2. Reduced flavins uncombined with proteins show very little acceptor specificity and react very rapidly with nearly all the commonly used acceptors. Their behaviour resembles that of dithionite very closely indeed, and it differs considerably from that of flavoproteins. Like dithionite, free reduced flavins reduce O2 quantitatively to H2O2; this oxidizes a further molecule of flavin.
3. H2O2 and cytochrome c react more slowly than most acceptors with reduced flavins. Nitrate and NDA+ do not act at all and require special activation.
4. Catalase can act as a catalyst for the aerobic oxidation of flavins by converting slowly-reacting H2O2 into rapidly-reacting O2.
5. In the absence of catalytic metals ascorbate reacts with acceptors much more slowly than reduced flavins do. 相似文献
2. It is best formed with an excess of O2 after reduction with a minimum amount of dithionite. It can also be formed at low O2 tension, but then contains some ferric oxidase.
3. Its formation from ferrocyanide-reduced oxidase remains incomplete and subsequent reduction by dithionite is also incomplete.
4. Cyanide does not inhibit its formation from ferrous oxidase. If only ferricytochrome a but no ferricytochrome a3 is reduced in the presence of cyanide by dithionite, there is no reaction with O2.
5. The anaerobic reduction of ‘oxygenated’ oxidase by dithionite is monophasic and fast. In contrast, that of ferric oxidase is biphasic, with an initial fast reduction of ferricytochrome a followed by a much slower reduction of ferricytochrome a3. The rate of cytochrome a, but not that of cytochrome a3 reduction depends on dithionite concentration.
6. In the presence of dissolved O2, the ferric oxidase reduction comes to a temporary standstill when one-third of the absorbance increase at 444 mμ has been reached.
7. Ethyl hydrogen peroxide reacting with ferrous oxidase forms a compound similar to the ‘oxygenated’ compound.
8. Hydrogen donors known to react with peroxidase-H2O2 complexes, particularly pyrogallol, accelerate the transformation of ‘oxygenated’ to ferric oxidase, though not at a rate comparable to that of cytochrome c.
9. These results strengthen the evidence for cytochromes a and a3 but indicate that this difference has disappeared in ‘oxygenated’ oxidase. 相似文献
2. The acceptor specificity of reduced flavin groups, when combined with apoenzyme proteins, is quite different from that of the same flavin groups in the free state (see Part II). Free flavins react very rapidly with a wide range of acceptors, but the same groups combined as flavoproteins have a severely restricted range of action.
3. There are remarkable differences between different flavoproteins. Nearly every flavoprotein fails altogether to react with at least one, and often several, of the acceptors, giving a specificity pattern which is different in each case. There seems to be no general acceptor for flavoproteins.
4. The effect of combination of a flavin with a particular apoenzyme is to inhibit specifically the reaction of the flavin with particular acceptors with which it would react very rapidly in the absence of the apoenzyme.
5. Each apoenzyme produces its own distinctive pattern of inhibitions. The degree of inhibition is often very high; the table shows over 50 cases of specific inhibitions that are essentially complete. Some of these are very difficult to explain.
6. There is no obvious parallelism between any acceptor and any other in its pattern of reactivity with a series of different flavoproteins.
7. In a few cases combination with apoenzyme specifically accelerates the reaction of the flavin with particular acceptors, so that the flavoprotein is oxidized faster than the free flavin.
8. Possible correlations are discussed between the effects of apoenzymes on the reactivity of flavins with acceptors and a number of special known features of different apoenzymes, but no adequate explanation of the differences in specificity has emerged.
9. In view of the interesting nature of the effects, a plea is made for a more intensive study of the acceptor side of flavoprotein specificity. 相似文献
2. The phosphate requirement could be met by orthophosphate, inorganic pyrophosphate, ATP or ADP but not by AMP. Approx. 3 molecules of O2 were evolved for each molecule of orthophosphate added and approx. 6 for each molecule of pyrophosphate.
