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1.
We have investigated ferrocytochrome c-induced proton ejection from reconstituted cytochrome c oxidase-containing vesicles using careful control of the number of enzyme turnovers. Ferrocytochrome c caused the appearance of protons at the vesicle exterior, and this could be abolished by using a protonophore. In addition, its decay was dependent on the permeability of the vesicle membranes to protons and the number of turnovers of the oxidase. These observations indicate that the ejection of protons was the result of genuine translocation. The possibility of this translocation occurring via a Mitchellian loop as a result of the presence of a reduced hydrogen carrier contaminating the enzyme was considered and excluded. Proton-translocating activity in this reconstituted system depended critically on the ratio of enzyme to lipid used in the reconstitution process and we propose a rationale to account for this. We conclude that our data provide strong support for the proposal that cytochrome c oxidase acts as a proton pump and that approx. 0.9 H+ is excluded per ferrocytochrome c molecule oxidized.  相似文献   

2.
A quantitative analysis of H+ extrusion by reconstituted cytochrome c oxidase vesicles is presented with particular regard to the decay kinetics of the extruded proton pulse and to the structural heterogeneity of the vesicle preparation. The decay of the extruded H+ pulse under conditions typical of those used for its measurement is much slower than expected from the passive proton permeability of the vesicle membranes. It is shown that this apparent anomaly results from insufficient transmembrane charge equilibration via valinomycin and K+ during oxidase turnover. This situation can be remedied by increasing the valinomycin concentration or by replacing this counterion system with 1 mM tetraphenylphosphonium. Under these latter conditions, the decay kinetics can be described as the sum of two exponential terms. To facilitate interpretation of the proton pump decay kinetics, a structural analysis of the oxidase vesicle preparation is presented. The bulk of the reconstituted vesicles (i.e., those representing approx. 80% of the total oxidase and lipid) are 30-62 nm in diameter. At least 70% of the reconstituted oxidase molecules are contained individually in separate vesicles, indicating that the enzyme monomer is competent in H+ translocation.  相似文献   

3.
T Nilsson  J Gelles  P M Li  S I Chan 《Biochemistry》1988,27(1):296-301
Cytochrome c oxidase in which the CuA site has been perturbed by extensive modification of the enzyme with the thiol reagent p-(hydroxymercuri)benzoate has been reconstituted into phospholipid vesicles. The reconstituted vesicles lack respiratory control, and the orientation of the enzyme in the vesicles is similar to that of the native cytochrome c oxidase. In the proton translocation assay, the vesicles containing the modified enzyme behave as if they are unusually permeable to protons. When the modified and native proteins were coreconstituted, a substantial portion of the latter became uncoupled as revealed by low respiratory control and low overall proton pumping activity. These results suggest that the modified enzyme catalyzes a passive transport of protons across the membrane. When milder conditions were used for the chemical modification, a majority of the thiols reacted while the CuA site remained largely intact. Reconstitution of such a partially modified cytochrome c oxidase produced vesicles with respiratory control and proton translocating activity close to those of reconstituted native enzyme. It thus appears that the appearance of a proton leak is related to the perturbation of the CuA site. These observations suggest that the structure of CuA may be related to the role of this site in the proton pumping machinery of cytochrome c oxidase.  相似文献   

4.
It has been shown previously that biogenic amines and a number of pharmaceutical agents can redistribute across vesicle membranes in response to imposed potassium ion or proton gradients. Surprisingly, drug accumulation is observed for vesicles exhibiting either a pH gradient (interior acidic) or a membrane potential (interior negative), implying that these compounds can traverse the lipid bilayer as either the neutral or charged species. This interpretation, however, is complicated by the fact that vesicles exhibiting a membrane potential (interior negative) accumulate protons in response to this potential, thereby creating a pH gradient (interior acidic). This raises the possibility that in both vesicle systems drug redistribution occurs in response to the proton gradient present. We have therefore compared the uptake of several lipophilic cations by reconstituted cytochromec oxidase vesicles and by similar vesicles exhibiting a potassium ion diffusion potential. While turnover of the oxidase generates a membrane potential of comparable magnitude to the potassium ion diffusion system, it is associated with a proton gradient of opposite polarity (interior basic). Both systems show rapid uptake of the permanently charged lipophilic cation, tetraphenylphosphonium, but only the potassium ion diffusion system accumulates the lipophilic amines doxorubicin and propranolol. This provides compelling evidence that such weak bases redistribute only in response to pH gradients and not membrane potential.  相似文献   

