Isolation and characterization of an Escherichia coli mutant lacking cytochrome d terminal oxidase.

A screening procedure was devised which permitted the isolation of a cytochrome d-deficient mutant by its failure to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine. Cytochrome a1 and probably cytochrome b558 were also missing in the mutant. Growth and oxygen uptake rates were similar for both parent and mutant strains. However, the strain lacking cytochrome d had an increased sensitivity to cyanide, indicating that cytochrome d confers some resistance to this respiratory inhibitor. The gene responsible for these phenotypes has been named cyd and maps between tolA and sucB.     

Isolation and characterization of an Escherichia coli mutant lacking the cytochrome o terminal oxidase.

A respiration-deficient mutant of Escherichia coli has been isolated which is unable to grow aerobically on nonfermentable substrates such as succinate and lactate. Spectroscopic and immunological studies showed that this mutant lacks the cytochrome o terminal oxidase of the high aeration branch of the aerobic electron transport chain. This strain carries a mutation in a gene designated cyo which is cotransducible with the acrA locus. Mutations in cyo were obtained by mutagenizing a strain that was cyd and, thus, was lacking the cytochrome d terminal oxidase. Strain RG99, which carries both the cyd- and cyo- alleles, grows normally under anaerobic conditions in the presence of nitrate. Introduction of the cyd+ allele into the strain restores the respiration function of the strain, indicating that the cytochrome o branch of the respiratory chain is dispensable under normal laboratory growth conditions.     

Immunological characterization of an Escherichia coli strain which is lacking cytochrome d.

The isolated membranes from an Escherichia coli mutant strain which lacks spectroscopically detectable levels of cytochromes d, a1, and b558 also have abnormally low levels of N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity. In this paper, it is shown that the material previously identified as the N,N,N',N'-tetramethyl-p-phenylenediamine oxidase is, in fact, the two-subunit cytochrome d complex. Antisera directed against the native cytochrome d complex as well as against each of two subunits apparent on sodium dodecyl sulfate-polyacrylamide gels were used to show that the mutant strain lacks both subunits of the cytochrome d complex. Introduction of F-prime F152 into the mutant strain restored the two subunits along with the spectroscopic and enzymatic activity associated with the cytochrome d complex.     

Escherichia coli strains carrying the cloned cytochrome d terminal oxidase complex are sensitive to near-UV inactivation.

To determine if membrane-bound cytochromes function as endogenous near-UV photosensitizers, strains containing the cloned cydA and cydB genes were tested for near-UV sensitivity. A strain containing both cloned genes overproduced cytochromes b558, b595, and d. Another strain containing only cloned cydB overproduced cytochrome b558. Both cytochrome-overproducing strains were hypersensitive to broad-spectrum near-UV inactivation. The presence of excess cytochromes did not affect sensitivity to far-UV radiation and provided protection against H2O2 inactivation.     

Characterization of the cytochrome d terminal oxidase complex of Escherichia coli using polyclonal and monoclonal antibodies

The cytochrome d terminal oxidase complex is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli. Previous work has shown by dodecyl sulfate-polyacrylamide gel electrophoresis that this enzyme contains two subunits (I and II) and three cytochrome components, b558 , a1, and d. Reconstitution studies have demonstrated that the enzyme functions as a ubiquinol-8 oxidase and catalyzes an electrogenic reaction, i.e. turnover is accompanied by a charge separation across the membrane bilayer. In this paper, monoclonal and polyclonal antibodies were used to obtain structural information about the cytochrome d complex. It is shown that antibodies directed against subunit I effectively inhibit ubiquinol-1 oxidation by the purified enzyme in detergent, whereas antibodies which bind to subunit II have no effect on quinol oxidation. The oxidation rate of N,N,N',N'-tetramethyl-p-phenylenediamine, in contrast, is unaffected by antisubunit I antibodies, but is inhibited by antibodies against subunit II. It is concluded that the quinol oxidation site is on subunit I, previously shown to be the cytochrome b558 component of the complex, and that N,N,N',N'-tetramethyl-p-phenylenediamine oxidation occurs at a secondary site on subunit II. The antibodies were also used to analyze the results of a protein cross-linking experiment. Dimethyl suberimidate was used to cross-link the subunits of purified, solubilized oxidase. Immunoblot analysis of the products of this cross-linking clearly indicate that subunit II probably exists as a dimer within the complex. Finally, it is shown that the purified enzyme contains tightly bound lipopolysaccharide. This was revealed after discovering that one of the monoclonal antibodies raised against the purified complex is actually directed against lipopolysaccharide. The significance of this finding is not known.     

