Abbreviations: TMPD; tetramethyl-p-phenylenediamine 相似文献
The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a Km of 27 μM for O2. This activity was also inhibited by KCN, NaN3 and NH2OH and by NO2−.
A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO2−. It was inhibited by smaller amounts of KCN, NaN3 and NH2OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a Km for O2 of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO2−.
The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller Km for O2 and would therefore be expected to function more efficiently especially at low concentrations of O2. 相似文献
2. Difference spectra of formate-reduced particles or intact cells demonstrated the presence of cytochromes of the c- and a-types like those of the NO2−-reduced material. Under anaerobic conditions NO3− or fumarate acted as an alternate electron acceptor in place of O2 in formate oxidation. Under aerobic conditions increasing NO3− concentrations resulted in (a) an increased role of NO3− as a terminal electron acceptor compared to O2, (b) a greater total enzymatic transfer of electrons from formate than if O2 were the sole electron acceptor, and (c) a partial inhibition of O2 uptake suggestive of a competition for electrons by the two acceptors. The formate oxidase system failed to catalyze consistently the transfer of electrons to either added mammalian cytochrome c or Fe(CN)63−. The marked sensitivity of the system to certain inhibitors implicated cytochrome oxidase as an integral part of the formate oxidase. The system was also inhibited significantly by a variety of chelating agents, indicating a metal component in the formate dehydrogenase or early portion of the electron transfer sequence.
3. The stoichiometry of the formate oxidase system was shown to approach the theoretical value of 2 moles of CO2 evolved per mole of O2 or per 2 moles of formate consumed.
4. To a limited extent, phosphorylation occurred concomittantly with the oxidation of formate in the presence of the cell-free particulate system. 相似文献
2. Fluoride shifts the γ-band of the enzyme from 423 to 421 nm and the -band from 597 to 598 nm. The difference spectrum (oxidized enzyme in the presence of fluoride minus oxidized enzyme) has peaks at 400, 453, 482, 605 and 638 nm and troughs at 430, 520, 552 and 674 nm. The changes in absorbance are small (about 3% at absorbance maxima) with respect to those of other hemoproteins.
3. On addition of fluoride to isolated cytochrome c oxidase 3 reactions can be distinguished: (I) a bimolecular binding reaction (Kon = 4 M−1 · s−1 and koff = 2.9 · 10−2s−1 at 25 °C, pH 7.4) contributing at 638 nm and 430 nm; (II) a first-order reaction (k = 2.4 · 10−2) s−1 at 22 °C, pH 7.2) visible mainly at 430 nm and (III) a very slow reaction with a half-time in the order of 10 min.
4. The spectroscopic dissociation constants for the fluoride binding, determined from Hill plots using the absorbance changes at 638 and 430 nm, are similar (7 and 10 mM, respectively, at 22 °C, pH 7.2).
5. A mechanism for the reaction is discussed in which the bimolecular binding reaction is followed by a conformational change of the enzyme-fluoride complex. 相似文献
2. Hydrated electrons do not readily reduce the heme of cytochrome c oxidase. This observation supports our previous conclusion that heme a is not directly exposed to the solvent.
3. In a mixture of cytochrome c and cytochrome c oxidase, cytochrome c is first reduced by hydrated electrons (k = 4 · 1010 M−1 · s−1 at 22 °C and pH 7.2) after which it transfers electrons to cytochrome c oxidase with a rate constant of 6 · 107 M−1 · s−1 at 22 °C and pH 7.2.
4. It was found that two equivalents of cytochrome c are oxidized initially per equivalent of heme a reduced, showing that one electron is accepted by a second electron acceptor, probably one of the copper atoms of cytochrome c oxidase.
5. After the initial reduction, redistribution of electrons takes place until an equilibrium is reached similar to that found in redox experiments of Tiesjema, R. H., Muijsers, A. O. and Van Gelder, B. F. (1973) Biochim. Biophys. Acta 305, 19–28. 相似文献
1. 1. The superoxide anion radical (O2−) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O2− and ferrocytochrome c.