5.
The H+/e- stoichiometry of reconstituted cytochrome c oxidase from bovine kidney, containing subunit VIaL (liver type), is 0.5 under standard conditions but 1.0 on addition of 1% cardiolipin to the lipid mixture (asolectin). Low concentrations of palmitate (half-maximal effect at 0.5 microm), but not laurate, myristate, stearate, oleate, 1-hexadecanol, palmitoyl glycerol and palmitoyl CoA, decreased the H+/e- ratio in the presence of cardiolipin from 1.0 to 0.5, accompanied by an increase of coupled, but not of uncoupled respiration of proteoliposomes. Cardiolipin and palmitate did not influence the H+/e- stoichiometry and respiration of reconstituted cytochrome c oxidase from bovine heart, containing subunit VIaH (heart-type). The H+/e- stoichiometry of the heart enzyme, however, is decreased from 1.0 to 0.5 by 5 mm intraliposomal ATP (instead of 5 mm ADP). It is assumed that palmitate binds to subunit VIaL. The partial uncoupling of proton pumping in cytochrome c oxidase is suggested to participate in mammalian thermogenesis.  相似文献   

6.
A study is presented on the pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of purified cytochrome c oxidase (COX) from beef heart reconstituted in phospholipid vesicles (COV). Protons were shown to be released from COV both in the oxidative and reductive phases. In the oxidation by O2 of the fully reduced oxidase, the H+/COX ratio for proton release from COV (R --> O transition) decreased from approximately 2.4 at pH 6.5 to approximately 1.8 at pH 8.5. In the direct reduction of the fully oxidized enzyme (O --> R transition), the H+/COX ratio for proton release from COV increased from approximately 0.3 at pH 6.5 to approximately 1.6 at pH 8.5. Anaerobic oxidation by ferricyanide of the fully reduced oxidase, reconstituted in COV or in the soluble case, resulted in H+ release which exhibited, in both cases, an H+/COX ratio of 1.7-1.9 in the pH range 6.5-8.5. This H+ release associated with ferricyanide oxidation of the oxidase, in the absence of oxygen, originates evidently from deprotonation of acidic groups in the enzyme cooperatively linked to the redox state of the metal centers (redox Bohr protons). The additional H+ release (O2 versus ferricyanide oxidation) approaching 1 H+/COX at pH < or = 6.5 is associated with the reduction of O2 by the reduced metal centers. At pH > or = 8.5, this additional proton release takes place in the reductive phase of the catalytic cycle of the oxidase. The H+/COX ratio for proton release from COV in the overall catalytic cycle, oxidation by O2 of the fully reduced oxidase directly followed by re-reduction (R --> O --> R transition), exhibited a bell-shaped pH dependence approaching 4 at pH 7.2. A mechanism for the involvement in the proton pump of the oxidase of H+/e- cooperative coupling at the metal centers (redox Bohr effects) and protonmotive steps of reduction of O2 to H2O is presented.  相似文献   

7.
Beef heart cytochrome c oxidase has been depleted of subunit III by treatment with chymotrypsin. The removal of subunit III has been evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel fluorography of preparations of the oxidase labeled with [14C]dicyclohexylcarbodiimide prior to proteolysis. Removal of subunit III resulted in a perturbation of the visible spectrum of reduced cytochrome oxidase. Subunit III-depleted oxidase is spectroscopically very similar to the oxidase from Paracoccus denitrificans. When reconstituted into liposomes, the depleted enzyme still pumped protons in response to a pulse of reduced cytochrome c. The H+/e- stoichiometry averaged 0.5. Redox-linked proton translocation could be observed only when respiratory control ratios were higher than 3 and the reductant pulse was of a magnitude that allowed for no more than 5 turnovers of the oxidase.  相似文献   

