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1.
1. Using stopped-flow technique we have investigated the electron transfer form cytochrome c to cytochrome aa3 and to the (porphyrin) cytochrome c-cytochromeaa3 complex.2. In a low ionic strength medium, the pre-steady state reaction occurs in a biphasic way with rate constants of at least 2 · 108 M?1 · s?1 and about 107 M?1 · s?1 (I = 8.8 mM, pH 7.0, 10° C), respectively.3. A comparison of the rate constants, determined in the presence of an excess of cytochrome c with those found in the presence of an excess of cytochrome aa3 reveals the existence of two slower reacting sites on the functional unit (2 hemes and 2 coppers) of cytochrome aa3. On basis of these results we discuss various models. If no site-site interactions are assumed (non-cooperative model) cytochrome aa3 has 2 high and 2 low affinity sites available for the reaction with ferrocytochrome c. If negative cooperativity occurs, cytochrome aa3 has 2 high affinity sites which change into 2 low affinity sites upon binding of one cytochrome c molecule. The latter model is favoured. 相似文献
2.
The xanthine-oxidizing enzyme of rat liver has been purified as an NAD+-dependent dehydrogenase (type D) and as the O2-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD+ with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The Km for xanthine is not significantly different for the two forms. 相似文献
3.
Efraim Racker 《The Journal of general physiology》1969,54(1):38-49
The problem of the resolution and reconstitution of the inner mitochondrial membrane has been approached at three levels. (1) Starting with phosphorylating submitochondrial particles, a "resolution from without" can be achieved by stripping of surface components. The most extensive resolution was recently obtained with the aid of silicotungstate. Such particles require for oxidative phosphorylation the addition of several coupling factors as well as succinate dehydrogenase. (2) Starting with submitochondrial particles that have been degraded by trypsin and urea a resolution of the inner membrane proper containing an ATPase has been achieved. These experiments show that at least five components are required for the reconstitution of an oligomycin-sensitive ATPase: a particulate component, F
1, Mg++, phospholipids, and Fc. Morphologically, the reconstituted ATPase preparations resemble submitochondrial particles. (3) Starting with intact mitochondria individual components of the oxidation chain have been separated from each other. The following components were required for the reconstitution of succinoxidase: succinate dehydrogenase, cytochrome b\, cytochrome c
1, cytochrome c, cytochrome oxidase, phospholipids and Q
10. The reconstituted complex had properties similar to those of phosphorylating submitochondrial particles; i.e., the oxidation of succinate by molecular oxygen was highly sensitive to antimycin. 相似文献
4.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2019,1867(6):595-603
Methanotrophs play a prominent role in the global carbon cycle, by oxidizing the potent greenhouse gas methane to CO2. Methane is first converted into methanol by methane monooxygenase. This methanol is subsequently oxidized by either a calcium-dependent MxaF-type or a lanthanide-dependent XoxF-type methanol dehydrogenase (MDH). Electrons from methanol oxidation are shuttled to a cytochrome redox partner, termed cytochrome cL. Here, the cytochrome cL homolog from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV was characterized. SolV cytochrome cGJ is a fusion of a XoxG cytochrome and a periplasmic binding protein XoxJ. Here we show that XoxGJ functions as the direct electron acceptor of its corresponding XoxF-type MDH and can sustain methanol turnover, when a secondary cytochrome is present as final electron acceptor. SolV cytochrome cGJ (XoxGJ) further displays a unique, red-shifted absorbance spectrum, with a Soret and Q bands at 440, 553 and 595 nm in the reduced state, respectively. VTVH-MCD spectroscopy revealed the presence of a low spin iron heme and the data further shows that the heme group exhibits minimal ruffling. The midpoint potential Em,pH7 of +240 mV is similar to other cytochrome cL type proteins but remarkably, the midpoint potential of cytochrome cGJ was not influenced by lowering the pH. Cytochrome cGJ represents the first example of a cytochrome from a strictly lanthanide-dependent methylotrophic microorganism. 相似文献
5.
