Abbreviations: TMPD; tetramethyl-p-phenylenediamine 相似文献
2. A particulate, heat-labile nitrite oxidase having an absolute requirement for O2 was prepared from N. agilis cells using sonic oscillation and differential centrifugation. The particles also possessed NADH oxidase, succinoxidase, formate oxidase and traces of NADPH oxidase activity. The stoichiometry of the nitrite oxidase reaction approached the theoretical value of 2 moles of NO2− consumed per mole of O2 consumed. The pH optimum of the nitrite oxidase system shifted to progressively more alkaline values as the NO2− concentration was increased, changing from a pH value of 6.8 at 0.6 mM KNO2 to pH 8.0 at 0.01 M KNO2 with apparent Km's of 0.2 and 1.2 mM NO2−, respectively. Computations of the HNO2 concentrations present under the above conditions showed an approx. 500-fold greater affinity for HNO2 which was independent of pH, suggesting the involvement of HNO2 as both a substrate and an inhibitor (at higher concentrations) of the nitrite oxidase system. The marked inhibition by NaN3, NaCN and Na2S, as well the light-reversible inhibition by CO, indicated the presence of cytochrome oxidase which was subsequently characterized. NO2− proved to be a competitive inhibitor of the nitrite oxidase system.
3. The particulate preparation also possessed a heat-labile nitrite-cytochrome c reductase activity which was energy independent and routinely measured under anaerobic conditions. As in the case of nitrite oxidase, the affinity of the enzyme for NO3− increased as the pH was lowered, but the pH optimum remained unaffected. In terms of calculated HNO2 concentration an approximately constant Km of about 0.2 μM was estimated at the several pH's examined. The inhibition by NO3− was shown to be competitive. The marked sensitivity of the reductase to several metal-binding agents implicated a metal component in the electron transport chain at the site prior to cytochrome c.
4. The membrane-like composition of the nitrite oxidase system is indicated. 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
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−.
By mixing the cell-free extracts of the two mutants chl A− and chl B− of Escherichia coli K12, previously freed from particle membranes, we achieved restoration of nitrate reductase activity. The activity is restored first in a soluble form, then in a particulate form. This mechanism is called “complementation”. In the soluble state, the purified enzyme reduces NO3− and ClO3−, using reduced benzyl viologen or FMNH2 as electron donors. It is sensitive to KCN, NaN3, p-hydroxymercuribenzoate (1 mM) and N-ethylmaleimide (0.1 mM)
The soluble form is sensitive neither to phospholipase C, nor to 2-n-heptyl-4-hydroxyquinoline-N-oxide; it associates with phospholipids and cytochrome b1 to form particles in which nitrate reductase activity is no longer sensitive to ethyl N-maleimide and p-hydroxymercuribenzoate, but, conversely, becomes sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide.
These results clearly demonstrate that it is possible to study the mechanism of integration of the enzyme leading to active membranes particles without any previous solubilisation of the original material. 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
The molecular weight of the cytochrome oxidase was calculated to be 119000 based on a sedimentation coefficient s° 20,w = 7.36 S, diffusion coefficient D 20,w = 5.36×10−7 cm2×s−1 and partial specific volume of 0.72 ml/g. The iron content of the enzyme (0.166 %) indicates that this entity contains four iron atoms per molecule. Succinylation of the enzyme produced two probably identical subunits containing both hemes c and d, having a sedimentation coefficient s° 20,w = 4.30 S and an approximate molecular weight of 65000. In dodecylsulphate-acrylamide gel electrophoresis the cytochrome oxidase also dissociates into two subunits with molecular weight of 63000. 相似文献
1. 1. Cyanide inhibits the catalytic activity of cytochrome aa3 in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·103 M−1·s−1 and a Ki that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3.
2. 2. When cyanide is added to the ascorbate-cytochrome c-cytochromeaa3−O2 system a biphasic reduction of cytochrome c occurs corresponding to an initial Ki of 0.8 μM and a final Ki of about 0.1 μM for the cytochrome aa3−cyanide reaction.
3. 3. The inhibited species (a2+a33+HCN) is formed when a2+a33+ reacts with HCN, when a2+a32+HCN reacts with oxygen, or when a3+a33+HCN (cyano-cytochrome aa3) is reduced. Cyanide dissociates from a2+a33+HCN at a rate of 2·10−3 s−1 at 22 °C, pH 7.3.
4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a2+a33+ than to a3+a33+.
Abbreviations: TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine 相似文献
Energization of the membrane can be demonstrated when NADH is oxidized by O2, ferricyanide or Q1, when succinate is oxidized by O2 or by oxidized diaminodurene, and during the oxidation of reduced diaminodurene.
Antimycin A completely inhibits energization between succinate and O2 or succinate and diaminodurene; however, it only inhibits partially NADH or succinate oxidases and energization between NADH and O2. KCN inhibits NADH oxidase in a biphasic way: the first level of inhibition is observed at concentrations which block the oxidation of exogenous cytochrome c or of diaminodurene and energization between succinate or ascorbate-diaminodurene and O2. The second level corresponds to the inhibition of the antimycin-insensitive oxidase.
The results are interpreted as evidence of the presence in these bacteria of a respiratory chain branching after the dehydrogenase system, one arm of the chain being sensitive to antimycin A and low concentrations of KCN and capable of energy conservation, the other being represented by a completely uncoupled 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
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
The activity exhibited an absolute requirement for Mg2+ in the neutral pH range, while Ca2+ was found able to activate ATPase at more alkaline pH. Optimal activity was observed at pH 7.5, with a Mg/ATP ratio of 0.5.
ADP was found to inhibit ATP hydrolysis and to transform the Michaelian ATP concentration dependence with a Km of 0.5 mM into a sigmoid curve with increasing Km and decreasing V.
In contrast ADP activated an ATP-ADP exchange process and this shift from hydrolysis to exchange was stimulated by high Mg2+ and by orthophosphate.
All nucleoside triphosphates tested interfered with ATP hydrolysis, all could be hydrolyzed and could donate their terminal phosphate group to ADP. The relative efficiencies of nucleoside triphosphates in these three processes varied in parallel with minor discrepancies.
ATP hydrolysis was inhibited by N,N′-dicyclohexylcarbodiimide (DCCD) Dio 9, NaN3 and pyrophosphate, the first two being ineffective against ATP-ADP exchange, the third being stimulatory and the last inhibitory.
ATP hydrolysis and ATP-ADP exchange are tentatively attributed to the terminal enzyme of oxidative phosphorylation. 相似文献
The reaction of ascorbate-reduced oxidase with CO has also been studied and is second order with a rate constant of 1.8 · 104 M−1 · s−1. The initial reaction with CO is followed by a slower reaction of significantly less magnitude. The equilibrium constant for the reaction with CO, calculated as a dissociation constant from titrimetric experiments with dithionite-reduced oxidase, is about 2.3 · 10−6 M. From these data a rate constant of 0.041 s−1 can be calculated for the dissociation of CO from the enzyme. 相似文献
The reduction of Fe3+ by the xanthine-xanthine oxidase system is different from that manifested by the NADPH-NADPH cytochrome c reductase system in the manner in which the former is carried out in the main by Ostaggered2staggered− · generated by the substrate - O2 - enzyme interaction.
Reduced iron, which is free in the solution, plays an important role for the initiation and propagation of the phospholipid peroxidation, monitored by malondialdehyde assay and light emission.
In the xanthine-induced lipoxygenation system, the ·OH radical, probably produced from hydrogen peroxide by the action of Fe2+, is not involved in the initiation of the peroxidative cleavage of phospholipid in microsomal lipoprotein. 相似文献