Recent studies of the cytochrome o terminal oxidase complex of Escherichia coli

The cytochrome o complex is the predominant terminal oxidase in the aerobic respiratory chain of Escherichia coli when the bacteria are grown under conditions of high aeration. The oxidase is a ubiquinol oxidase and reduces molecular oxygen to water. Electron transport through the enzyme is coupled to the generation of a protonmotive force. The purified cytochrome o complex contains four or five subunits, two protoheme IX (heme b) prosthetic groups, plus at least one Cu. The subunits are all encoded by the cyo operon. Sequence comparisons show that the cytochrome o complex is closely related to the aa3-type cytochrome c oxidase family. Gene fusions have been used to define the topology of each of the gene products. Subunits I, II, III and IV are proposed to have 15, 2, 5 and 3 transmembrane spans, respectively. The fifth gene product (cyoE) encodes a protein with 7 membrane spanning segments, and this may also be a subunit of this enzyme. Fourier transform infrared spectroscopy has been used to monitor CO bound in the active site where oxygen is reduced. These data provide definitive proof that the cytochrome o complex has a heme-copper binuclear center, similar to that present in the aa3-type cytochrome c oxidases. Site-directed mutagenesis is being utilized to define which amino acids are ligands to the heme iron and copper prosthetic groups.     

Identification of subunit I as the cytochrome b558 component of the cytochrome d terminal oxidase complex of Escherichia coli

The cytochrome d terminal oxidase complex was recently purified from Escherichia coli membranes (Miller, M. J., and Gennis , R. B. (1983) J. Biol. Chem. 258, 9159-1965). The complex contains two polypeptides, subunits I and II, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and three spectroscopically defined cytochromes, b558 , a1, and d. A mutant that failed to oxidize N,N,N',N'-tetramethyl-p-phenylenediamine was obtained which was lacking this terminal oxidase complex and was shown to map at a locus called cyd on the E. coli genome. In this paper, localized mutagenesis was used to generate a series of mutants in the cytochrome d terminal oxidase. These mutants were isolated by a newly developed selection procedure based on their sensitivity to azide. Two classes of mutants which map to the cyd locus were obtained, cydA and cydB . The cydA phenotype included the lack of all three spectroscopically detectable cytochromes as well as the absence of both polypeptides, determined by immunological criteria. Strains manifesting the cydB phenotype lacked cytochromes a1 and d, but had a normal amount of cytochrome b558 . Immunological analysis showed that subunit I (57,000 daltons) was present in the membranes, but that subunit II (43,000 daltons) was missing. These data justify the conclusion that subunit I of this two-subunit complex can be identified as the cytochrome b558 component of the cytochrome d terminal oxidase complex.     

Analysis of the topology of the cytochrome d terminal oxidase complex of Escherichia coli by alkaline phosphatase fusions

The cytochrome d complex of Escherichia coli is a heterodimer located in the bacterial cytoplasmic membrane, where it functions as a terminal oxidase of the aerobic respiratory chain. The topology of each of the two subunits of the cytochrome d complex was analysed by the genetic method involving alkaline phosphatase gene fusions. These fusions were generated by both an in vivo method using the transposon TnphoA and an in vitro method of construction. A total of 48 unique fusions were isolated and the whole-cell alkaline phosphatase-specific activities were determined. Data from these fusions, in combination with information from other studies, provide the basis for two-dimensional models for each of the two subunits, defining the way in which the subunits fold in the inner membrane of E. coli.     

