Properties of purified kidney microsomal NADPH-cytochrome c reductase

NADPH-cytochrome c reductase, solubilized by lipase digestion of microsomes prepared from perfused porcine kidney cortex, was purified about 3600-fold to give a turnover number of 1230 nmoles cytochrome c reduced per min per nmole flavin. The kinetic determination of K_m and V with respect to NADPH, cytochrome c, and NADH, resulted in values similar to those obtained with purified liver reductase. The kidney microsomal enzyme also exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction.Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN per mole of reductase. The molecular weight was estimated by Sephadex G-200 gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 68,000 and 71,000 g per mole, respectively.Immunochemical techniques, including Ouchterlony double-diffusion studies and inhibition of catalytic activity by antibody to the liver microsomal NADPH-cytochrome c reductase, established the similarity of the purified liver and kidney reductases.     

The influence of in vitro procedures which diminish NADPH cytochrome c reductase activity on oxidative drug metabolism in lung and liver microsomes

Rabbit lung and liver microsomes were subjected to three procedures which decreased NADPH cytochrome c reductase activity; flavoprotein antibody, trypsin and subtilisin digestion. The effects on benzphetamine and p-nitroanisode demethylation and amine metabolic-intermediate complex formation were investigated. In general, the proteolytic digestion had a greater inhibitory effect on oxidation reactions for a given loss of NADPH cytochrome c reductase activity than did flavoprotein antibody; and of the two proteases, subtilisin, which also diminises the cytochrome b₅ reduction pathway, had a greater inhibitory effect than trypsin. Subtilisin digestion had similar effects in both liver and lung microsomes; a loss of flavoprotein without a loss of cytochrome P-450; but whereas all three oxidative reactions decreased in unison as the flavoprotein was lost in the liver, benzphetamine demethylation was less susceptible to flavoprotein depletion than the other two reactions in lung microsomes. With trypsin digestion flavoprotein was removed without loss of cytochrome P-450 only in lung microsomes; in liver microsomes the cytochrome P-450 was susceptible to tryptic degradation. In lung microsomes, benzphetamine and p-nitroanisole demethylations were less susceptible to flavoprotein loss than metabolic-intermediate complex formation.     

Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.     

Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420

The presence of cytochromes b₅, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c reductases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b₅. In addition, cytochrome b₅ fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with M_r values in the range 10 000–13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.     

Characteristcs of microsomal enzyme controls in primary cultures of rat hepatocytes.

Cytochrome P-450, NADPH-cytochrome c reductase, biphenyl hydroxylase, and epoxide hydratase have been compared in intact rat liver and in primary hepatocyte cultures. After 10 days in culture, microsomal NADPH-cytochrome c reductase and epoxide hydratase activities declined to a third of the liver value, while cytochrome P-450 decreased to less than a tenth. Differences in the products of benzo[a]pyrene metabolism and gel electrophoresis of the microsomes indicated a change in the dominant form(s) of cytochrome P-450 in the cultured hepatocytes. Exposure of the cultured cells to phenobarbital for 5 days resulted in a threefold induction in NADPH-cytochrome c reductase and epoxide hydratase activities which was typical of liver induction of these enzymes. Exposure of the cells to 3-methylcholanthrene did not affect these activities. Cytochrome P-450 was induced over two times by phenobarbital and three to four times by 3-methylcholanthrene. The λ_max of the reduced carbon monoxide complex (450.7 nm) and analysis of microsomes by gel electrophoresis showed that the phenobarbital-induced cytochrome P-450 was different from the species induced by 3-methylcholanthrene (reduced carbon monoxide λ_max = 447.9 nm). However, metabolism of benzo[a]pyrene (specific activity and product distribution) was similar in microsomes of control and phenobarbital- and 3-methylcholan-threne-induced hepatocytes and the specific activity per nmole of cytochrome P-450 was higher than in liver microsomes. The activities for 2- and 4-hydroxylation of biphenyl were undetectable in all hepatocyte microsomes even though both activities were induced by 3-methylcholanthrene in the liver. Substrate-induced difference spectra and gel electrophoresis indicated an absence in phenobarbital-induced hepatocytes of most forms of cytochrome P-450 which were present in phenobarbital-induced rat liver microsomes. It is concluded that the control of cytochrome P-450 synthesis in these hepatocytes is considerably different from that found in whole liver, while other microsomal enzymes may be near to normal. Hormonal deficiencies in the culture medium and differential hormonal control of the various microsomal enzymes provide a likely explanation of these effects.     