8.
The existence of a proton pump associated with bovine cytochrome c oxidase (EC 1.9.3.1) has over the last few years been a matter of considerable dispute. In an attempt to resolve some of the problems with the measuring system we have synthesized fluorescein-phosphatidylethanolamine which when reconstituted with cytochrome c oxidase into phospholipid vesicles provided a reliable indicator of the intravesicular pH. It was observed that cytochrome c oxidase catalyzed the abstraction of almost 2 protons from the intravesicular medium/molecule of ferrocytochrome c oxidized. In parallel experiments whereby the extravesicular pH was measured with an electrode it was found that the enzyme appeared to be responsible for the appearance of almost 1.0 proton/molecule of ferrocytochrome c oxidized. Taken together these data unequivocally demonstrate that cytochrome c oxidase behaves as a proton pump. Furthermore, the other proton which was abstracted is believed to be used for the process of the reduction of oxygen. Similar experiments were performed with a cytochrome c oxidase preparation which was devoid of subunit III. Under these circumstances the enzyme appeared to be unable to translocate protons across the vesicular membrane but was competent to abstract protons from the intravesicular medium for the reduction of oxygen.  相似文献   

9.
Discontinuous sucrose gradient ultracentrifugation was used to separate liposomes containing Rhodobacter sphaeroides cytochrome c oxidase (pCOV) from liposomes devoid of the enzyme, and the biophysical and biochemical properties of pCOV were compared to unpurified liposomes containing cytochrome c oxidase (COV). Isolated and purified R. sphaeroides cytochrome c oxidase (COX) was reconstituted into asolectin phospholipid vesicles by cholate dialysis, and this preparation was purified further on a discontinuous sucrose gradient to isolate only those vesicles which contained the enzyme (pCOV). After centrifugation at 300,000g for 22h, 80% of the enzyme recovered was in a single band. The number of COX molecules per pCOV liposome was estimated by measuring the visible absorbance spectrum of cytochrome c oxidase (for heme aa(3)) and inorganic phosphate concentration (for phospholipid). The number of COX molecules incorporated per pCOV was estimated to be approximately one (0.72+/-0.19-1.09+/-0.28). The pCOV exhibited similar physical properties as COV; respiratory control ratios (indicators of endogenous proton permeability) and maximum enzymatic turnover number at pH 7.4 were comparable (6.0+/-1.3 and 535+/-130s(-1)). Furthermore, proton pumping activities of the pCOV were at least 70% of COV, indicating that discontinuous sucrose gradient centrifugation is a useful technique for functional experiments in R. sphaeroides cytochrome c oxidase. Our results suggest that the monomeric form of R. sphaeroides COX when reconstituted into a phospholipid bilayer is completely functionally active in its ability to perform electron transfer and proton pumping activities of the enzyme.  相似文献   

10.
To enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-terminal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Characterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values for cytochrome c that are 6-14-fold higher for the truncated and his-tagged forms than for the wildtype. This lowered ability to bind cytochrome c indicates a previously undetected role for the C-terminus in cytochrome c binding and is mimicked by reduced affinity for an FPLC anion exchange column. The elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Paracoccus denitrificans oxidase, does not produce the same enzyme as the native processing reaction. MALDI-TOF MS data show the true C-terminus of subunit II is at serine 290, three amino acids longer than expected. When the his-tagged form is reconstituted into lipid vesicles and further purified by metal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results are attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.  相似文献   

