Abbreviations: PMS, N-methylphenazonium methosulfate; CCCP, carbonylcyanide m-chlorophenylhydrazone; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; TMPD, N,N,N′,N′-tetramethyl-p-phenylendiamine; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea 相似文献
The addition of ascorbate to the PMS phosphorylating system decreases the short-wavelength (<680 mμ) phosophorylation activity but increases the long-wavelength (>690 mμ) phosphorylation activity. The quantum yield profile of this system, showing a long-wavelength rise in phosphorylation efficiency is obtained with or without the addition of 3(3,4-dichlorophenyl)-1, 1-dimethylurea.
These experiments have been interpreted as indicating two separate electrontransfer processes catalyzed by PMS, one in which PMS acts at a reduced efficiency as a Hill oxidant, and the other in which PMS acts as an electron donor and acceptor in a cyclic fashion in sensitizing and essentially long-wavelength phosphorylation process. 相似文献
2. Induction kinetics of delayed light varied depending on temperature. It was found to be composed of two phases; one was an initial rapid rise followed by a rather fast decline to a low steady state level (fast phase), and the other was a slow increase after the initial rapid rise to the maximum followed by an insignificant slow decrease to a high steady state level (slow phase). The fast phase existed between −175 and 40 °C with the maximum at −40 °C, while the slow phase, between 0 and 40 °C with the maximum at 25 °C.
3. The intensity of delayed light at −175 °C was found to be less than one fiftieth that at 0 °C, and no delayed light emission was observed at −196 °C within experimental accuracy. This is in contrast to the results reported by Tollin, G., Fujimori, E. and Calvin, M. ((1958) Proc. Natl. Acad. Sci. U.S. 44, 1035–1047) in which the intensity of delayed light measured at −170 °C was about a half that at 0 °C.
4. The induction of delayed light measured at −96 °C was found to be significantly suppressed by the preillumination at −196 °C. This finding suggests that the primary photochemical event still survives at −196 °C without emission of delayed light.
5. Decay kinetics of delayed light after the flash excitation revealed the presence of at least two decay components. A slow decay component with a half decay time of several tens of milliseconds was observed at temperatures higher than 0 °C. A fast decay component with a half decay time of about 0.2 ms was observed at temperatures between −120 and 25 °C. The decay rate of this component was slightly retarded on cooling.
6. The System II particles derived from spinach chloroplasts with digitonin treatment showed a temperature dependence of delayed light similar to that of the chloroplasts. System I particles, on the other hand, scarcely emitted the delayed light at any temperature between 40 and −196 °C. 相似文献
1. (1) Reduced PMS at the concentration of 1 μM inhibits the back reaction as effectively as hydroxylamine, suggesting an electron donating function of reduced PMS for System II.
2. (2) The inhibition of the back reaction by CCCP is regenerated to a high degree by oxidized PMS which led to assume a cyclic System II electron flow catalysed by PMS.
3. (3) At concentrations of reduced PMS higher than 1 μM it is shown that both the fast initial emission and more significantly the variable emission are quenched.
Both HP700 fragments show the characteristic rapid absorbance changes of P700 upon illumination. The fluorescence properties of the HP700 fragments at −196° are different from those of the original membrane fragments. At −196° the long wavelength fluorescence peak is located at a shorter wavelength (724 mμ) in the diphenylamine-Anabaena HP700 fragment and is lower in intensity than that observed with the membrane fragment. Long wavelength fluorescence at −196° is low in the flurorescence spectra of the membrane fragments of Scenedesmus mutant 6E and is barely observable in the HP700 fragment. The fluorescence spectra of the HP700 fragments of both diphenylamine-Anabaena and Scenedesmus mutant 6E at −196° show a shoulder or peak at 700 nm. The data on fluorescence properties of the HP700 fragments suggest that 730 nm fluorescence does not originate from P700. 相似文献
Light-minus-dark difference spectrum indicated that, in the presence of 10 mM ferrocyanide and 50 μM ferricyanide, only cytochrome c-555 was photooxidized. In the presence of 30 mM ascorbate or at Eh values lower than about 0 mV, both cytochrome c-555 and cytochrome c-552 were photooxidized. The quantum yield of cytochrome c-555 photooxidation was calculated to be about 0.4.
