Bo-type Oxidase Research Articles

The Energetic Conversion Competence of Escherichia coli during Aerobic Respiration Studied by 31P NMR Using a Circulating Fermentation System

To determine the actual potential of the energetic conversion efficiency of Escherichia coli during aerobic respiration, apparent P/O ratios (P/O(app)) under either limited or standard glucose-feeding conditions were estimated. The previously reported circulating fermentation system (CFS) was used, and (31)P NMR saturation-transfer (ST) techniques were employed. By coupling with on-line NMR observations, CFS allowed us to evaluate cellular energetics directly, with both the dissolved oxygen tension and glucose feeding precisely controlled to prevent the effect of substrate-level phosphorylation based on aerobic or anaerobic acidogenesis in E. coli cells. Phosphate consumption rates under standard and limited glucose-conditions were estimated as 4.62 +/- 0.46 and 1.99 +/- 0.11 micromol/s g of dry cell weight (DCW), respectively. Using simultaneously assessed O(2) consumption rates, the P/O(app) values under these two conditions were estimated as 1.4 +/- 0.3 and 1.5 +/- 0.1, respectively. To correlate the obtained P/O(app) values with the potential efficiency of respiratory enzymes, we determined the activities of two NADH dehydrogenases (NDH 1 and 2) and two ubiquinol oxidases (bo- and bd-type) during the periods when ST was performed. NDH-1 activities in standard or limited glucose cultures were maintained at 57% or 58% of the total NADH oxidizing activity. The percentages of bo-type oxidase activity in relation to the total ubiqinol oxidizing activity under the standard and limited glucose conditions were 32% and 36%, respectively. These percentages of enzymatic activities represent the respiratory competence of E. coli cells, suggesting that, during the NMR observatory period, the enzymatic activity was not at a maximum, which could also explain the estimated P/O(app) values. If this is the case, enhancing the expression of the bo-type oxidase or disrupting of the bd-type oxidase gene could be effective approach to increasing both the P/O ratio and cellular yields.

Energy conservation in aerobically grown Staphylococcus aureus

The present studies provide new data on the involvement of menaquinol oxidases in substrate oxidation and energy conservation in aerobically grown, resting cells of Staphylococcus aureus 17810R, starved of endogenous energy reserves and supplemented with glutamate or L-lactate. These cells were energetically competent, since they oxidized both substrates, generated an electrochemical proton gradient (Δμ H + ) and synthesized ATP via oxidative phosphorylation. Studies with KCN showed that: (i) L-lactate oxidation occurred via two terminal menaquinol oxidases – the ba 3-type sensitive to low KCN and the bo-type insensitive to cyanide, (ii) glutamate oxidation proceeded via the bo-type oxidase, and (iii) ATP synthesis with glutamate or L-lactate was coupled only to the bo-type oxidase. Also in glucose-grown cells oxidizing L-lactate, ATP synthesis was coupled to the highly repressed bo-type oxidase. It is suggested that in the respiratory chain of strain 17810R two energy coupling sites may be present: in the complex of NADH-menaquinone oxidoreductase and in the complex of the bo-type menaquinol oxidase. The rate of ATP synthesis was similar with both substrates, but the rate of their oxidation differed significantly: the P/O ratios were 1.5 and 0.03 with glutamate and L-lactate, respectively. CCCP accelerated glutamate oxidation by 50% but was without effect on L-lactate oxidation. In cell lysates, the rates of NADH and L-lactate oxidation were equal. It is concluded that in whole cells of S. aureus 17810R oxidation of NADH derived from glutamate breakdown is tightly coupled to phosphorylation, while L-lactate oxidation seems to be rather loosely coupled.

Facilitated intramolecular electron transfer in cytochrome bo-type ubiquinol oxidase initiated upon reaction of the fully reduced enzyme with dioxygen

Flow-flash and double-flash studies of the reaction of fully reduced bo-type quinol oxidase with oxygen have revealed that a single turnover of the enzyme proceeds much faster than mammalian cytochrome c oxidase. Facilitated intramolecular electron transfer in the bo-type oxidase with k ⪢ 5 × 10 4 s −1 at pH 7.4 and 20°C is responsible for this fast turnover. The kinetics of this reaction indicates that the oxygen reduction does not require electron exchange between quinol oxidase molecules, each having three metal centers. Thus, a bound quinol in the fully reduced enzyme is suggested to be an electron source for complete reduction of dioxygen into water supplementing electrons provided by the metal centers. A single turnover of the quinol oxidase yields a novel spectral species with a Soret maximum at 415 nm corresponding to a ‘pulsed’ state of mammalian cytochrome c oxidase.