3. With CO2 as the sole added substrate the extent of the initial lag in O2 evolution was not greatly affected by small quantities of added orthophosphate but as the concentration of orthophosphate was increased there was a progressive increase in the lag and a progressive decrease in the maximum rate. Pyrophosphate failed to produce these effects at a 100 times the concentration and in the presence of pyrophosphate the orthophosphate inhibition was less severe. There was little or no orthophosphate inhibition in the presence of substrate quantities of 3-phosphoglycerate or ribose 5-phosphate and CO2.
4. There was also a requirement for phosphate by chloroplasts evolving O2 in the presence of 3-phosphoglycerate or ribose 5-phosphate plus CO2. In the presence of endogenous phosphate only, added ribose 5-phosphate suppressed the O2 evolution which normally followed the addition of 3-phosphoglycerate.
5. The results provide direct support for the proposed phosphate requirement of the photosynthetic carbon cycle and are discussed in this context. They also imply that orthophosphate, ribose 5-phosphate and 3-phosphoglycerate can penetrate the intact chloroplast envelope with considerable rapidity. 相似文献
2. In the presence of methyl viologen, all the blue-green algae except Anabaena flos-aquae show a light-dependent O2 consumption as well as a post-illumination O2 evolution. The rate of O2 consumption is stimulated by 1 mM KCN, an inhibitor of catalase, but the dark O2 evolution becomes suppressed.
3. A. flos-aquae shows a light-dependent methyl viologen-catalyzed O2 uptake which is not affected by 1 mM KCN. Furthermore, there is no release of O2 in the dark following illumination.
4. With C. reinhardi, the cells do not show any net O2 exchange during or after illumination. Addition of 1 mM KCN, however, results in an immediate O2 uptake in the light.
5. Based on the mechanism postulated for the Mehler reaction in isolated chloroplasts, it was deduced that the differences in the kinetics of the O2 exchange catalyzed by methyl viologen reflect differences in the endogenous catalase activity in these algae. Cells of A. flos-aquae are deficient in catalase activity whereas those of the other blue-green algae possess catalase, although at low activity. C. reinhardi, on the other hand, has high catalase activity in vivo.
6. These findings are corroborated by results obtained from O2 electrode measurements of catalase activity in cell-free extracts of these algae.
7. The possible roles of catalase in algae and the implications of these results are also discussed. 相似文献
2. The initial phase of H+ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H+ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.
3. Dibromothymoquinone reduction, observed as O2 evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O2 evolution agreeing well with the steady-state rate observed under continuous illumination.
4. Such measurements of the initial rates of O2 evolution and H+ uptake yield an H+/e− ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H+/e− ratio of 1.7 at pH 7.6.)
5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H+ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, Pi and hexokinase, when added individually, have no effect on proton uptake in this system.
6. The involvement of a proton uptake reaction with an H+/e− ratio of 0.5 in the Photosystem II partial reaction H2O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e2) and proton uptake (H+/e−) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling. 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. 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. Oxygen evolution by reconstituted chloroplasts with 3-phosphoglycerate as substrate was also inhibited by the addition of ADP or following uncoupling by added NH4Cl.
3. In all cases the inhibitory effects of ADP were reversed by addition of phosphocreatine and creatine phosphokinase activity.
4. The stoichiometry of 3-phosphoglycerate reduction to NADPH oxidation in chloroplast extracts was 1:1 and there was negligible turnover of the Benson-Calvin cycle in either chloroplast extracts or in reconstituted chloroplasts under the particular conditions employed.
5. The maximum rate of 3-phosphoglycerate-dependent O2 evolution by reconstituted chloroplasts was ultimately limited by NADP reduction and photo-phosphorylation, and was similar to the maximum rate of oxygen evolution under optimal conditions by intact chloroplasts. In the presence of sufficient ADP phosphorylating activity, the rate of enzymic 3-phosphoglycerate reduction was relatively high. The inhibition of this reaction by ADP may represent a control mechanism in photosynthesis. 相似文献
The influence of very low O2 concentration on the NADP+ reduction by isolated spinach chloroplasts has been studied.