2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 106 M−1 · s−1 and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O2− and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O2− reacts with the form present below pH 7.45 with k = 1.4 · 106 M−1 · s−1, the form above pH 7.45 with k = 3.0 · 105 M−1 · s−1, and the form present above pH 9.2 with k = 0.
3. 3. The reaction has an activation energy of 20 kJ mol−1 and an enthalpy of activation at 25 °C of 18 kJ mol−1 both above and below pH 7.45. It is suggested that O2− may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.
4. 4. No reduction of ferricytochrome c by HO2 radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO2 radicals oxidize ferrocytochrome c with a rate constant of about 5 · 105–5 · 106 M−1 · s−1
. 相似文献
1. 1. The preparations catalysed ATP synthesis coupled to O2 uptake or NO3− reduction. With NADH or succinate as the electron donors the P:O ratios were about 1.5 and 0.5, respectively; and the P:NO3− ratios were about 0.9 and 0.06, respectively.
2. 2. Addition of ADP or Pi to the reaction mixture increased the rates of NADH-dependent O2 uptake and NO3− reduction. Addition of 1 mM 2,4-dinitrophenol, which inhibited phosphorylation by 50–60%, increased the basal rates of electron transport.
3. 3. Evidence derived from spectrophotometry and from the differential inhibition by antimycin A of O2 and NO3− reduction leads to the conclusion that the nitrate reductase interacted with the respiratory chain in the region of the b-type cytochrome, and that the c-type cytochrome present was not involved in the reduction of NO3− to NO2−.
2. Redox potentials of haem c in the presence of 2.5 M pyridine were determined in the pH range 1.5–13; it was found necessary to add cetyl trimethyl ammonium bromide (CTAB) to prevent precipitation in the acid range below about pH 4. The Em vs pH curve shows three slopes (−dE/dpH) of value, 0.18, 0.01 and 0.06 with points of inflexion at pH 3.8 and 10.6. The potentials are intermediate between those of protohaem and mesohaem obtained under similar conditions.
3. With constant haem c concentration (a) 10−4 M and (b) 10−5 M and varying pyridine concentration (0.12–5 M) it was found at pH 9.0 that Em values increased as the pyridine concentration was increased and there was a tendency to reach a plateau value. The explanation appears to be that pyridine binds more firmly to ferroporphyrin c than to ferriporhyrin c.
4. When the pyridine concentration was kept constant (2.5 M) and the haem c concentration was varied in the range 7 · 10−4–7 · 10−6 M, it was found that a decrease in haem c concentration brought about an increase in redox potential. The results are explained as being due to dimerization of the oxidized form.
5. The results are discussed in comparison with a number of related haem systems. 相似文献
In this paper a simple model system was used: unsaturated phosphatidylcholine (PC) vesicles were treated with HOCl in the presence of varying NaNO2 concentrations and the yield of reaction products was determined by MALDI-TOF MS: the extent of chlorohydrin generation was significantly reduced in the presence of NaNO2 because HOCl is consumed by the oxidation of NO2− to NO3−.
Similar results were obtained when HOCl was generated by the myeloperoxidase (MPO)/H2O2/Cl− system or the experiments were carried out in the presence of a simple peptide. It is concluded that the transient products of the reaction between HOCl and NO2− do not have a sufficient reactivity to modify PL. 相似文献
2. Freeze-drying of the bacterial membranes causes a selective detachment of DPNH dehydrogenase (DPNH: (acceptor) oxidoreductase, EC 1.6.99.3) from the membranes. This solubilization is accompanied by a decrease of Km(K3Fe(CN)6) from 2.0 to 0.25 mM, while no change is detected in Km(DPNH). This enzyme is not the DPNH diaphorase found in the bacteria.
3. DPNH dehydrogenase of E. coli is a metalloflavoprotein, containing non-heme iron, labile sulfide, FMN and FAD.
4. Reduction of the enzyme with DPNH in the absence of electron acceptor (ferricyanide or DCIP) causes a rapid and irreversible change to a less active state, Form II. Form II is characterized by a higher Km(DPNH) and slower vmax., while the Km(K3Fe(CN)6) remains unchanged.