Immunochemical evidence for the participation of cytochrome b5 in microsomal stearyl-CoA desaturation reaction

A rabbit antiserum was prepared against rat liver microsomal cytochrome b₅, and utilized in demonstrating the participation of this cytochrome in the microsomal stearyl-CoA desaturation reaction. The antiserum inhibited the NADH-cytochrome c reductase activity of rat liver microsorncs, but it did not inhibit either NADH-ferricyanide or NADPH-cytochrome c reductase activity of the microsomes. Thus, the inhibitory effect of the antiserum on the microsomal electron-transferring reactions seemed to be specific to those which require the participation of cytochrome b₅.The NADH-dependent and NADPH-dependent desaturations of stearyl CoA by rat liver microsomes were strongly inhibited by the antiserum. The reduction of cytochrome b₅ by NADH-cytochrome b₅ reductase as well as the reoxidation of the reduced cytochrome b₃ by the desaturase, the terminal cyanide-sensitive factor of the desaturation system, was also strongly inhibited by the antiserum. When about 90%, of cytochrome b₅ was removed from rat liver microsomes by protease treatment, the desaturation activity of the microsomes became much more sensitive to inhibition by the antiserum. These results confirmed our previous conclusion that the reducing equivalent for the desaturation reaction is transferred from NAD(P)H to the cyanidesensitive factor mainly via cytochrome b₅ in the microsomal membranes.     

Effects of anti-NADPH-cytochrome c reductase and anti-cytochrome b5 antibodies on the hepatic and pulmonary microsomal metabolism and covalent binding of the pulmonary toxin 4-ipomeanol.

An antibody prepared against purified rat liver NADPH-cytochrome $\text{c}$ reductase inhibited both the pulmonary and hepatic microsomal covalent binding of 4-ipomeanol as well as the respective NADPH-cytochrome $\text{c}$ reductase activities, findings which are consistent with previous studies which indicated the participation of cytochrome P450 in the metabolic activation of the toxin. An antibody prepared against purified rat liver cytochrome b₅, which strongly inhibited both the rat hepatic and pulmonary NADH-dependent cytochrome $\text{c}$ reductases, and was inactive against the respective NADPH-dependent cytochrome $\text{c}$ reductases, had little effect on metabolic activation of 4-ipomeanol by hepatic microsomes, but strongly inhibited both the NADH-supported and the NADPH-supported pulmonary microsomal metabolism and covalent binding of the compound. These results suggest that metabolic activation of 4-ipomeanol involves a two-electron transfer in which transfer of the second electron via cytochrome b₅ is rate-limiting in lung microsomes.     

Thyroxine stimulation of rate liver microsomal NADH-cytochrome c reductase in vitro

Rat liver microsomal NADH-cytochrome $\text{c}$ reductase activity is stimulated by 20 μM thyroxine $\text{in}$$\text{vitro}$. Thyroxine does not influence microsomal NADH-dichlorophenolindophenol reductase, NADPH-cytochrome $\text{c}$ reductase, or NADPH-dichlorophenolindophenol reductase activity. Stimulation of NADH-cytochrome $\text{c}$ reductase activity is not mediated by super-oxide and is likely due to enhanced reduction or oxidation of cytochrome $\text{b}$5.     