11.
Han D  Morgan JE  Gennis RB 《Biochemistry》2005,44(38):12767-12774
Cytochrome c oxidase uses the free energy of oxygen reduction to establish a transmembrane proton gradient. The proton-conducting D-channel in this enzyme is the major input pathway for protons which go to the binuclear center for water formation ("chemical protons") and likely the only input pathway for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel starts at an acidic residue near the protein surface (D132, Rhodobacter sphaeroides numbering) and leads to another acidic residue near the binuclear center. Recent studies have shown that mutants that introduce an additional acidic residue in the channel (N139D) have the remarkable effect of accelerating steady-state oxidase activity but completely eliminating proton pumping. In this work, an aspartic acid was introduced at the position of glycine 204, G204D, which is also within the D-channel, and the effects were examined. In contrast to N139D, the G204D mutation results in a dramatic decrease of the steady-state oxygen reductase activity (<2% of wild type) [Aagaard, A., and Brzezinski, P. (2001) FEBS Lett. 494, 157-160]. The residual activity is not coupled to the proton pump, and furthermore, in reconstituted vesicles the mutant enzyme exhibits a reverse respiration control ratio; i.e., the mutant oxidase activity is stimulated rather than inhibited when working against a protonmotive force. Hence, the mutant behaves very much like the D132N, which blocks proton uptake through the D-channel. Single-turnover experiments show that the rate-limiting step in the reaction of O2 with the fully reduced G204D mutant is the F --> O transition, similar to the D132N mutant. The block of the D-channel in the D132N mutant can be partly bypassed by biochemically removing subunit III from the enzyme, indicating that removal of the subunit reveals an alternate entrance for protons to the channel. However, this is not observed with the G204D mutant. This suggests that the cryptic entrance to the D-channel that is revealed by the removal of subunit III is between the levels of G204 and D132.  相似文献   

12.
Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) were characterized for electron transfer and proton translocating activities in the presence of the mobile potassium ionophore, valinomycin, and the channel-forming ionophore, gramicidin, in order to determine if the ionophores modify the functional properties of the enzyme. In agreement with previous work, incubation of COV with valinomycin resulted in a perturbation of the absorbance spectrum of oxidized heme aa3 in the Soret region (430 nm); gramicidin had no effect on the heme aa3 absorbance spectrum. Different concentrations of the two ionophores were required for maximum respiratory control ratios in COV; 40- to 70-fold higher concentrations of valinomycin were required to completely uncouple electron transfer activity when compared to gramidicin. The proton translocating activity of COV incubated with each inophore gave a similar apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) of 0.66 +/- 0.10. However, COV treated with low concentrations of gramicidin (0.14 mg/g phospholipid) exhibited 1.5- to 2.5-fold higher rates of alkalinization of the extravesicular media after the initial proton translocation reaction than did COV treated with valinomycin, suggesting that gramicidin allows more rapid equilibration of protons across the phospholipid bilayer during the proton translocation assay. Moreover, at higher concentrations of gramicidin (1.4 mg/g phospholipid), the observed H+/e- ratio decreased to 0.280 +/- 0.020, while the rate of alkalinization increased an additional 2-fold, suggesting that at higher concentrations, gramicidin acts as a proton ionophore. These results support the hypothesis that cytochrome c oxidase is a redox-linked proton pump that operates at similar efficiencies in the presence of either ionophore. Low concentrations of gramicidin dissipate the membrane potential in COV most likely by a channel mechanism that is different from the carrier mechanism of valinomycin, yet does not make the phospholipid bilayer freely permeable to protons.  相似文献   