Identity of the axial ligand of the high-spin heme in cytochrome oxidase: Spectroscopic characterization of mutants in the bo-type oxidase of Escherichia coli and the aa3-type oxidase of Rhodobacter sphaeroides

Prokaryotic and eukaryotic cytochrome c oxidases and several bacterial ubiquinol oxidases compose a superfamily of heme-copper oxidases. These enzymes are terminal components of aerobic respiratory chains, the principal energy-generating systems of aerobic organisms. Two such heme-copper oxidases are the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides and the bo-type ubiquinol oxidase of Escherichia coli. These enzymes catalyze the reduction of oxygen to water at a heme-copper binuclear center. Energy conservation is accomplished by coupling electron transfer through the metals of the oxidases to proton translocation across the cellular membrane. The Rb. sphaeroides and E. coli enzymes have previously been utilized in site-directed mutagenesis studies which identified two histidines which bind the low-spin heme (heme a), as well as additional histidine residues which are probable ligands for copper (CuB). However, the histidine that binds the heme of the binuclear center (heme a3) could not be unequivocally identified between two residues (His284 and His419). Additional characterization by Fourier transform infrared spectroscopy of the CO-bound forms of the E. coli enzyme in which His284 is replaced by glycine or leucine demonstrates that these mutations cause only subtle changes to CO bound to the heme of the binuclear center. Resonance Raman spectroscopy of the Rb. sphaeroides enzyme in which His284 is replaced by alanine shows that the iron-histidine stretching mode of heme a3 is maintained, in contrast with the loss of this mode in mutants at His419. These results demonstrate that His284 is not the heme a3 ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Energetic efficiency of Escherichia coli: effects of mutations in components of the aerobic respiratory chain

The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type). The amounts of each of these enzymes present in the E. coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration. Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force. The same is true for the two ubiquinol oxidases. Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized. In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated. The results are compatible with the proton translocation values of the various components reported from in vitro measurements. The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.

Site-directed mutants of the cytochrome bo ubiquinol oxidase of Escherichia coli: amino acid substitutions for two histidines that are putative CuB ligands.

The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of structurally related heme-copper respiratory oxidases. The members of this family, which also includes the aa3-type cytochrome c oxidases, contain at least two heme prosthetic groups, a six-coordinate low-spin heme, and a high-spin heme. The high-spin heme is magnetically coupled to a copper, CuB, forming a binuclear center which is the site of oxygen reduction to water. Vectorial proton translocation across the membrane bilayer appears to be another common feature of this superfamily of oxidases. It has been proposed previously that the two adjacent histidines in putative transmembrane helix VII (H333 and H334 in the E. coli sequence) of the largest subunit of the heme-copper oxidases are ligands to CuB. Previously reported mutagenesis studies of the E. coli bo-type oxidase and the aa3-type oxidase of Rhodobacter sphaeroides supported this model, as substitutions at these two positions produced nonfunctional enzymes but did not perturb the visible spectra of the two heme groups. In this work, six different amino acids, including potential copper-liganding residues, were substituted for H333 and H334 of the E. coli oxidase. All of the mutations resulted in inactive, but assembled, oxidase with both of the heme components present. However, cryogenic Fourier transform infrared (FTIR) spectroscopy of the CO adducts revealed that dramatic changes occur at the binuclear center as a result of each mutation and that CuB appears to be absent.(ABSTRACT TRUNCATED AT 250 WORDS)

Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity.

The cytochrome o complex is a bo-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. This complex has a close structural and functional relationship with the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. The specific activity, subunit composition, and metal content of the purified cytochrome o complex are not consistent for different preparative protocols reported in the literature. This paper presents a relatively simple preparation of the enzyme starting with a strain of Escherichia coli which overproduces the oxidase. The pure enzyme contains four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Partial amino acid sequence data confirm the identities of subunit I, II, and III from the SDS-PAGE analysis as the cyoB, cyoA, and cyoC gene products, respectively. A slight modification of the purification protocol yields an oxidase preparation that contains a possible fifth subunit which may be the cyoE gene product. The pure four-subunit enzyme contains 2 equivs of iron but only 1 equiv of copper. There is no electron paramagnetic resonance detectable copper in the purified enzyme. Hence, the equivalent of CuA of the aa3-type cytochrome c oxidases is absent in this quinol oxidase. There is also no zinc in the purified quinol oxidase. Finally, monoclonal antibodies are reported that interact with subunit II. One of these monoclonals inhibits the quinol oxidase activity of the detergent-solubilized, purified oxidase. Hence, although subunit II does not contain CuA and does not interact with cytochrome c, it still must have an important function in the bo-type ubiquinol oxidase.

Nucleotide Sequence of the Gene Coding for Four Subunits of Cytochrome c Oxidase from the Thermophilic Bacterium PS31

The gene coding for four subunits of cytochrome aa3-type oxidase was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced. The N-terminus of each subunit was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequences contained 615 amino acid residues for subunit I (CO1/caaB product), 333 residues for subunit II (CO2/caaA product), 207 residues for subunit III (CO3/caaC product), and 109 residues for subunit IV (CO4/caaD product) after processing. Re-examination of the sequencing of caa revealed a longer open reading frame for CO1, which contains 14 transmembrane segments instead of 12 [Sone et al. (1988) J. Biochem. 103, 606-610], although the main portions of the sequences constituting cytochrome a (FeA), cytochrome a3 (FeB), and CuB are correct. PS3 CO2 has an additional sequence for cytochrome c after the CuA binding protein portion with 2 transmembrane segments, which is homologous to the mitochondrial counterpart. PS3 CO3 has DCCD-binding glutamyl residues but contains only 5 transmembrane segments, unlike the mitochondrial counterpart, which has 7 segments. The subunits of PS3 cytochrome oxidase (aa3-type) show clear similarity in amino acid sequences with those of cytochrome bo-type oxidase from Escherichia coli as well, in spite of the difference of hemes. PS3 CO3 and CO4 are much more similar to E. coli CO3 and CO4 than to mitochondrial CO3 and CO4, respectively.