Effects of methylbenzenes on microsomal enzymes in rat liver,kidney and lung

The effects of pretreatment with toluene, o-, m-, p-xylene and mesitylene were investigated on the microsomal enzymes of liver, kidney and lung in rats. The activities of aminopyrine N-demethylase, aryl hydrocarbon hydroxylase, aniline hydroxylase, NADPH-cytochrome c reductase, as well as the concentrations of cytochrome P-450 and cytochrome b₅ were determined. The effects were most marked in the liver, where toluene caused increase in aniline hydroxylase and cytochrome P-450; o-xylene in aminopyrine N-demethylase and cytochrome b₅; m-xylene and mesitylene in all the enzymes investigated. In kidneys, all the compounds increased the activity of aniline hydroxylase; m-xylene induced cytochrome P-450 and b₅ as well as NADPH-cytochrome c reductase; p-xylene induced cytochrome P-450, and mesitylene cytochrome P-450 and b₅. Aminopyrine N-demethylase activity was decreased by toluene. In lungs, only mesitylene caused any significant differences from the controls: increase in aminopyrine N-demethylase and aryl hydrocarbon hydroxylase, decrease in aniline hydroxylase. The methylbenzenes tested induced the microsomal enzymes in a rough correlation to the number of their methyl groups and their hydrophobic properties.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.     

Oxidative enzyme activities in rat brain subcellular fractions. The effect of deoxycholate, freeze-thaw, and water

(1) The distributions of four oxidative enzymes were studied in crude brain fractions. (2) Freeze-thaw cycle treatment and frozen storage of homogenate fractions gave apparent enhancement of cytochrome oxidase and NADH cytochrome c reductase activities. (3) Deoxycholate released cytochrome oxidase and NADH cytochrome c reductase activities from low-speed precipitates. The NADH diaphorase was enhanced to a small degree while NADPH cytochrome c reductase was not affected by deoxycholate. (4) Distilled water coupled with a single homogenization released trapped soluble enzymes and mitochondria and gave nearly maximal cytochrome oxidase activity as judged by deoxycholate treatment. The total distilled water activity of NADH cytochrome c reductase was much less than that of deoxycholate-stimulated fractions. The activities of other enzymes were not markedly affected by distilled water although their distribution was changed.     

The effect of enzyme induction on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes

The effect of pretreatment with phenobarbitone, rifampicin, β-naphthoflavone, antipyrine and spironolactone on the irreversible binding of ethynyloestradiol to guinea pig liver microsomes has been examined and the corresponding changes in microsomal P-450 content and cytochrome $\text{c}$ reductase activity measured. Rifampicin produced the greatest increase (220%) in irreversible binding while phenobarbitone produced the greatest increase in both microsomal P-450 content (172%) and cytochrome $\text{c}$ reductase activity (210%). There was no correlation of irreversible binding with either microsomal P-450 content or with cytochrome $\text{c}$ reductase activity.     

Investigation of the transverse topology of the microsomal membrane using combinations of proteases and the non-penetrating reagent diazobenzene sulfonate

Intact microsomal vesicles from rat liver were subjected to combined treatment with trypsin and an unspecific protease and were also examined after reaction with the chemical probe p-diazobenzene sulfonate. In addition, the latency of various enzymes in intact microsomal vesicles has been investigated. All microsomal electron transport enzymes studied, i.e. NADH-ferricyanide and cytochrome c reductases, cytochrome b₅, NADPH-cytochrome c reductase and cytochrome P-450, were either solubilized or inactivated by one or both treatments. The experimental data indicate that UDPglucuronyl-transferase is also localized at the outer surface of microsomes. In contrast, a number of hydrolytic enzymes are apparently located inside the permeability barrier of the membrane and presumably at the inner surface. Under conditions where the levels of electron transport enzyme activities or amounts are changed, such as in newborn rats and rats treated with phenobarbital or methylcholanthrene, the intramembranous position of these enzymes is the same as in control adult rats. This indicates that the enzyme molecules are not relocated after their insertion into the membrane.     

Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA

A cDNA clone was isolated from a maize (Zea mays L. cv W64A×W183E) scutellum λgt11 library using maize leaf NADH:nitrate reductase Zmnr1 cDNA clone as a hybridization probe; it was designated Zmnr1S. Zmnr1S was shown to be an NADH:nitrate reductase clone by nucleotide sequencing and comparison of its deduced amino acid sequence to Zmnr1. Zmnr1S, which is 1.8 kilobases in length and contains the code for both the cytochrome b and flavin adenine dinucleotide domains of nitrate reductase, was cloned into the EcoRI site of the Escherichia coli expression vector pET5b and expressed. The cell lysate contained NADH:cytochrome c reductase activity, which is a characteristic partial activity of NADH:nitrate reductase dependent on the cytochrome b and flavin adenine dinucleotide domains. Recombinant cytochrome c reductase was purified by immunoaffinity chromatography on monoclonal antibody Zm2(69) Sepharose. The purified cytochrome c reductase, which had a major size of 43 kilodaltons, was inhibited by polyclonal antibodies for maize leaf NADH:nitrate reductase and bound these antibodies when blotted to nitrocellulose. Ultraviolet and visible spectra of oxidized and NADH-reduced recombinant cytochrome c reductase were nearly identical with those of maize leaf NADH:nitrate reductase. These two enzyme forms also had very similar kinetic properties with respect to NADH-dependent cytochrome c and ferricyanide reduction.     

NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

Purification and Characterization of Cytochrome P450 Reductase from Liver Microsomes of Feral Leaping Mullet (Liza saliens)

NADPH-cytochrome P450 reductase was purified to electrophoretic homogeneity from detergent-solubilized liver microsomes from the leaping mullet (Liza saliens). The purified reductase was characterized with respect to spectral, electrophoretic, and biocatalytic properties. In addition, effects of pH, ionic strength, and the substrate concentration on the NADPH-dependent cytochrome c reductase activity of the purified fish liver cytochrome P450 reductase were studied. Cytochrome P450 reductase was purified 438-fold with a yield of 17.5% with respect to the initial amount present in the fish liver microsomes. The specific activity of the enzyme was found to be 52.6 μmol cytochrome c reduced per minute per mg protein. The monomer molecular weight of the purified enzyme was calculated to be 77,000 ± 1000 when electrophoresed on polyacrylamide gels under the denaturing conditions in the presence of SDS. The absorption spectrum of fish reductase showed two peaks at 378 and 455 nm. NADPH-dependent cytochrome c reductase activity of the purified Liza saliens liver cytochrome P450 reductase was found to be maximal when pH was between 7.4 and 7.8. The apparent K_m of the purified enzyme was found to be 7.69 μM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer, pH 7.7, at room temperature, and the enzyme was fully saturated by its substrate, cytochrome c, when the substrate concentration was at or above the 70 μM. Furthermore, the purified enzyme was biocatalytically active in reconstituting the 7-ethoxyresorufin O-deethylase activity in the reconstituted system containing purified mullet liver cytochrome P4501A1 and lipid. These results suggested that the purified fish liver cytochrome P450 reductase is similar to its mammalian counterparts with respect to spectral, electrophoretic, and biocatalytic properties. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 103–113, 1998     

Steroid 17,20-lyase from testis microsomes: participation of NADPH cytochrome c reductase

The enzymic activity of testis microsomes which mediates cleavage of the 2-carbon side chain from the 17-position of 21-carbon steroids is a mixed-function oxidase and recent reports have suggested that cytochrome P-450 is a participant in this reaction. The studies reported in this communication were intended to demonstrate that the flavoprotein, NADPH cytochrome c reductase, is also a participant in the reaction.The cleavage activity (referred to here as 17,20-lyase) from rat testis microsomes was shown to be inhibited by a number of agents which are electron acceptors for NADPH cytochrome c reductase. This finding indicates that the lyase activity is inhibited by diversion of electron flow and, more specifically, that the point of interruption is at the reductase. Cytochrome c, itself, is a noncompetitive inhibitor of lyase activity when NADPH is substrate. Lyase and reductase activity were diminished to an equal extent by heating a microsomal suspension. An antibody to cytochrome e reductase was prepared after purification of the reductase from rat liver. This antibody caused a parallel and equal reduction of activity of the lyase and reductase. These data show that the reductase and lyase activities are closely linked and suggest that the reductase functions as an electron carrier for the reduction of P-450.     