The purification and characterization of the cytochrome d terminal oxidase complex of the Escherichia coli aerobic respiratory chain

The aerobic respiratory chain of Escherichia coli is branched. In aerobically grown cells harvested in midexponential phase, a respiratory chain containing only b-type cytochromes is predominant. This chain contains a terminal oxidase which is a b-type cytochrome, referred to as cytochrome o. However, when the bacteria are grown under conditions of oxygen limitation, additional components of the respiratory chain are induced, as evidenced by the appearance of new spectroscopic species. These include a new b-type cytochrome, cytochrome b558, as well as cytochrome a1 and cytochrome d. In this paper, a purification protocol and the initial characterization of the terminal oxidase complex containing cytochrome d are reported. Solubilization of the membrane is effected by Zwittergent 3-12, and purification is accomplished by chromatography with DEAE-Sepharose CL-6B and hydroxyapatite. The complex contains cytochrome b558, a1, and d. Analysis by sodium dodecyl sulfate-polyacrylamide gels indicates that the complex contains only two types of polypeptides with the molecular weights estimated to be 57,000 and 43,000. The purified complex has oxidase activity in the presence of detergents, utilizing substrates including ubinquinol-1, N,N,N',N'-tetramethyl-p-phenylenediamine, and 2,3,5,6-tetramethyl-p-phenylenediamine. The cytochrome d complex contains protoheme IX and iron, but does not contain nonheme iron or copper. Approximately half of the cytochromes which are thought to participate in E. coli aerobic respiration are accounted for by this single complex. These results suggest that the E. coli aerobic respiratory chain is organized around a relatively small number of cytochrome-containing complexes.     

Cloning of the cyo locus encoding the cytochrome o terminal oxidase complex of Escherichia coli.

The structural genes encoding the cytochrome o terminal oxidase complex (cyo) of Escherichia coli have been subcloned into the multicopy plasmid pBR322 after the Mu-mediated transposition of the gene locus from the bacterial chromosome onto the conjugative R plasmid RP4. Introduction of cyo plasmids into strains (cyo cyd) lacking both terminal oxidases restored the ability of the strains to grow aerobically on nonfermentable substrates. Strains carrying the cyo plasmids produced 5 to 10 times more cytochrome o oxidase than did control strains. The gene products encoded by the cyo plasmids could be immunoprecipitated with monospecific antibodies raised against cytochrome o. The cloned genes will be valuable for studying the structure, function, and regulation of the cytochrome o terminal oxidase complex.     

Reconstitution of the Ubiquinone-dependent pyruvate oxidase system of Escherichia coli with the cytochrome o terminal oxidase complex

The aerobic respiratory chain of Escherichia coli is branched and contains two terminal oxidases. The chain predominant when the cells are grown with low aeration terminates with the cytochrome d terminal oxidase complex, and the branch present under high aeration ends with the cytochrome o terminal oxidase complex. Previous work has shown that cytochrome d complex functions as a ubiquinol-8 oxidase, and that a minimal respiratory chain can be reconstituted in proteoliposomes with a flavoprotein dehydrogenase (pyruvate oxidase), ubiquinone-8, and the cytochrome d complex. This paper demonstrates that the cytochrome o complex functions as an efficient ubiquinol-8 oxidase in reconstituted proteoliposomes, and that ubiquinone-8 serves as an electron carrier from the flavoprotein to the cytochrome complex. The maximal turnover (per cytochrome o) achieved in reconstituted proteoliposomes is at least as fast as observed in E. coli membrane preparations. Electron flow from the flavoprotein to oxygen in the reconstituted proteoliposomes generates a transmembrane potential of at least 120 mV, negative inside, which is sensitive to ionophore uncouplers and inhibitors of the terminal oxidase. These data demonstrate the minimal composition of this respiratory chain as a flavoprotein dehydrogenase, ubiquinone-8, and the cytochrome o complex. Previous models have suggested that cytochrome b556, also a component of the E. coli inner membrane, is required for electron flow to cytochrome o. This is apparently not the case. It now is clear that both of the E. coli terminal oxidases act as ubiquinol-8 oxidases and, thus, ubiquinone-8 is the branch point between the two respiratory chains.     

