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1.
Cytochrome bd is a bacterial respiratory oxidase carrying three hemes but no copper. We show that nitric oxide (NO) reacts with the intermediate F of cytochrome bd from Azotobacter vinelandii: (i) with a 1:1 stoichiometry, (ii) rapidly (k=1.2 +/- 0.1 x 10(5)M(-1)s(-1) at 20 degrees C), and (iii) yielding the oxidized enzyme with nitrite bound to heme d at the active site. Unexpectedly, the NO reaction mechanism of this catalytic intermediate in the Cu(B)-lacking cytochrome bd appears similar to that of beef heart cytochrome c oxidase, where Cu(B) was proposed to play a key role.  相似文献   

2.
Bacterial bd-type quinol oxidases, such as cytochrome bd from Escherichia coli, contain three hemes, but no copper. In contrast to heme-copper oxidases and similarly to globins, single electron-reduced cytochrome bd forms stable complexes with O(2), NO and CO at ferrous heme d. Kinetics of ligand dissociation from heme d(2+) in the single electron- and fully-reduced cytochrome bd from E. coli has been investigated by rapid mixing spectrophotometry at 20 degrees C. Data show that (i) O(2) dissociates at 78 s(-1), (ii) NO and CO dissociation is fast as compared to heme-copper oxidases and (iii) dissociation in the single electron-reduced state is hindered as compared to the fully-reduced enzyme. Presumably, rapid ligand dissociation requires reduced heme b(595). As NO, an inhibitor of respiratory oxidases, is involved in the immune response against microbial infection, the rapid dissociation of NO from cytochrome bd may have important bearings on the patho-physiology of enterobacteria.  相似文献   

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Cytochrome bd is a prokaryotic terminal oxidase catalyzing O2 reduction to H2O. The oxygen-reducing site has been proposed to contain two hemes, d and b595, the latter presumably replacing functionally CuB of heme-copper oxidases. We show that NO, in competition with O2, rapidly and potently (Ki = 100 +/- 34 nM at approximately 70 microM O2) inhibits cytochrome bd isolated from Escherichia coli and Azotobacter vinelandii in turnover, inhibition being quickly and fully reverted upon NO depletion. Under anaerobic reducing conditions, neither of the two enzymes reveals NO reductase activity, which is proposed to be associated with CuB in heme-copper oxidases.  相似文献   

5.
Escherichia coli GK100, with deletions in the operons encoding its two terminal oxidases, cytochrome bo and ctyochrome bd, was complemented for growth on succinate by a recombinant plasmid (pMS100) containing a 3.4-kb region of DNA from alkaliphilic Bacillus firmus OF4. The complementing DNA was predicted to encode five proteins, but neither sequence analysis nor complementation experiments with subclones provided insight into the basis for the complementation. Cytochrome difference spectra of everted membrane vesicles from the transformed strain had characteristics of a cytochrome bd spectrum but with features different from those observed for alkaliphile membranes. To determine the bacterial source and identity of the structural genes for the cytochrome bd in the transformed mutant, the complex was extracted and partially purified. On sodium dodecyl sulfate-polyacrylamide gels, two polypeptides were resolved from the preparation, 43 (subunit I) and 27 (subunit II) kDa. An internal peptide from subunit I was sequenced, and it yielded the same primary sequence as is found in positions 496 to 510 of E. coli appC. Consistent with the microsequencing results pMS100 failed to complement a triple mutant of E. coli carrying a deletion in appB as well as in the cyo and cyd loci. The deduced sequence of AppBC had been predicted to be very similar to the sequence of CydAB (J. Dassa et al., Mol. Gen. Genet. 229:341-352, 1991) but this is the first demonstration that the former is indeed a cytochrome bd terminal oxidase. The enzyme catalyzed oxygen uptake coupled to quinol or N,N,N',N'-tetramethyl-p-phenylenediamine oxidation, and the activity was sensitive to cyanide. No cross-reactivity to subunit-specific polyclonal antibodies directed against the two individual subunits of cyd-encoded cytochrome bd was detected. Since this is the second cytochrome bd discovered in E. coli, it is proposed that the two complexes be designated cytochrome bd-I (cydAB-encoded enzyme) and cytochrome bd-II (appBC-encoded enzyme). In addition, cbdAB is suggested as a more appropriate gene designation for cytochrome bd than either appBC or cyxAB.  相似文献   