- The dye-linked methanol dehydrogenase from Paracoccus denitrificans grown aerobically on methanol has been purified and its properties compared with similar enzymes from other bacteria. It was shown to be specific and to have high affinity for primary alcohols and formaldehyde as substrate, ammonia was the best activator and the enzyme could be linked to reduction of phenazine methosulphate.
- Paracoccus denitrificans could be grown anaerobically on methanol, using nitrate or nitrite as electron acceptor. The methanol dehydrogenase synthesized under these conditions could not be differentiated from the aerobically-synthesized enzyme.
- Activities of methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, nitrate reductase and nitrite reductase were measured under aerobic and anaerobic growth conditions.
- Difference spectra of reduced and oxidized cytochromes in membrane and supernatant fractions of methanol-grown P. denitrificans were measured.
- From the results of the spectral and enzymatic analyses it has been suggested that anaerobic growth on methanol/nitrate is made possible by reduction of nitrate to nitrite using electrons derived from the pyridine nucleotide-linked dehydrogenations of formaldehyde and formate, the nitrite so produced then functioning as electron acceptor for methanol dehydrogenase via cytochrome c and nitrite reductase.
6.
(1) The kinetic behaviour of the prosthetic groups and the semiquinones in QH2:cytochrome c oxidoreductase has been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) In the absence of antimycin, cytochrome b-562 is reduced in two phases separated by a lag time. The initial very rapid reduction phase, that coincides with the formation of the antimycin-sensitive Q?in, is ascribed to high-potential cytochrome b-562 and the slow phase to low-potential cytochrome b-562. The two cytochromes are present in a 1:1 molar ratio. The lag time between the two reduction phases decreases with increasing pH. Both the [2 Fe-2 S] clusters and cytochrome c1 are reduced monophasically under these conditions, but at a rate lower than that of the initial rapid reduction of cytochrome b-562. (3) In the presence of antimycin and absence of oxidant, cytochrome b-562 is still reduced biphasically, but there is no lag between the two phases. No Q?in is formed and both the Fe-S clusters and cytochrome c1 are reduced biphasically, one-half being reduced at the same rate as in the absence of antimycin and the other half 10-times slower. (4) In the presence of antimycin and oxidant, the recently described antimycin-insensitive species of semiquinone anion, Q?out (De Vries, S., Albracht, S.P.J., Berden, J.A. and Slater, E.C. (1982) J. Biol. Chem. 256, 11996–11998) is formed at the same rate as that of the reduction of all species of cytochrome b. In this case cytochrome b is reduced in a single phase. (5) The reversible change of the line shape of the EPR spectrum of the [2Fe-2S] cluster 1 is caused by ubiquinone bound in the vicinity of this cluster. (6) The experimental results are consistent with the basic principles of the Q cycle. Because of the multiplicity, stoicheiometry and heterogeneous kinetics of the prosthetic groups, a Q cycle model describing the pathway of electrons through a dimeric QH2:cytochrome c oxidoreductase is proposed. 相似文献
7.
8.
Michael Egert Angela Hamann Ralf Kömen Cornelius G. Friedrich 《Archives of microbiology》1993,159(4):364-371
With choline as carbon source Thiosphaera pantotropha GB17 grew with a doubling time (td) of 6 h. The cellular yield was 55.8 g dry cell weight per mol of choline, indicating that its methyl moieties were used for growth. However, T. pantotropha was unable to grow with methanol or with methylamine as carbon source. Mutants were isolated from liquid or from solid media able to grow with methanol (Mox+) as carbon or methylamine as nitrogen source (Mam+). The Mox+ mutant GB17M grew with a mean td of 11.7h and a growth yield of 8.9 g dry cell weight per mol of methanol. Diauxic growth of strain GB17M was observed with mixtures of pyruvate and methanol as substrates in batch culture. Methanol led to the formation of methanol dehydrogenase, formate dehydrogenase, ribulosebisphosphate carboxylase and of a soluble cytochrome c-551.5. Tn5-insertional mutants defective in the thiosulfate oxidizing enzyme system or in hydrogenase acquired the Mox+ phenotype. However, Tn5-insertional mutants defective in either a c-type cytochrome or the molybdenum cofactor did not mutate to the Mox+ phenotype, indicating common functions in thiosulfate and in methanol metabolism. 相似文献
9.