The results obtained in the present study are compared with other investigators' and the possibility of the presence of two types of associations between the cytochromes and reaction-center bacteriochlorophyll is discussed. 相似文献
A specific effort was made to measure the difference spectrum for the photooxidation of P680 under conditions (chloroplasts frozen to −196 °C in the presence of ferricyanide) where a stable, Photosystem II mediated EPR signal, attributed to P680+ has been reported. The difference spectra, however, did not show that P680+ was stable at −196 °C under any conditions tested. Absorbance measurements induced by saturating flashes at −196 °C (in the presence or absence of ferricyanide) indicated that all of the P680+ formed by the flash was reduced in the dark either by a secondary electron donor or by a backreaction with the primary electron acceptor. We conclude that P680+ is not stable in the dark at −196 °C: if the normal secondary donor at −196 °C is oxidized by ferricyanide prior to freezing, P680+ will oxidize other substances. 相似文献
2. Absorbance changes of cytochrome b-559 were not detected in untreated leaves or in most preparations of isolated chloroplasts. After treatment of leaves or chloroplasts with carbonyl cyanide m-chlorophenylhydrazone, high rates of photooxidation of cytochrome b-559 were obtained, both in far-red (>700 nm) and red actinic light. Cytochrome f was photooxidized in far-red light, but in red light it remained mainly in the reduced state. The initial rates of photooxidation of cytochrome b-559 in leaves or chloroplasts treated with carbonyl cyanide m-chlorophenylhydrazone were considerably decreased by 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea.
3. A slow photoreduction of cytochrome b-559 was observed in aged mutant pea chloroplasts in red light.
4. The results do not support the view that cytochrome b-559 is a component of the electron transport chain between the light reactions. It is suggested that cytochrome b-559 is located on a side path from Photosystem II, but with a possible additional link to Photosystem I. 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
Since the reduced iron-sulfur protein signals can only be detected at very low temperature, their decay kinetics cannot be continuously monitored at higher temperatures. Therefore, exposure at appropriate temperatures and reaction times were selected according to the decay kinetics of P700+, after which decay was stopped by lowering the temperature to 13 °K and the P700+ and reduced iron-sulfur protein signals were recorded and compared. In the temperature range (225-13 °K) studied, the decay of P700+ and reduced iron-sulfur protein signals appears identical, suggesting that the two oppositely charged species recombine in the dark. These experiments support the view that iron-sulfur protein(s) is the reaction partner of P700 in the primary photochemical act of Photosystem I. 相似文献
2. The distribution of finger skin temperatures was quite similar at ambient temperature ranges 0 to −10 °C and −10 to −20 °C, while at −20 to −30 °C the finger temperatures were clearly lower.
3. At different ambient temperature ranges, 20–69% of finger temperatures were low enough to cause cold thermal sensations.
4. Sensation of cold was experienced at a finger temperature of 11.6±3.7 °C (mean±SD). 相似文献
2. In the presence of a Co/N2 mixture, the apparent E′0 of cytochrome b270 shifts markedly towards higher potentials (+355 mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain.
3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c2 and of virtually all cytochrome cc′. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 nor the CO-binding cytochrome cc′ are involved in this pathway.
4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in CO-difference spectra and with an band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome “o” and considered the alternative CO-sensitive oxidase. 相似文献
2. Through an electrostatic interaction of the bacteriochlorophyll reaction-center complex and mammalian cytochrome c, an intimate contact between the two components resulted, and a collision-independent electron-transfer with a halftime of 25 μsec can be attained by laser-flash excitation. The absorbance changes at 870 and 550 nm indicated a good stoichiometry of the reaction. The oxidation of the c-type cytochrome in cells of Rps. spheroides (R-26 mutant) has a halftime of 12 μsec.
3. The portion of P870 which recovered rapidly was closely related to the mole ratio of cytochrome/P870. Complete recovery with a halftime of 25 μsec occurred when the cytochrome/P870 ratio was above approx. 10. At cytochrome/P870 ratios lower than 10, only the fraction of the reaction-center complex which have cytochromes bound at the active site can recover with the rapid decay time. Ultrafiltration measurements showed that each particle of the reaction-center complex can bind approx. 24 cytochrome molecules.
4. An electro static interaction is expected simply from the large difference between the isoelectric points of cytochrome c ( 10) and that of the reaction-center complex (4.1 measured by electro-focusing). The electro static interaction was further evidenced by the effects of pH, ionic strength, and by polylysine displacement of binding sites on the coupled oxidation of ferrocytochrome c by P870. From the limiting polylysine concentration giving complete blocking of cytochrome coupling, it was calculated that each reaction-center complex with a particle weight of 6.5×105 contained approx. 500 negative charges.
5. Arrhenius plot of the first-order rate constants vs. the reciprocal absolute temperature yielded an activation energy of 12 kcal/mole for the cytochrome/P870 reaction, which is presumably the energy needed for cytochrome to achieve the most favorable orientation for the rapid electron transfer. Below the freezing temperature of the sample, the cytochrome reaction appeared to be uncoupled. The temperature dependence is consistent with the effect of viscosity on the reaction rate.
6. Double flash excitations spaced 200 μsec apart showed that at a cytochrome/P870 ratio of 24, the first flash caused maximum oxidation, indicating that all the reaction-center particles have at least one cytochrome attached to the active site. However, only 60% of the particles have a second cytochrome closely attached and capable of undergoing the rapid electron transport. 相似文献
1. 1. It was found that respiring mitochondria catalyze the insertion of Fe(II) and Co(II) into deuteroporphyrin. When Fe(III) was used as the metal donor, the reaction revealed an absolute requirement for a supply of reducing equivalents supported by the respiratory chain.
