Oxidase and periplasmic cytochrome assembly in Escherichia coli K-12: CydDC and CcmAB are not required for haem-membrane association. (1/51)

The mechanism(s) that bacteria use to transport haem into and across the cytoplasmic membrane to complete the assembly of periplasmic cytochromes is unknown. The authors have tested directly the role(s) of two ATP-binding cassette (ABC) transporters - the cydDC and ccmAB gene products - in Escherichia coli by measuring haem uptake in everted (inside-out) membrane vesicles. If haem is exported to the periplasm in vivo, the same process should result in active accumulation in such everted vesicles. [14C]Haemin (chloride) with bovine serum albumin (BSA) as a carrier protein was accumulated in intact everted membrane vesicles by an energy-independent mechanism. The kinetics of this process were biphasic: rapid uptake/binding was followed by a slower uptake of haem, which was inhibited by a large excess of unlabelled haemin-BSA, but not by BSA. However, accumulated haemin was not chased out of the vesicles by unlabelled haemin-BSA, suggesting specific binding of haemin with the membrane or transport into the lumen of the vesicle. Neither ATP nor a protonmotive force (delta(p)) generated by lactate oxidation was required for haemin binding or subsequent transport, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), sodium vanadate and monensin had no effect on haemin transport. The rate of haemin uptake following the initial rapid binding was proportional to the external haemin concentration, suggesting that the uptake process was driven by the haemin concentration gradient across the cell membrane. The kinetics of [14C]haemin uptake were similar in wild-type and cydD1 or delta(ccmA) mutants, suggesting that the activity of neither the CydDC nor CcmAB transporters is essential for haem export to the periplasm. Cytochrome d levels were unaffected by mutations in trxB (encoding thioredoxin reductase), trxA (thioredoxin), or grx (glutaredoxin), suggesting that the CydDC transporter does not export these components of reducing pathways for cytochrome assembly.  (+info)

The binding of cyanide to cytochrome d in intact cells, spheroplasts, membrane fragments and solubilized enzyme from Salmonella typhimurium. (2/51)

This investigation focused on the kinetics of cyanide binding to oxidized and reduced cytochrome d in Salmonella typhimurium intact cells, spheroplasts, membrane fragments and solubilized enzyme, and on the effect of pH on this binding. Cyanide bound to the oxidized form of cytochrome d under all experimental conditions, inducing a trough at 649 nm in the oxidized-cyanide-minus-oxidized difference absorption spectra. V(max) of cyanide binding to oxidized cytochrome d at pH 7.0 was 14.0+/-2.0 pmol/min/mg protein (prot.) in intact cells, 37.0+/-3.5 pmol/min/mg prot. in spheroplasts, 125.0+/-6.0 pmol/min/mg prot. in membrane fragments, and 538.0+/-8.5 pmol/min/mg prot. in solubilized cytochrome d. The pseudo-first order rate constants were 0.004 s(-1) for intact cells, 0.005 s(-1) for spheroplasts, 0.007 s(-1) for membrane fragments and 0.025 s(-1) for the solubilized enzyme. The V(max) value was highest at pH 7.0 for intact cells and solubilized cytochrome d and at pH 8.0 for both spheroplasts and membrane fragments. The K(s) of binding at pH 7.0 was around 4 mM in intact cells, spheroplasts and membrane fragments, but was 10.5 mM in solubilized cytochrome d. This difference between the K(s) values suggested a change in conformation, upon solubilization, leading to a decrease in the affinity of cyanide for the solubilized enzyme. The K(s) value was nearly the same at all pH investigated (pH 5-10). Cyanide was found to also bind to the reduced form of cytochrome d in membrane fragments (K(s)=18+/-3 mM, V(max)=377+/-28 pmol/min/mg prot. at pH 7) and the solubilized enzyme (K(s)=18+/-1.2 mM, V(max)=649+/-45 pmol/min/mg prot. at pH 7) with a lower affinity of cyanide for the reduced cytochrome d than for the oxidized enzyme. Pseudo-first order rate constants were 0.025 s(-1) and 0.042 s(-1) respectively for membrane fragments and solubilized enzyme. The value of V(max) for cyanide binding to the reduced cytochrome d, whether membrane-bound or solubilized, increased slightly with pH (for pH 6-10) while the K(s) value dropped significantly with increasing pH. The pH dependence observed here might be interpretable as a possible role for conformational transition associated with energy transduction. Finally, this investigation pointed to the influence of the microenvironment of a protein within the cell on its reactivity.  (+info)

pH dependence of proton translocation by Escherichia coli. (3/51)