Location of heme axial ligands in the cytochrome d terminal oxidase complex of Escherichia coli determined by site-directed mutagenesis

The cytochrome d terminal oxidase complex is one of two terminal oxidases which are components of the aerobic respiratory chain of Escherichia coli. This membrane-bound enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Enzyme turnover generates proton and voltage gradients across the bilayer. The oxidase is a heterodimer containing 2 mol of protoheme IX and 1 or 2 mol of heme d per mol of complex. To explain the functional properties of the enzyme, a simple model has been proposed in which it is speculated that the heme prosthetic groups define two separate active sites on opposite sides of the membrane at which the oxidation of quinol and the reduction of water, respectively, are catalyzed. This paper represents an initial effort to define the axial ligands of each of the three or four hemes within the amino acid sequence of the oxidase subunits. Each of the 10 histidine residues has been altered by site-directed mutagenesis with the expectation that histidine residues are likely candidates for heme ligands. Eight of the 10 histidine residues are not essential for enzyme activity, and 2 appear to function as heme axial ligands. Histidine 186 in subunit I is required for the cytochrome b558 component of the enzyme. This residue is likely to be located near the periplasmic surface of the membrane. Histidine 19, near the amino terminus of subunit I also appears to be a heme ligand. It is concluded that two of the four or five expected heme axial ligands have been tentatively identified, although further work is required to confirm these conclusions. A minimum of two additional axial ligands must be residues other than histidine.     

Identification of the cydC locus required for expression of the functional form of the cytochrome d terminal oxidase complex in Escherichia coli.

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases which are differentially regulated. The cytochrome o complex predominates under growth conditions of high aeration, whereas the cytochrome d complex predominates when the oxygen tension is low. Either terminal oxidase will support aerobic growth. The goal of the work presented in this paper was to identify genes required for the expression of the functional form of the cytochrome d complex, other than the genes encoding the polypeptide components of the oxidase complex (cyd locus). A strain lacking the cytochrome o complex (cyo mutant strain) was mutagenized by using a lambda-Mu hybrid hopper bacteriophage, lambda placMu53, which inserts randomly into the chromosome and carries a kanamycin resistance marker. Strains were isolated and examined which were unable to grow aerobically, i.e., which lacked functional cytochrome d complex, and which could not be complemented by introduction of the cyd gene on F-prime episomes. One strain was selected for characterization. The phage insert was mapped to min 18.9 on the genetic linkage map, defining a new genetic locus, cydC. Evidence described in the text suggests that the gene product is probably required for the synthesis of the unique heme d component of the cytochrome d complex.     

Reconstitution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with the cytochrome d terminal oxidase

Pyruvate oxidase is a flavoprotein dehydrogenase located on the inner surface of the Escherichia coli cytoplasmic membrane and coupled to the E. coli aerobic respiratory chain. In this paper, the role of quinones in the pyruvate oxidase system is investigated, and a minimal respiratory chain is described consisting of only two pure proteins plus ubiquinone 8 incorporated in phospholipid vesicles. The enzymes used in this reconstitution are the flavoprotein and the recently purified E. coli cytochrome d terminal oxidase. The catalytic velocity of the reconstituted liposome system is about 30% of that observed when the flavoprotein is reconstituted with E. coli membranes. It is also shown that electron transport from pyruvate to oxygen in the liposome system generates a transmembrane potential of at least 180 mV (negative inside), which is sensitive to the uncouplers carbonyl cyanide p-(tri-chloromethoxy)phenylhydrazone and valinomycin. A trans-membrane potential is also generated by the oxidation of ubiquinol 1 by the terminal oxidase in the absence of the flavoprotein. It is concluded that (1) the flavoprotein can directly reduce ubiquinone 8 within the phospholipid bilayer, (2) menaquinone 8 will not effectively substitute for ubiquinone 8 in this electron-transfer chain, and (3) the cytochrome d terminal oxidase functions as a ubiquinol 8 oxidase and serves as a "coupling site" in the E. coli aerobic respiratory chain. These investigations suggest a relatively simple organization for the E. coli respiratory chain.     

Use of an azido-ubiquinone derivative to identify subunit I as the ubiquinol binding site of the cytochrome d terminal oxidase complex of Escherichia coli