2. 2. A close correlation was found between the disappearance of porphyrin and the formation of heme which allows an accurate estimate of the extinction coefficient for the porphyrin to heme conversion. The value Δ (mM−1 · cm−1) = 3.5 for the wavelength pair 498 509 nm, is considerably lower than previously reported.
3. 3. The maximal rate of deuteroheme synthesis was found to be approx. 1 nM · min−1 · mg−1 of protein at 37 °C, pH 7.4 and optimal substrate concentrations, i.e. 75 μM Fe(III) and 50 μM deuteroporphyrin.
4. 4. Provided the mitochondria are supplemented with an oxidizable substrate, the presence of oxygen has no effect on the rate of deuteroheme synthesis.
Abbreviations: EPPS, (4-(2-hydroxyethyl)-1-piperazine propane sulphonic acid); HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid; PIPES, piperazine-N,N′-2-bis(2-ethanesulphonic acid) 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 DT-10 fragment showed only traces of photochemical activity with water as electron donor, but it was active in a Photosystem II reaction with 2,6-dichlorophenolindophenol as electron acceptor and diphenyl carbazide as donor. Photoreduction of NADP+ with diphenyl carbazide as donor was negligible. There was some photoreduction of NADP+ with ascorbate plus 2,6 dichlorophenolindophenol as donor but this activity could be accounted for by contamination with Photosystem I. These results are consistent with the Z-scheme of photosynthesis with Photosystems I and II operating in series for the reduction of NADP+ from water. DT-10 subchloroplast fragments showed a light-induced rise in fluorescence yield at 20 °C in the presence of diphenyl carbazide. A light-induced fluorescence increase also was observed at 77 °K.
3. During the preparation of the DT-10 fragment, the high potential form of cytochrome b-559 was largely converted to a form of lower potential and C-550 was converted to the reduced state. A photoreduction of C-550 was observed at liquidnitrogen temperature, provided the C-550 was oxidised with ferricyanide prior to cooling. Some photooxidation of cytochrome b-559 was obtained at 77 °K if the preparation was reduced prior to cooling, but the degree of photooxidation was variable with different preparations. C-550 does not appear to be identical with the primary fluorescence quencher, Q.
4. Photosystem I subchloroplast fragments (D-144) released by the action of digitonin were compared with Photosystem I fragments (DT-144) released from D-10 fragments by Triton X-100. There were no significant differences between D-144 and DT-144 fragments either in chlorophyll a/b ratio or in P700 content. 相似文献
2. Uptake of the pyruvate anion by an electrogenic carrier is suggested to be the major mode of transport. Chloroplast swelling was observed in potassium pyruvate plus valinomycin and uptake of [14C]pyruvate was inhibited by membrane-permeant anions. Valinomycin reduced uptake in the absence of external potassium and the inhibition could be reversed by addition of external potassium.
3. Uptake of pyruvic acid (or a pyruvate −/OH− antiport) is ruled unlikely since [14C]pyruvate uptake was relatively independent of the pH gradient across the envelope and addition of pyruvate to chloroplasts did not result in an alkalization of the medium. The low rate of swelling observed in ammonium pyruvate may be due to non-mediated permeation of pyruvic acid, which is possible only at high pyruvate concentrations.
4. The concentration of pyruvate in the stroma increased with external concentration over the range tested (up to 40 mM) but the concentration ratio (internal/external) was always less than one. The steady-state concentration of [14C]pyruvate in the stroma was dependent on the ionic strength of the medium, with saturation at roughly I = 0.04 M, while accumulation of the membrane-permeant cation tetraphenylmethylphosphonium decreased with increasing ionic strength. This suggests that ionic strength modifies a membrane potential (inside negative) across the envelope and that pyruvate uptake responds to the magnitude and direction of that potential (−80 mV at low ionic strength).
5. Chloride and inorganic phosphate were potent inhibitors of [14C]pyruvate uptake. Of the sulfhydryl reagents tested, N-ethylmaleimide was not inhibitory while mersalyl completely blocked [14C]pyruvate uptake and swelling in potassium pyruvate plus valinomycin. Pyruvate uptake, as measured by valinomycin induced swelling in potassium pyruvate, was highly temperature sensitive, with an energy of activation of 39 kcal/mol above 9 °C.
6. Phenylpyruvate, -ketoisovalerate, -ketoisocaproate, -cyano-4-hydroxycinnamic acid and -cyanocinnamic acid inhibited [14C]pyruvate but not [14C]-acetate uptake in the dark and also reduced pyruvate metabolism by the chloroplasts in the light. 相似文献
2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a2). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a2 from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a1). The ratio of the fluorescence yields of chlorophyll a1 and chlorophyll a2 is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.
3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP+ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP+ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.
4. The constancy of the fluorescence yield of chlorophyll a1 in the small fragments, under conditions at which P 700 is oxidized and NADP+ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a1 is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a1 to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.
5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments. 相似文献