5. The transformation of the enzyme to Form II is accompanied by the reduction of the non-heme iron component. The role of non-heme iron in the enzymic reaction is discussed. 相似文献
2. The near-ultraviolet difference spectrum of whole cells reveals an absorption peak at 315 mμ with a shoulder around 350 mμ.
3. Both the endogenous respiration and motility of spermatozoa are completely blocked by 0.2 mM CN− and by 0.2 μM antimycin A. 2,4-Dinitrophenol and pentachlorophenol completely inhibit motility at the maximal stimulation of respiration. Rotenone strongly inhibits NADH oxidase of spermatozoa, although it has no effect on the respiration of whole cells.
4. It is concluded that the motility of mussel spermatozoa is tightly coupled to respiration, and the respiratory chain phosphorylating process is the only energy-supplying system for motility. 相似文献
2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·108 M−1·s−1 and 6.5·108 M−1·s−1 for the reactions of paraquat radical with O2 and O2−·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O2 and O2−· (− 0.59 V and + 1.12 V, respectively).
3. These rate constants, together with that for the self-reaction of O2−·, have been used to calculate the steady-state concentration of O2−· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O2−· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O2−· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O2−· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants. 相似文献
2. Preparations of cytochrome b active in reconstitution contained 5–28% native cytochrome b, as adjudged by reducibility with succinate in the reconstituted preparation and by lack of reaction with CO. Preparations of cytochrome b containing no native cytochrome b according to this criterion were inactive in reconstitution.
3. With a fixed amount of cytochrome b, the activity of the reconstituted preparation increased with increasing amounts of cytochrome c1 until a ratio of about 2b (total): 1c1 (allowing for the cytochrome c1 present in the cytochrome b preparation) was reached.
4. The amount of antimycin necessary for maximal inhibition of the reconstituted enzyme is a function of the amount of the cytochrome b and is independent of the amount of cytochrome c1. It is equal to about one half the amount of native cytochrome b.
5. Preparations of intact or reconstituted succinate-cytochrome c reductase or of cytochrome b completely quench the fluorescence of added antimycin, until an amount of antimycin equal to onehalf the amount of native cytochrome b present was added. Antimycin added in excess of this amount fluoresces with normal intensity. The quenching is only partial in the presence of Na2S2O4. Denatured cytochrome b does not quench the fluorescence.
6. Since preparations of cytochrome b active in reconstitution contained cytochrome c1 in an amount exceeding one half the amount of native cytochrome b present in the preparation, there is no evidence that native cytochrome b has been resolved from cytochrome c1. The stimulatory action of cytochrome c1 may be due to the restoration of a damaged membrane conformation.
7. Based on the assumption that the bc1 segment of the respiratory chain contains 2b:1c1:1 antimycin-binding sites, the specific quenching of antimycin fluorescence by binding to cytochrome b enables an accurate determination of the absorbance coefficients of cytochromes b and c1. These are 25.6 and 20.1 mM−1×cm−1 for the wavelength pairs 563–577 nm and 553–539 nm, respectively, in the difference spectrum reduced minus oxidized. 相似文献
2. The rate of the peroxidation reaction is proportional to the concentration of H2O2 and ferricytochrome c but is independent of the concentration of ferrocytochrome c in the concentration ranges studied.
3. Integration of the rate equation, d[c3+]/dt = k[c3+][H2O2], gives a theoretical expression which fits the experimental time courses for the ferrocytochrome c peroxidation reaction.
4. No direct spectral evidence was found for the formation of a catalytically active ferricytochrome c-H2O2 derivative. Kinetic evidence is presented, however, which indicates the existence of such an intermediate.
5. Ferricytochrome c was more susceptible than ferrocytochrome c to an apparent degradation reaction caused by excess H2O2, thus supporting the idea that the cytochrome c heme iron is more accessible in the oxidized form. 相似文献