure of the chromaffin granule membrane has been probed using a number of different spin labels. Both the effect of temperature and high levels of calcium have been studied. 1. The results from three positional isomers of the stearic acid spin label demonstrate that a substantial part of the membrane lipid (that is sensed by the probe) is in a bilayer structure which undergoes a structural transition at 32-36 degrees C, characterized by an increase in the population of gauche isomers in the lipid chains. A possible mechanism for this transition would be the preferential segregation of cholesterol. 2. The covalently bound iodoacetamide spin label reveals a transition within the protein component of the membrane or its immediate lipid environment at 32 degrees C. This transition corresponds to an increased degree of motional freedom of the spin label above the transition temperature. 3. The lipid-soluble spin label 2,2,6,6-tetramethyl-piperidine-1-oxyl exhibits a break at 34 degrees C in the temperature-dependence of its partitioning into the membrane. This could correspond to the onset of a lateral separation in the membrane lipid, again possible involving a re-distribution of cholesterol. 4. Calcium abolishes, diminishes or shifts the transition observed by the spin label and decreases the amplitude of motion of the stearic acid spin labels, again possibly involving a redistribution of cholesterol and also lysolecithin. The temperatures of the structural transition agree well with the changes in the enzymic activity of the membrane ATPase and NADH oxidase functions and also with the results from fluorescent probes [Bashford et al., Eur. J. Biochem. 67, 105-114(1976)]. It is possible that triggering of the transition either by calcium or some other stimulus may play a role in catecholamine release and membrane fusion.     

Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. I. Membrane stability in cells of differing growth phase

Physical properties of Escherichia coli membrane lipids in logarithmic- and stationary-phase cells were studied by measuring the fluorescence polarization change of cis- and trans-parinaric acid as a function of temperature. In aqueous dispersions of phospholipids extracted from cytoplasmic and outer membranes of cells of differing growth phase, a similar polarization increase was observed over the range from physiological temperature to below 0 degrees C, and nearly the same transition ratios were obtained in all samples. The cytoplasmic membrane of both of the growth-phase cells showed a higher polarization ratio above the transition temperatures, compared to that in the aqueous dispersion of phospholipids. The polarization ratios below the transition temperatures of these specimens were lower than the value obtained with the lipids, especially in the stationary-phase specimens. The outer membrane specimens showed a similar polarization change but the transition temperature ranges were considerably higher both in the logarithmic- and the stationary-phase specimens, compared to those in the cytoplasmic membrane specimens. Freeze-thawing of logarithmic-phase cells showed the emergence of activity of certain enzymes which are known to be located in the membranes. The stationary-phase cells did not suffer from any such deleterious effect and maintained a high level of cell viability in a similar treatment. These results indicate that in the stationary-phase cell membranes lipids are in a highly ordered state, and the lipid state causes a membrane stability which results in the high resistance of the cell to freeze-thawing.     

Interaction of n-alkanes with respiration and oxidative phosphorylation in rabbit heart mitochondria: n-nonane

Studies "in vitro" on the effect of n-nonane on coupled rabbit heart mitochondria with both succinate and glutamate as substrates show that the hydrocarbon examined makes the membrane permeable to protons (uncoupling), to some matrix enzymes and to exogenous NADH. The effect increases with increasing n-alkane concentration (from 0 to 160 microgram/mg mitochondrial protein) and temperature (from 15 degrees to 38 degrees C). Furthermore at higher concentrations and temperatures NADH oxidase inhibition is observed, whereas on succinate oxidase a biphasic effect (activation at lower concentrations and inhibition at higher concentrations) is produced. However the results, qualitatively similar to those observed with n-hexane, exhibit features probably due to a longer chain and that can be ascribed to perturbations of the physical state of membrane lipids.     

Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilizes complex I in Paracoccus denitrificans

Stable supercomplexes of bacterial respiratory chain complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) have been isolated as early as 1985 (Berry, E. A., and Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458-2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete "respirasome." Inactivation of the gene for tightly bound cytochrome c552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.