G M Bartoli T Galeotti G Palombini G Parisi A Azzi 《Archives of biochemistry and biophysics》1977,184(1):276-281
Liver microsomes of adult rats produce, by an NADPH-dependent pathway, O2? radicals, as detected by the epinephrine cooxidation to adrenochrome (24.8 nmol/min/mg of protein). This production has also been measured during liver development (from 1 to 20 days after birth) and correlated to the enzyme content (NADPH-cytochrome c reductase, cytochrome b5, and cytochrome P-450), with the aim of establishing the level at which Superoxide radicals are formed in the electron transport system. At 1 day the adrenochrome formation and the activity of NADPH-cytochrome c reductase are about 50 and 40% of those of the adult, respectively, whereas those of cytochromes b5 and P-450 are approximately 10%. After 20 days of development cytochrome b5 and the dehydrogenase reach the adult level, while cytochrome P-450 is about 80%. At this age O2? radicals have a 30% increment and reach only 60% of those of the adult; H2O2 production is also 60% and the N-demethylation of aminopyrine is only 30%. Thus, at birth the formation of O2? radicals is almost entirely dependent on the activity of the flavoprotein. The close correlation between the slight increase in the demethylase activity and adrenochrome formation from 1 to 20 days suggests that a portion of O2? radicals produced by the NADPH-dependent electron transfer is directly involved in the mixed function oxidation. Since about 50% of the radicals are formed at the flavoprotein level, these results indicate that in the adult liver the remaining amount may be generated at the level of cytochrome P-450. 相似文献
10.
A highly purified reduced ubiquinone-cytochrome c reductase preparation (the b-c1III complex) has been made. The b-c1III complex is not reconstitutively active with succinate dehydrogenase. When the complex at about 10 mg/ml is reduced by succinate in the presence of catalytic (nanomolar) amounts of SDH and a ubiquinone protein (required in the succinate dehydrogenase region i.e, OP-S), a ubisemiquinone radical(s) has been detected using EPR measurements. The formation of the radical(s) is concurrent with the reduction of cytochrome b after the complete reduction of cytochrome c1. All these rates are dependent on the amounts of succinate dehydrogenase and QP-S used. The maximal concentration of the radical formed is independent of the amounts of succinate dehydrogenase and QP-S added but dependent on the amount of succinate present. The formation of the radical and the reduction of b and c1 by succinate requires the presence of phospholipids. Addition of thenoyltrifluoroacetone not only prevents the formation of the ubisemiquinone but also abolishes the prior formed radical and causes the reoxidation of b. Antimycin A also diminishes the radical intensity but causes only slight reoxidation of prior reduced cytochrome b. Treatment of the b-c1III complex with α-chymotrypsin results in the diminishing of the radical formation. Consideration of all these results presented collectively indicates the existence of a ubiquinone binding protein in the b-c1III complex preparation. 相似文献
11.
Extracts of Pseudomonas C grown on methanol as sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD+) and formate dehydrogenase (NAD+) were also detected in the extracts.The addition of d-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD+ or NADP+. In addition, the oxidation of [14C]formaldehyde to CO2, by extracts of Pseudomonas C, increased when d-ribulose 5-phosphate was present in the assay mixtures.The amount of radioactivity found in CO2, was 6.8-times higher when extracts of methanol-grown Pseudomona C were incubated for a short period of time with [1-14C]glucose 6-phosphate than with [U-14C]glucose 6-phosphate.These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO2 both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant. 相似文献
12.
The respiratory chain-linked external NADH dehydrogenase has been isolated from Candida utilis in highly purified form. The enzyme is soluble and has a molecular weight of approx. 1.5 · 106. The enzyme contains two moles of FMN per mole of enzyme and is composed of two large subunits of mol. wt. 270 000 and eight smaller subunits of mol. wt. 135 000. Iron and copper are present in the preparations, but appear to be contaminants. The enzyme catalyzes the oxidation of NADH and NADPH at nearly equal rates and reacts readily with 2,6-dichlorophenolindophenol, CoQ6 and CoQ1 derivatives as acceptors. Rotenone (10?5 M) and seconal (10?3 M) do not inhibit enzymatic activity. 相似文献
13.