Proton translocation in spheroplasts from Escherichia coli has been studied in two mutants, one of which expresses cytochrome o and the other cytochrome d as the terminal oxidase. Using the O2 pulse method, the H+/e- ratio of proton translocation associated with cytochrome o was confirmed to be near 2 at neutral pH, but was found to decrease considerably when the medium pH was raised above 8. At high pH there was an increase in H+/OH- permeability of the cell membrane, but this was not sufficient to explain the decline in proton ejection. The pH effect was confined to cytochrome o-linked activity. It was not present when cytochrome d generated the electrochemical proton gradient. This makes it improbable that the Na+/H+ antiporter is responsible. The most likely explanation for our finding is that there is a "slip" in the proton-pumping mechanism of cytochrome o at high pH.  (+info)

arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochrome d operon. (4/51)

In a screen for Escherichia coli genes whose products are required for high-temperature growth, we identified and characterized a mini-Tn10 insertion that allows the formation of wild-type-size colonies at 30 degrees C but results in microcolony formation at 36 degrees C and above (Ts- phenotype). Mapping, molecular cloning, and DNA sequencing analyses showed that the mini-Tn10 insertion resides in the cydB gene, the distal gene of the cydAB operon (cytochrome d). The Ts- growth phenotype was also shown to be associated with previously described cyd alleles. In addition, all cyd mutants were found to be extremely sensitive to hydrogen peroxide. Northern (RNA) blot analysis showed that cyd-specific mRNA levels accumulate following a shift to high temperature. Interestingly, this heat shock induction of the cyd operon was not affected in an rpoH delta background but was totally absent in an arcA or arcB mutant background. Extragenic suppressors of the Cyd Ts- phenotype are found at approximately 10(-3). Two extragenic suppressors were shown to be null alleles in either arcA or arcB. One interpretation of our results is that in the absence of ArcA or ArcB, which are required for the repression of the cyo operon (cytochrome o), elevated levels of Cyo are produced, thus compensating for the missing cytochrome d function. Consistent with this interpretation, the presence of the cyo gene on a multicopy plasmid suppressed the Ts- and hydrogen peroxide-sensitive phenotypes of cyd mutants.  (+info)

Evidence for multiple terminal oxidases, including cytochrome d, in facultatively alkaliphilic Bacillus firmus OF4. (5/51)

The terminal oxidase content of Bacillus firmus OF4, a facultative alkaliphile that grows well over the pH range of 7.5 to 10.5, was studied by difference spectroscopy. Evidence was found for three terminal oxidases under different growth conditions. The growth pH and the stage of growth profoundly affected the expression of one of the oxidases, cytochrome d. The other two oxidases, cytochrome caa3 and cytochrome o, were expressed under all growth conditions tested, although the levels of both, especially cytochrome caa3, were higher at more alkaline pH (P.G. Quirk, A.A. Guffanti, R.J. Plass, S. Clejan, and T.A. Krulwich, Biochim. Biophys. Acta, in press). These latter oxidases were identified in everted membrane vesicles by reduced-versus-oxidized difference spectra (absorption maximum at 600 nm for cytochrome caa3) and CO-reduced-versus-reduced difference spectra (absorption maxima at 574 and 414 nm for cytochrome o). All three terminal oxidases were solubilized from everted membranes and partially purified. The difference spectra of the solubilized, partially purified cytochrome caa3 and cytochrome o complexes were consistent with these assignments. Cytochrome d, which has not been identified in a Bacillus species before, was tentatively assigned on the basis of its absorption maxima at 622 and 630 nm in reduced-versus-oxidized and CO-reduced-versus-reduced difference spectra, respectively, resembling the maxima exhibited by the complex found in Escherichia coli. The B. firmus OF4 cytochrome d was reducible by NADH but not by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine in everted membrane vesicles. Cytochrome d was expressed under two conditions: in cells growing exponentially at pH 7.5 (but not at pH 10.5) and in cells stationary phase at either pH 7.5 or 10.5. Protein immunoblots with antibodies against subunit I of the E. coli cytochrome d complex reacted only with membrane vesicles that contained spectrally identifiable cytochrome d. Additional evidence that this B. firmus OF4 cytochrome is related to the E. coli complex was obtained with a solubilized, partially purified fraction of cytochrome d that also reacted with antibodies against the subunits of the E. coli cytochrome d.  (+info)