13.
We have investigated the covalent binding of dicyclohexylcarbodiimide (DCCD) to cytochrome c oxidase in relation to its inhibition of ferrocytochrome c-induced H+ translocation by the enzyme reconstituted in lipid vesicles. DCCD bound to the reconstituted oxidase in a time- and concentration-dependent manner which appeared to correlate with its inhibition of H+ translocation. In both reconstituted vesicles and intact beef heart mitochondria, the DCCD-binding site was located in subunit III of the oxidase. The apolar nature of DCCD and relatively minor effects of the hydrophilic carbodiimide, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, on H+ translocation by the oxidase indicate that the site of action of DCCD is hydrophobic. DCCD also bound to isolated cytochrome c oxidase, though in this case subunits III and IV were labeled. The maximal overall stoichiometries of DCCD molecules bound per cytochrome c oxidase molecule were 1 and 1.6 for the reconstituted and isolated enzymes, respectively. These findings point to subunit III of cytochrome c oxidase having an important role in H+ translocation by the enzyme and indicate that DCCD may prove a useful tool in elucidating the mechanism of H+ pumping.  相似文献   

14.
The aerobic respiratory chain of Escherichia coli contains two terminal oxidases, the cytochrome o complex and the cytochrome d complex. These both function as ubiquinol-8 oxidases and reduce molecular oxygen to water. Electron flux is funneled from a variety of dehydrogenases, such as succinate dehydrogenase, through ubiquinone-8, to either of the terminal oxidases. A strain was examined which lacks the intact cytochrome d complex, but which overproduces one of the two subunits of this complex, cytochrome b558. This cytochrome, in the absence of the other subunit of the oxidase complex, does not possess catalytic activity. It is shown that the extent of reduction of cytochrome b558 in the E. coli membrane monitors the extent of reduction of the quinone pool in the membrane. The activity of each purified oxidase was examined in phospholipid vesicles as a function of the amount of ubiquinone-8 incorporated in the bilayer. A ratio of ubiquinol-8:phospholipid as low as 1:200 is sufficient to saturate each oxidase. The maximal turnover of the oxidases in the reconstituted system is considerably faster than observed in E. coli membranes, demonstrating that the rate-limiting step in the E. coli respiratory chain is at the dehydrogenases which feed electrons into the system.  相似文献   

15.
In this paper a study is presented of the characteristics of redox-linked proton ejection exhibited by isolated beef-heart cytochrome c oxidase incorporated in asolectin vesicles. The enzyme was 90% oriented 'right-side out' as in the mitochondrial membrane. The effects on the H+/e- stoichiometry of the modalities of activation of electron flow, the pH of the medium and its ionic composition were investigated. The results obtained show that, whilst ferrocytochrome c pulses of the aerobic oxidase vesicles at neutral pH and in the presence of saturating concentrations of valinomycin and K+ to ensure charge compensation produced H+/e- ratios around 1 (as has been shown previously), oxygen pulses of reduced anaerobic vesicles supplemented with cytochrome c, gave H+/e- ratios around 0.3. The H+/e- ratios exhibited, with both reductant and oxidant pulses, a marked pH dependence. Maximum values were observed at pH 7.0-7.7, which decreased to negligible values at acidic pH with apparent pKa of 6.7-6.3. Mg2+ and Ca2+ caused a marked depression of the H+/e- ratio, which in the presence of these cations and after a few ferrocytochrome pulses, became negligible. Analysis of cytochrome c oxidation showed that the modalities of activation of electron flow and divalent cations exerted profound effects on the kinetics of cytochrome c oxidation by oxidase vesicles. The observations presented seem to provide interesting clues for the nature and mechanism of redox-linked proton ejection in reconstituted cytochrome c oxidase.  相似文献   