Comparison of unitrophic and mixotrophic substrate metabolism by acetate-adapted strain of Methanosarcina barkeri 总被引:22,自引:12,他引:10
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We examined the unitrophic metabolism of acetate and methanol individually and the mixotrophic utilization of these compounds by using detailed 14C-labeled tracer studies in a strain of Methanosarcina barkeri adapted to grow on acetate as the sole carbon and energy source. The substrate consumption rate and methane production rate were significantly lower on acetate alone than during the unitrophic or mixotrophic metabolism of methanol. Cell yields (in grams per mole of substrate) were identical during exponential growth on acetate and exponential growth on methanol. During unitrophic metabolism of acetate, the methyl moiety accounted for the majority of the CH4 produced, but 14% of the CO2 generated originated from the methyl moiety. This correlated with the concurrent reduction of equivalent amounts of the C-1 of acetate to CH4. 14CH4 was also produced from added 14CO2, although to a lesser extent than from reduction of the C-1 of acetate. During mixotrophic metabolism, methanol and acetate were catabolized simultaneously. The rates of 14CH4 and 14CO2 generation from [2-14C]acetate were logarithmic and higher in mixotrophic than in unitrophic cultures at substrate concentrations of 50 mM. A comparison of the oxidoreductase activities in cell extracts of the acetate-adapted strain grown on acetate and of strain MS grown on methanol or on H2 plus CO2 indicated that the pyruvate, α-ketoglutarate, and isocitrate dehydrogenase activities remained constant, whereas the CO dehydrogenase activity was significantly higher (5,000 nmol/min per mg of protein) in the acetate-adapted strain. These results suggested that a significant intramolecular redox pathway is possible for the generation of CH4 from acetate, that energy metabolism from acetate by M. barkeri is not catabolite repressed by methanol, and that the acetate-adapted strain is a metabolic mutant with derepressed CO dehydrogenase activity. 相似文献
14.
Sousa PM Silva ST Hood BL Charro N Carita JN Vaz F Penque D Conrads TP Melo AM 《Biochimie》2011,93(3):418-425
The organization of respiratory chain complexes in supercomplexes has been shown in the mitochondria of several eukaryotes and in the cell membranes of some bacteria. These supercomplexes are suggested to be important for oxidative phosphorylation efficiency and to prevent the formation of reactive oxygen species.Here we describe, for the first time, the identification of supramolecular organizations in the aerobic respiratory chain of Escherichia coli, including a trimer of succinate dehydrogenase. Furthermore, two heterooligomerizations have been shown: one resulting from the association of the NADH:quinone oxidoreductases NDH-1 and NDH-2, and another composed by the cytochrome bo3 quinol:oxygen reductase, cytochrome bd quinol:oxygen reductase and formate dehydrogenase (fdo). These results are supported by blue native-electrophoresis, mass spectrometry and kinetic data of wild type and mutant E . coli strains. 相似文献
15.
The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria 总被引:12,自引:4,他引:8
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1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase and NADH–cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP+-linked) and NADPH–cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD+- and NADP+-linked), α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD+-linked), glutamate–oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, α-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD+- and NADP+-linked), glutamate dehydrogenase (NAD+-linked), glutamate–oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed. 相似文献
16.
《Archives of biochemistry and biophysics》1987,252(1):91-96
Allantoin in the presence of calcium ions has been implicated as a potential toxic agent in Reye's syndrome. An investigation of possible alternative sources of allantoin in humans, which lack the enzyme uricase, has been initiated. Urate is a strong reducing agent which can reduce cytochrome c nonenzymatically, with the concomitant production of CO2 and H+. The stoichiometries measured for the various reactants and products were 1 urate:2 cytochrome c:1 H+:1 CO2. The initial reaction rate depended on the concentrations of both urate and cytochrome c, with reaction kinetics that were first order with respect to urate and second order with respect to cytochrome c. The participation of molecular oxygen in this reaction could not be detected. The pH and ionic strength optima for this reaction were determined to be 9.5–10.5 and 10−5m, respectively. Based on the results reported here, the following balanced equation can be written: urate−2 + 2 cytochrome c+3 + 2 H2O → allantoin + 2 cytochrome c+2 + H+ + HCO3−. We propose that allantoin can be generated from the oxidation of urate by cytochrome c+3, and that this is a potential source of allantoin in human tissues. 相似文献
17.