Isolation and characterization of a new class of cytochrome d terminal oxidase mutants of Escherichia coli. (6/51)

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.  (+info)

Cloning, characterization, and expression in Escherichia coli of the genes encoding the cytochrome d oxidase complex from Azotobacter vinelandii. (7/51)

Azotobacter vinelandii is a free-living nitrogen-fixing bacterium that has one of the highest respiratory rates of all aerobic organisms. Based on various physiological studies, a d-type cytochrome has been postulated to be the terminal oxidase of a vigorously respiring but apparently uncoupled branch of the electron transport system in the membranes of this organism. We cloned and characterized the structural genes of the two subunits of this oxidase. The deduced amino acid sequences of both subunits of the A. vinelandii oxidase have extensive regions of homology with those of the two subunits of the Escherichia coli cytochrome d complex. Most notably, the histidine residues proposed to be the axial ligands for the b hemes of the E. coli oxidase and an 11-amino-acid stretch proposed to be part of the ubiquinone binding site are all conserved in subunit I of the A. vinelandii oxidase. The A. vinelandii cytochrome d was expressed in a spectrally and functionally active form in the membranes of E. coli, under the control of the lac or tac promoter. The spectral features of the A. vinelandii cytochrome d expressed in E. coli are very similar to those of the E. coli cytochrome d. The expressed oxidase was active as a quinol oxidase and could reconstitute an NADH to oxygen electron transport chain.  (+info)

Transcriptional regulation of cytochrome d in nitrogen-fixing Azotobacter vinelandii. Evidence that up-regulation during N2 fixation is independent of nifA but dependent on ntrA. (8/51)

Cytochrome d has been postulated to be the "respiratory protection" oxidase of Azotobacter vinelandii, allowing this organism to fix nitrogen under aerobic growth conditions. We have previously cloned and characterized the structural genes for the A. vinelandii cytochrome d (cydA and cydB). The cyd genes are co-transcribed, yielding an mRNA of approximately 3.6 kilobase pairs. The level of the cyd message was 2-3-fold higher in cells that were fixing nitrogen, as compared with non-nitrogen-fixing cells. RNase protection analysis was used to determine the transcriptional start site at 275 bases upstream of the initiator ATG of cydA, and this start site was the same for nitrogen-fixing and non-nitrogen-fixing cells. The cyd promoter has sequence similarities to the canonical Escherichia coli promoters, which are transcribed by the major sigma 70 form of RNA polymerase. Plasmid-borne lacZ transcriptional fusions were constructed, using approximately 650 base pairs of 5'-upstream sequences of the cyd structural genes. This region had a strong promoter activity which was further up-regulated 1.5-2.5-fold upon the induction of nitrogen fixation. The cyd-lacZ fusions were characterized in a nifA- as well as an ntrA- background. Mutations in neither of these nif regulatory genes affected the constitutive expression of cyd under non-nitrogen-fixing conditions. However, the up-regulation of this promoter during the induction of nitrogen fixation was abolished only in the ntrA- background. Based on these results, the cytochrome d promoter of A. vinelandii belongs to a new class of nitrogen-regulated promoters which, unlike the authentic nif genes, does not require the ntrA gene product for its expression. The up-regulation of this promoter during nitrogen fixation, however, requires the ntrA gene product.  (+info)