6.
Cytochrome d was spectroscopically detected in membrane fractions of the amino-acid-fermenting, high-G+C gram-positive bacterium Corynebacterium glutamicum. Inhibition of NADH oxidase activity in the membranes by cyanide suggested that the main terminal respiratory oxidase during the stationary phase was a type of cytochrome bd. Cytochrome bd-type quinol oxidase, purified from the membranes, was composed of two subunits. Its reduced form showed absorption peaks at 627, 595, and 560 nm, which were due to haem d, high-spin protohaem, and low-spin protohaem, respectively. The air-oxidised form showed a peak at 645 nm, which might be due to oxygenated ferrous haem d. The spectral features and the size of subunit I are more similar to the properties of cytochromes bd from Proteobacteria, such as Escherichia coli, than to those of cytochrome bd from low-G+C gram-positive bacteria, such as Bacillus stearothermophilus. The menaquinol oxidase activity of the purified cytochrome bd was low, but was enhanced about fivefold by pre-incubating the enzyme with menaquinones. The order of effectiveness of quinols as oxidase substrates was clearly different from that of quinones as the activators of enzyme activity. Furthermore, activation was destroyed by ultraviolet irradiation of the pre-incubated enzyme and then restored by a second incubation with menaquinone. These results indicate that the enzymatic properties of this new oxidase are more similar to the properties of cytochromes bd from low-G+C gram-positive bacterial than to those of proteobacterial counterparts. They also suggest that the enzyme has a second quinone-binding site essential for full activity, in addition to the active centre for substrate oxidation. By using probes based on partial peptide sequences of the subunits, the genes for the two subunits of C. glutamicum cytochrome bd were cloned. The deduced amino acid sequence demonstrated that subunit I lacks the C-terminal half of the Q loop and that the primary structure of C. glutamicum cytochrome bd is more similar to that of other gram-positive bacteria than to proteobacterial cytochromes bd.  相似文献   

7.
The rate of reaction of trioxodinitrate with reduced cytochrome oxidase d in membrane particles from Escherichia coli at pH 7 and 25 degrees C depends linearly upon [HN2O3-] over the concentration range studied (up to 0.05 mM) and is also first-order in cytochrome d. The known rate of decomposition of trioxodinitrate to give NO- and NO2- is about 4.5-times faster than the rate of reaction of reduced cytochrome d with trioxodinitrate, implying that cytochrome d reacts directly with NO-, with a trapping ratio of between 0.20 and 0.25, rather than with trioxodinitrate. The implications of the facile formation of the NO(-)-nitrosyl complex of cytochrome d for the mechanism of denitrification are discussed with particular reference to the mechanism of N-N bond formation. The reaction of reduced cytochrome d with nitrite (a decomposition product of trioxodinitrate) under these conditions is much slower than that with trioxodinitrate. The kinetics show a biphasic dependence of initial rate upon nitrite concentration. The rate data at low [NO2-] are consistent with saturation of a high affinity site for nitrite, having Vmax = 4.29.10(-9) M s-1 and Km = 0.034 mM. The existence of two binding sites for nitrite is consistent with the suggestion that the cytochrome bd complex contains two cytochrome d haems.  相似文献   

8.
The virulences of many pathogens depend on their abilities to detoxify the immune antimicrobial nitric oxide (NO?). The functions of bacterial NO? detoxification machinery depend on oxygen (O2), with O2 inhibiting some enzymes, whereas others use it as a substrate. Previously, Escherichia coli NO? detoxification was found to be highly attenuated under microaerobic conditions and metabolic oscillations were observed. The oscillations in [NO?] and [O2] were found to result from the inhibitory action of NO? on aerobic respiration, the catalytic inactivation of NO? by Hmp (an NO? dioxygenase), and an imbalanced competition for O2 between Hmp and cytochrome terminal oxidase activity. Here the authors investigated the role of the ArcAB two component system (TCS) in microaerobic NO? detoxification. The authors observed that wild‐type, ΔarcA , and ΔarcB had comparable initial NO? clearance times; however, the mutant cultures failed to exhibit [NO?] and [O2] oscillations. Using an approach that employed experimentation and computational modeling, the authors found that the loss of oscillations in ΔarcA was due to insufficient induction of cytochrome bd ‐I expression. Collectively, these results establish ArcAB as a TCS that influences NO? detoxification in E. coli within the physiologically‐relevant microaerobic regime.  相似文献   