18.
Biochemical heterogeneity of rat liver mitochondria separated by rate zonal centrifugation 总被引:2,自引:2,他引:0
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1. Rat liver mitochondria were separated on the basis of their sedimentation coefficients in an iso-osmotic gradient of Ficoll–sucrose by rate zonal centrifugation. The fractions (33, each of 40ml) were collected in order of decreasing density. Fractions were analysed by spectral analysis to determine any differences in the concentrations of the cytochromes and by enzyme analyses to ascertain any differences in the activities of NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. 2. When plotted as% of the highest specific concentration, the contents of cytochrome a+a3 and cytochrome c+c1 were constant in all fractions but cytochrome b was only 65% of its maximal concentration in fraction 7 and increased with subsequent fractions. As a result, the cytochrome b/cytochrome a+a3 ratio almost doubled between fractions 7 and 25 whereas the cytochrome c+c1/cytochrome a+a3 ratio was unchanged. 3. Expression of the dehydrogenase activities as% of highest specific activity showed the following for fractions 6–26: NADH dehydrogenase activity remained fairly constant in all fractions; succinate dehydrogenase activity was 62% in fraction 6 and increased steadily to its maximum in fraction 18 and then decreased; the activity of α-glycerophosphate dehydrogenase was only 53% in fraction 6 and increased slowly to its peak in fractions 22 and 24. 4. These differences did not result from damaged or fragmented mitochondria or from microsomal contamination. 5. These results demonstrate that isolated liver mitochondria are biochemically heterogeneous. The importance of using a system for separating biochemically different mitochondria in studies of mitochondrial biogenesis is discussed. 相似文献
19.
A periplasmic location for methanol dehydrogenase from Paracoccus denitrificans: implications for proton pumping by cytochrome aa3 总被引:13,自引:0,他引:13
Evidence is presented that methanol dehydrogenase from has a periplasmic location. The implications for the mechanism of proton translocation during electron flow from methanol to oxygen via cytochromes c and aa3, or to nitrite via cytochrome c and nitrite reductase, are discussed. 相似文献
20.
《Journal of steroid biochemistry》1980,12(8):861-867
Addition of pregnenolone to guinea-pig adrenal microsomes produces a slowly developing difference spectrum with peaks at about 425 and 557 nm and a trough at about 410 nm. The spectral change is similar to that resulting from the reduction of cytochrome b5 by NADH or NADPH. In the presence of sufficient quantities of NADH to fully reduce cytochrome b5, pregnenolone produces a typical type I difference spectrum (ΔA385–420 nm). Pregnenolone is converted to progesterone by adrenal microsomes without addition of cofactor (NAD+) for the 3β-hydroxysteroid dehydrogenase (HSD) reaction. The rate of conversion is increased 2–3 fold by NAD+ and inhibited by NADH. Accompanying the metabolism of pregnenolone (with or without added NAD+) is the production of NADH and reduction of cytochrome b5. Addition of pregnenolone alone to adrenal microsomes results in 60–80% reduction of cytochrome b5. The reduction of cytochrome b5 is maintained for at least as long as pregnenolone is being metabolized. Inhibition of pregnenolone metabolism changes the pregnenolone-induced spectral change to a type I and prevents the reduction of cytochrome b5. The results suggest that the oxidation-reduction state of cytochrome b5 in adrenal microsomes is controlled in part by pregnenolone metabolism which in turn influences the pregnenolone-induced difference spectrum. Oxidation of NADH by cytochrome b5 may serve to prevent NADH inhibition of HSD activity and to generate additional NAD+ as cofactor for the reaction. 相似文献