16.
The heme-copper oxidases convert the free energy liberated in the reduction of O(2) to water into a transmembrane proton electrochemical potential (protonmotive force). One of the essential structural elements of the enzyme is the D-channel, which is thought to be the input pathway, both for protons which go to form H(2)O ("chemical protons") and for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel contains a chain of water molecules extending about 25 A from an aspartic acid (D132 in the Rhodobacter sphaeroides oxidase) near the cytoplasmic ("inside") enzyme surface to a glutamic acid (E286) in the protein interior. Mutations in which either of these acidic residues is replaced by their corresponding amides (D132N or E286Q) result in severe inhibition of enzyme activity. In the current work, an asparagine located in the D-channel has been replaced by the corresponding acid (N139 to D; N98 in bovine enzyme) with dramatic consequences. The N139D mutation not only completely eliminates proton pumping but, at the same time, confers a substantial increase (150-300%) in the steady-state cytochrome oxidase activity. The N139D mutant of the R. sphaeroides oxidase was further characterized by examining the rates of individual steps in the catalytic cycle. Under anaerobic conditions, the rate of reduction of heme a(3) in the fully oxidized enzyme, prior to the reaction with O(2), is identical to that of the wild-type oxidase and is not accelerated. However, the rate of reaction of the fully reduced enzyme with O(2) is accelerated by the N139D mutation, as shown by a more rapid F --> O transition. Whereas the rates of formation and decay of the oxygenated intermediates are altered, the nature of the oxygenated intermediates is not perturbed by the N139D mutation.  相似文献   

17.
18.
The kinetics of cytochrome oxidase reconstituted into small phospholipid vesicles (COV) has been followed by transient optical spectroscopy under steady-state and pre-steady-state conditions, in the presence and absence of ionophores. The effect of valinomycin on the activity of reconstituted cytochrome oxidase is shown to depend on the absolute concentration of the ionophore and on the number of turnovers elapsed by the enzyme; this novel observation, which escaped previous investigations, may account for important differences in results and therefore in interpretation of the mechanism of control of the enzyme activity as between Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), Gregory and Ferguson-Miller (Gregory, L., and Ferguson-Miller, S. (1989) Biochemistry 28, 2655-2662) and Capitanio et al. (Capitanio, N., De Nitto, E., Villani, G., Capitanio, G., and Papa, S. (1990) Biochemistry 29, 2939-2944). Quantitative analysis of the optical spectra acquired within 10 ms over a large wavelength and time range (500-650 nm and 5 ms to 60 s) under different experimental conditions, indicates that the electrical component of the transmembrane electrochemical gradient controls the rate of the internal electron transfer from cytochrome a-CuA to cytochrome a3-CuB as well as the cytochrome c to cytochrome a electron transfer. The slow down of cytochrome oxidase activity observed in the presence of valinomycin after several (greater than 10) turnovers is attributed to alkalinization of the vesicle interior, which affects the internal electron transfer rate. These two mechanisms of control act most likely independently. A "cubic scheme," which illustrates the effect of the electrochemical gradient on two states of cytochrome oxidase characterized by different redox and proton pumping activities is presented and discussed.  相似文献   

19.
When the carbon monoxide complex of fully reduced cytochrome c oxidase, reconstituted into liposomes, is mixed with oxygen-containing buffer, complex kinetic progress curves are observed. This pattern is seen irrespective of whether the oxidase used in reconstitution is the dimeric or monomeric (subunit III-depleted) enzyme. These findings are interpreted in the light of similar experiments on the detergent-solubilized enzyme reported by Gibson and Greenwood (Gibson, Q.H., and Greenwood, C. (1963) Biochem. J. 86, 541-554) and confirmed by ourselves. We conclude that reconstitution of monomeric (subunit III-less) enzyme yields, preferentially, vesicles containing more than one functional unit, possibly associated as dimers. This result is of significance to our understanding of the relationships between aggregation state and proton pumping capacity of cytochrome oxidase.  相似文献   

20.
Changes in pH during the reactions of the fully reduced and mixed-valence cytochrome oxidase with molecular oxygen have been followed in flow-flash experiments, using the pH indicator phenol red. Solubilized enzyme as well as enzyme reconstituted into phospholipid vesicles has been studied. With the solubilized enzyme, a biphasic uptake of one proton from the medium was observed, whereas the reconstituted enzyme gave release of 1.3 protons to the extravesicular medium. It is concluded from these results that a total of two to three protons are taken up during oxidation of the fully reduced enzyme. Kinetic analysis suggests that the proton uptake is initiated by the transfer of the third electron to the oxygen binding site. A reaction scheme that integrates proton transfers and oxygen chemistry is presented.  相似文献   

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