9.
The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.  相似文献   

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Cytochrome bd is one of the two terminal quinol oxidases in the respiratory chain of Escherichia coli. The enzyme catalyzes charge separation across the bacterial membrane during the oxidation of quinols by dioxygen but does not pump protons. In this work, the reaction of cytochrome bd with O(2) and related reactions has been studied by time-resolved spectrophotometric and electrometric methods. Oxidation of the fully reduced enzyme by oxygen is accompanied by rapid generation of membrane potential (delta psi, negative inside the vesicles) that can be described by a two-step sequence of (i) an initial oxygen concentration-dependent, electrically silent, process (lag phase) corresponding to the formation of a ferrous oxy compound of heme d and (ii) a subsequent monoexponential electrogenic phase with a time constant <60 mus that matches the formation of ferryl-oxo heme d, the product of the reaction of O(2) with the 3-electron reduced enzyme. No evidence for generation of an intermediate analogous to the "peroxy" species of heme-copper oxidases could be obtained in either electrometric or spectrophotometric measurements of cytochrome bd oxidation or in a spectrophotometric study of the reaction of H(2)O(2) with the oxidized enzyme. Backflow of electrons upon flash photolysis of the singly reduced CO complex of cytochrome bd leads to transient generation of a delta psi of the opposite polarity (positive inside the vesicles) concurrent with electron flow from heme d to heme b(558) and backward. The amplitude of the delta psi produced by the backflow process, when normalized to the reaction yield, is close to that observed in the direct reaction during the reaction of fully reduced cytochrome bd with O(2) and is apparently associated with full transmembrane translocation of approximately one charge.  相似文献   

12.
Photosynthesis is one of the most fundamental and complex mechanisms in nature. It is a well-studied process, however, some photosynthetic mechanisms are yet to be deciphered. One of the many proteins that take part in photosynthesis, cytochrome bd, is a terminal oxidase protein that plays a role both in photosynthesis and in respiration in various organisms, specifically, in cyanobacteria. To clarify the role of cytochrome bd in cyanobacteria, a system for the incorporation of an unnatural amino acid into a genomic membrane protein cytochrome bd was constructed in Synechococcus sp. PCC7942. N-propargyl- l -lysine (PrK) was incorporated into mutants of cytochrome bd. Incorporation was verified and the functionality of the mutant cytochrome bd was tested, revealing that both electrochemical and biochemical activities were relatively similar to those of the wild-type protein. The incorporation of PrK was followed by a highly specific labeling and localization of the protein. PrK that was incorporated into the protein enabled a “click” reaction in a bio-orthogonal manner through its alkyne group in a highly specific manner. Cytochrome bd was found to be localized mostly in thylakoid membranes, as was confirmed by an enzyme-linked immunosorbent assay, indicating that our developed localization method is reliable and can be further used to label endogenous proteins in cyanobacteria.  相似文献   

13.
Recent proteome studies on the Escherichia coli membrane proteins suggested that YhcB is a putative third subunit of cytochrome bd-type ubiquinol oxidase (CydAB) (F. Stenberg, P. Chovanec, S.L. Maslen, C.V. Robinson, L.L. Ilag, G. von Heijne, D.O. Daley, Protein complexes of the Escherichia coli cell envelope. J. Biol. Chem. 280 (2005) 34409-34419). We isolated and characterized cytochrome bd from the DeltayhcB strain, and found that the formation of the CydAB heterodimer, the spectroscopic properties of bound hemes, and kinetic parameters for the ubiquinol-1 oxidation were identical to those of cytochrome bd from the wild-type strain. Anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis showed that YhcB was not associated with the cytochrome bd complex. We concluded that YhcB is dispensable for the assembly and function of cytochrome bd. YhcB, which is distributed only in gamma-proteobacteria, may be a part of another membrane protein complex or may form a homo multimeric complex.  相似文献   