Microsomal marker enzymes of Manduca sexta (L) midgut

The subcellular distribution of four enzymes (glucose-6-phosphatase, phosphodiesterase I, NADPH-cytochrome c reductase, and p-nitroanisole O-demethylase) in the midgut of “wandering” fifth-instar larvae of the tobacco hornworm, Manduca sexta (L), was determined and the composition of mitochondrial and microsomal pellets was examined by electron microscopy. Most of the glucose-6-phosphatase activity and one-third of the phosphodiesterase I activity were found in the high-speed supernatant. NADPH-cytochrome c reductase activity was marginal and O-demethylase activity was undetectable in the supernatant. The highest specific activities for phosphodiesterase I, NADPH-cytochrome c reductase, and p-nitroanisole O-demethylase were measured in microsomes, but the relative specific activity of phosphodiesterase I was only half that obtained with the latter two enzymes. In all subcellular preparations the relative specific activities of NADPH-cytochrome c reductase and p-nitroanisole O-demethylase were closely correlated. It is concluded that glucose-6-phosphatase and phosphodiesterase I are not microsomal marker enzymes in the midgut, but the activities of NADPH-cytochrome c reductase and p-nitroanisole O-demethylase are quantitative measures of microsomal content.     

Microsomal electron transport. I. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and cytochrome P-450 as electron carriers in microsomal NADPH-peroxidase activity

An electron transport system that catalyzes the oxidation of NADPH by organic, hydroperoxides has been discovered in microsomal fractions. A tissue distribution study revealed that the microsomal fraction of rat liver was particularly effective in catalyzing the NADPH-peroxidase reaction whereas microsomes from adrenal cortex, lung, kidney, and testis were weakly active. The properties of the hepatic microsomal NADPH-peroxidase enzyme system were next examined in detail.The rate of NADPH oxidation by hydroperoxides was first-order with respect to microsomal protein concentration and a K_m value for NADPH of less than 3 μm was obtained. Examination of the hydroperoxide specificity revealed that cumene hydroperoxide and various steroid hydroperoxides were effective substrates for the enzyme system. Using cumene hydroperoxide as substrate, the reaction rate showed saturation kinetics with increasing concentrations of hydroperoxide and an apparent K_m of about 0.4 mm was obtained. The NADPH-peroxidase reaction was inhibited by potassium cyanide, half-maximal inhibition occurring at a cyanide concentration of 2.2 mm. NADH was able to support the NADPH-dependent peroxidase activity synergistically.Evidence compiled for the involvement of NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) in the NADPH-peroxidase reaction included: (1) an identical pH optimum for both activities; (2) stimulation of NADPH-peroxidase activity by increasing ionic strength; (3) inhibition by 0.05 mm, p-hydroxymercuribenzoate with partial protection by NADPH; (4) inhibition by NADP⁺; and (5) inactivation by antiserum to NADPH-cytochrome c reductase. In contrast, antibody to cytochrome b₅ did not inhibit the NADPH-peroxidase activity. Evidence for the participation of cytochrome P-450 in the NADPH-peroxidase reaction included inhibition by compounds forming type I, type II, and modified type II difference spectra with cytochrome P-450; inhibition by reagents converting cytochrome P-450 to cytochrome P-420; and marked stimulation by in vivo phenobarbital administration. The NADPH-reduced form of cytochrome P-450 was oxidized very rapidly by cumene hydroperoxide under a CO atmosphere.It was concluded that the NADPH-peroxidase enzyme system of liver microsomes is composed of the same electron transport components which function in substrate hydroxylation reactions.     

The role of individual lysine residues of horse cytochrome <Emphasis Type="Italic">c</Emphasis> in the formation of reactive complexes with components of the respiratory chain

A number of mutant forms of horse cytochrome c with single or double substitutions of lysine residues near the heme cavity involved in interaction of mitochondrial cytochrome c with ubiquinol:cytochrome c reductase (EC 1.10.2.2) (complex III) and cytochrome c oxidase (EC 1.9.3.1) (complex IV) were prepared.. The succinate:cytochrome c reductase and cytochrome c oxidase activities of mitoplasts of rat liver were measured in the presence of mutant forms of cytochrome c. The lysine residues in positions 8, 27, 72, 86, and 87 were shown to be the main contribution to the formation of a reactive complex with ubiquinol:cytochrome c reductase of the respiratory chain, whereas the lysine residues in positions 13, 79, 86, and 87 were predominantly responsible for the formation of a complex with cytochrome c oxidase.