The radiolabeled, photoreactive azido-ubiquinone derivative (azido-Q), 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl-[3H]octyl)- 1,4-benzoquinone, was used to investigate the active site of ubiquinol oxidase activity of the cytochrome d complex, a two-subunit terminal oxidase of Escherichia coli. The azido-Q, when reduced by dithioerythritol, was shown to support enzymatic oxygen consumption by the cytochrome d complex that was 8% of the rate observed with ubiquinol-1. This observation provided the rationale behind further studies of the possible photoinactivation and labeling of the active site by this azido-Q. Ten min of photolysis of the purified cytochrome d complex in the presence of the azido-Q resulted in a 60% loss of the ubiquinol-1 oxidase activity. Uptake of the radiolabeled azido-Q by the cytochrome d complex was correlated to the photoinactivation of the ubiquinol-1 oxidase activity. Both increased linearly during the first 4 min of photolysis and reached 90% of the maximum within 10 min. Photolysis times longer than 10 min resulted in no increase in the maximum of 2 mol of azido-Q incorporated per mol of enzyme. The rate of azido-Q uptake by subunit I, but not subunit II, correlated well with the rate of loss of ubiquinol oxidase activity. Use of ubiquinol-0, which is not oxidized by the enzyme, to competitively inhibit radiolabeling of nonspecific binding sites, resulted in a significant decrease (42%) of azido-Q labeling of subunit II while it did not affect the labeling of subunit I. After photolysis for 4 min, the ratio of radiolabeled azido-Q in subunits I to II of the complex was 4.3 to 1.0. These observations support the conclusion that the ubiquinol substrate binding site is located on subunit I of the cytochrome d complex.     

Isolation and characterization of a new class of cytochrome d terminal oxidase mutants of Escherichia coli.

Cytochrome d terminal oxidase mutants were isolated by using hydroxylamine mutagenesis of pNG2, a pBR322-derived plasmid containing the wild-type cyd operon. The mutagenized plasmid was transformed into a cyo cyd recA strain, and the transformants were screened for the inability to confer aerobic growth on nonfermentable carbon sources. Western blot analysis and visible-light spectroscopy were performed to characterize three independent mutants grown both aerobically and anaerobically. The mutational variants of the cytochrome d complex were stabilized under anaerobic growth conditions. All three mutations perturb the b595 and d heme components of the complex. These mutations were mapped and sequenced and are shown to be located in the N-terminal third of subunit II of the cytochrome d complex. It is proposed that the N terminus of subunit II may interact with subunit I to form an interface that binds the b595 and d heme centers.     

Identification of a ferryl intermediate of Escherichia coli cytochrome d terminal oxidase by resonance Raman spectroscopy.

The 680-nm-absorbing "peroxide state" of the Escherichia coli cytochrome d terminal oxidase complex, obtained by addition of excess hydrogen peroxide to the enzyme, is shown to be a ferryl intermediate in the catalytic cycle of the enzyme. This ferryl intermediate is also created by aerobic oxidation of the fully reduced enzyme. Resonance Raman spectra with 647.1-nm excitation show an FeIV = O stretching band at 815 cm-1, a higher frequency than noted in any other ferryl-containing enzyme to date. The band shows an 16O/18O frequency shift of -46 cm-1, larger than that observed for any porphyrin ferryl species. The FeIV = O formulation was unambiguously established by oxidations of the reduced enzyme with 16O2, 18O2, and 16O18O. Only the use of a mixed-isotope gas permitted discrimination between a ferryl and a peroxo structure. A catalytic cycle for the cytochrome d terminal oxidase complex is proposed, and possible reasons for the high v(Fe = O) frequency are discussed.     

Immunological characterization of the cytochrome o terminal oxidase from Escherichia coli

The cytochrome o terminal oxidase from Escherichia coli was immunochemically purified and monospecific antiserum toward cytochrome o was obtained. This antiserum is able to precipitate 100% of the ubiquinol-1 oxidase activity in Triton X-100 extracts of membranes from an E. coli strain in which cytochrome o is the only terminal oxidase. Cytochrome o was analyzed and quantitated using crossed immunoelectrophoresis, rocket immunoelectrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that cytochrome o is composed of four subunits of approximate equimolar stoichiometry with molecular weights of 51,000, 28,500, 18,000, and 12,700. The low temperature (77 K) reduced - oxidized spectrum of the immunoprecipitate shows two peaks at 555 and 562 nm, indicating b-type cytochromes. With the anti-cytochrome o and antiserum toward the cytochrome d terminal oxidase complex which was previously obtained, it is possible to immunochemically assay for all the cytochromes in the cytoplasmic membrane of aerobically grown E. coli. Preliminary results indicate that the biosynthesis of cytochrome o is repressed when cytochrome d is induced by lowering the dissolved oxygen concentration during cell growth.