The results show that in the presence of very low O2 concentration (< 0.3%) NADP+ reduction is partially inhibited. This inhibition may be partially reversed under some conditions, especially when, in spite of the presence of an O2 trap (glucose plus glucose oxidase (EC 1.1.3.4)) an O2 evolution is observed. 相似文献
2. The amounts of cytochromes c1 and aa3 are similar in the mutant and wild type. Cytochrome b-566 could not be detected in low-temperature spectra after reduction with various substrates or dithionite. A b-558 is, however, present.
3. The b-cytochromes in the mutant are not reduced by NADH or succinate during the steady state even after addition of ubiquinone-1. QH2-3: cytochrome c reductase activity is very low and succinate oxidation is highly stimulated by phenazine methosulphate.
4. Antimycin does not bind to either oxidized or reduced mitochondrial particles of the mutant.
5. In contrast to the b-cytochromes of the wild type, b-558 in the mutant reacts with CO.
6. Cytochromes aa3, c and c1 are partly reduced in aerated submitochondrial particles isolated from the mutant and the EPR signal of Cu (II), measured at 35°K, is detectable only after the addition of ferricyanide. In the mutant, a signal with a trough at g = 2.01 is found, in addition to the signal at g = 1.98 found in the wild type.
7. The ATPase activity of particles isolated from the mutant is much lower than in the wild type but is still inhibited by oligomycin. 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 chloroplasts isolated in MES-sorbitol media exhibited induction phenomena which were similar to those shown by chloroplasts isolated in orthophosphate-sugar mixtures. Added ribose 5-phosphate shortened the lags which preceded the attainment of maximal rates of CO2 fixation and O2 evolution. O2 evolution reached its maximum rate almost immediately in the presence of 3-phosphoglycerate. Induction periods were shortened by pre-illumination of the parent tissue prior to separation of the chloroplasts.
3. In the absence of added substrate (other than CO2) lags exhibited by chloroplasts isolated in MES-sorbitol were shorter than those observed with chloroplasts prepared in orthophosphate-sorbitol. These shorter lags could be extended by briefly exposing the chloroplasts to sugar media containing orthophosphate, malate or acetate or to Tris-NaCl.
4. The results are discussed in relation to photosynthetic induction phenomena and current methods of chloroplast isolation. 相似文献
2. A convenient electrochemical preparation of large amounts of MV.+ has been developed.
3. A commercial stopped-flow apparatus was modified in order to obtain a high degree of anaerobicity.
4. The reaction of MV.+ with O2 produced H2O2 (k > 5 · 106 M−1 · s−1, pH 7.5, 25 °C). H2O2 subsequently reacted with excess MV.+ (k = 2.3 · 103 M−1 · s−1, pH 7.5, 25 °C) to produce water. The kinetics of this reaction were complex and have only been interpreted over a limited range of concentrations.
5. The results support the theory that the herbicidal action of methyl viologen (Paraquat, Gramoxone) is due to H2O2 (or radicals derived from H2O2) induced damage of plant cell membrane. 相似文献
The influence of O2 concentrations on the Hill reactions in the presence of p-benzoquinone, ferricyanide, NADP+, NADP+ plus ferredoxin has been studied with isolated spinach chloroplasts.
Because of the partial reoxidation of the hydroquinone, which is depending upon the O2 concentration, it does not seem possible to localize a site of action for O2.
With ferricyanide the influence of O2 is weak. However, the rate of ferricyanide reduction is increased in the presence of O2. The observed stimulation is greater for 21% O2 than for 70% O2. Bicarbonate stimulates the ferricyanide reduction and decreases the stimulating effect of 21% O2.
O2 decreases the rate of NADP+ reduction. Ferredoxin as well as bicarbonate stimulate the NADP+ reduction and reduce the O2 inhibition.
These results seem to indicate that O2 may enter the electron transport chain at a site situated near Photosystem I and before the ferredoxin's site.
The inhibitory effect of O2 on the Hill reactions with p-benzoquinone and NADP+ is depending upon the plants' growth conditions. It is greater with plants grown under weak light. 相似文献