14.
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16.
Nitric oxide reductases (NORs) that are found in bacteria belong to the large enzyme family which includes cytochrome oxidases. Two types of bacterial NORs have been characterised. One is a cytochrome bc-type complex (cNOR) that receives electrons from soluble redox protein donors, whereas the other type (qNOR) lacks the cytochrome c component and uses quinol as the electron donor. The latter enzyme is present in several pathogens that are not denitrifiers. We summarise the current knowledge on bacterial NORs, and discuss the evolutionary relationship between them and cytochrome oxidases in this review.  相似文献   

17.
Corynebacterium glutamicum possesses two terminal oxidases, cytochrome aa3 and cytochrome bd. Cytochrome aa3 forms a supercomplex with the cytochrome bc1 complex, which contains an unusual diheme cytochrome c1. Both the bc1 -aa3 supercomplex and cytochrome bd transfer reducing equivalents from menaquinol to oxygen; however, they differ in their proton translocation efficiency by a factor of three. Here, we analyzed the role of cytochrome bd for growth and lysine production. When cultivated in glucose minimal medium, a cydAB deletion mutant of C. glutamicum ATCC 13032 grew like the wild type in the exponential phase, but growth thereafter was inhibited, leading to a biomass formation 40% less than that of the wild type. Constitutive overproduction of functional cytochrome bd oxidase in ATCC 13032 led to a reduction of the growth rate by approximately 45% and of the maximal biomass by approximately 35%, presumably as a consequence of increased electron flow through the inefficient cytochrome bd oxidase. In the L-lysine-producing C. glutamicum strain MH20-22B, deletion of the cydAB genes had only minor effects on growth rate and biomass formation, but lysine production was increased by approximately 12%. Thus, the respiratory chain was shown to be a target for improving amino acid production by C. glutamicum.  相似文献   

18.
The cydDC operon of Escherichia coli encodes an ATP-dependent transporter of unknown function that is required for cytochrome bd synthesis. Strains containing defects in either the cydD or cydC gene also demonstrate hypersensitivity to growth at high temperatures and the inability to exit the stationary phase at 37 degrees C. We wished to determine what is responsible for these hypersensitive phenotypes and whether they are due to a lack of the CydDC proteins or a defect of the cytochrome bd encoded by the cydAB genes. Using both K-12- and B-type strains of E. coli, we have compared the phenotypes of isogenic cydAB mutants and cydC mutants. In both K-12- and B-type backgrounds, the hypersensitive phenotypes are due to defects of cytochrome bd activity and not defects of the cydDC genes. We also found that the temperature-sensitive growth phenotypes can be suppressed by exogenous reducing agents, such as glutathione and cysteine. Strikingly, even the enzymes catalase and superoxide dismutase, when added exogenously, can correct the temperature-sensitive and stationary phase arrest phenotypes. We propose that the temperature sensitive growth phenotypes are due to a buildup of diffusible oxygen radicals brought on by the absence of cytochrome bd.  相似文献   

19.
The genome of Azotobacter vinelandii contains DNA sequences homologous to the structural genes for the Escherichia coli cytochrome bd terminal oxidase complex. Two recombinant clones bearing cydA- and cydB-like sequence were isolated from an A. vinelandii gene library and subcloned into the plasmid vector pACYC184. Physical mapping demonstrated that the cydA- and cydB-like regions in A. vinelandii are contiguous. The cydAB and flanking DNA was mutagenized by the insertion of Tn5-B20. Mutations in the cydB-hybridizing region resulted in the loss of spectral features associated with cytochromes b595 and d. A new locus, cydB, encoding cytochromes b595 and d in A. vinelandii is proposed. A second region adjacent to cydB was also involved in expression of the cytochrome bd complex in A. vinelandii, since mutations in this region resulted in an increase in the levels of both cytochrome b595 and cytochrome d. The regions involved in expression of the cytochrome bd complex and cydB are transcribed in the same direction. Mutants deficient in cytochromes b595 and d were unable to grow on N-deficient medium when incubated in air but could fix nitrogen when the environmental O2 concentration was reduced to 1.5% (vol/vol). It is proposed that the branch of the respiratory chain terminated by the cytochrome bd complex supports the high respiration rates required for the respiratory protection of nitrogenase.  相似文献   

20.
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