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1.
Chemical modification of plastocyanin was carried out using ethylenediamine plus a water-soluble carbodiimide, which has the effect of replacing a negatively charged carboxylate group with a positively charged amino group at pH 6–8. The conditions were adjusted to produce a series of singly and doubly modified forms of plastocyanin. Differences in charge configuration allowed separation of these forms on a Pharmacia fast protein liquid chromatograph using a Mono Q anion exchange column. These forms were used to study the interaction of plastocyanin with its reaction partner cytochrome f. The rate of cytochrome f oxidation was progressively inhibited upon incorporation of increasing numbers of ethylenediamine moieties indicating a positively charged binding site on cytochrome f. However, differential inhibition was obtained for the various singly modified forms allowing mapping of the binding site on plastocyanin. The greatest inhibition was found for forms modified at negatively charged residues Nos. 42–45 and Nos. 59–61 which comprise a negative patch surrounding Tyr-83. In contrast, the form modified at residue No. 68, on the opposite side of the globular plastocyanin molecule, showed the least inhibition. It can be concluded that the binding site for cytochrome f is located in the vicinity of residues Nos. 42–45 and Nos. 59–61. Modification of plastocyanin at residues Nos. 42–45 showed no effect on the rate of P-700 + reduction, suggesting that these residues are not involved in the binding of Photosystem I. However, an increase in the rate of P-700 + reduction was observed for plastocyanins modified at residue No. 68 or Nos. 59–61, which is consistent with the idea that the reaction domain of Photosystem I is negatively charged and Photosystem I binds at the top of the molecule and accepts electrons via His-87 in plastocyanin. These results raise the possibility that plastocyanin can bind both cytochrome f and Photosystem I simultaneously. The effect of ethylenediamine modification on the formal potential of plastocyanin was also examined. The formal potential of control plastocyanin was found to be +372 ± 5 mV vs. normal hydrogen electrode at pH 7. All modified forms showed a positive shift in formal potential. Singly modified forms showed increases in formal potentials between +8 and +18 mV with the largest increases being observed for plastocyanins modified at residues Nos. 42–45 or Nos. 59–61. 相似文献
2.
An electrometric technique was used to investigate electron transfer between spinach plastocyanin (Pc) and photooxidized primary electron donor P700 in photosystem I (PS I) complexes from the cyanobacterium Synechocystis sp. PCC 6803. In the presence of Pc, the fast unresolvable kinetic phase of membrane potential generation related to electron transfer between P700 and the terminal iron–sulfur acceptor F B was followed by additional electrogenic phases in the microsecond and millisecond time scales, which contribute approximately 20% to the overall electrogenicity. These phases are attributed to the vectorial electron transfer from Pc to the protein-embedded chlorophyll dimer P700 + within the PsaA/PsaB heterodimer. The observed rate constant of the millisecond kinetic phase exhibited a saturation profile at increasing Pc concentration, suggesting the formation of a transient complex between Pc and PS I with the dissociation constant Kd of about 80 μM. A small but detectable fast electrogenic phase was observed at high Pc concentration. The rate constant of this phase was independent of Pc concentration, indicating that it is related to a first-order process. 相似文献
3.
Electrostatic characteristics of the membrane surface in the vicinity of P-700 were estimated by analyzing the salt and detergent effects on its reaction rate with ionic reagents using the Gouy-Chapman diffuse double layer theory in various preparations of chloroplasts. Upon disruption of thylakoid membranes by sonic treatment or by treatment with digitonin, the reaction rate markedly increased, while the estimated surface charge density became smaller. It was concluded that the membrane surface which determines the reaction rate between P-700 and the ionic reagents changed as the disruption of thylakoid structure. The outer thylakoid surface had more negative charges than the inner one. Changes in the electrical potential profile across the thylakoid membrane during the illumination were also discussed from these results. 相似文献
4.
Electron paramagnetic resonance (EPR) power saturation and saturation recovery methods have been used to determine the spin lattice, T1, and spin-spin, T2, relaxation times of P-700 + reaction-center chlorophyll in Photosystem I of plant chloroplasts for 10 K T 100 K. T1 was 200 μs at 100 K and increased to 900 μs at 10 K. T2 was 40 ns at 40 K and increased to 100 ns at 10 K. T1 for 40 K T 100 K is inversely proportional to temperature, which is evidence of a direct-lattice relaxation process. At T = 20 K, T1 deviates from the 1/ T dependence, indicating a cross relaxation process with an unidentified paramagnetic species. The individual effects of ascorbate and ferricyanide on T1 of P-700 + were examined: T1 of P-700 + was not affected by adding 10 mM ascorbate to digitonin-treated chloroplast fragments (D144 fragments). The P-700 + relaxation time in broken chloroplasts treated with 10 mM ferricyanide was 4-times shorter than in the untreated control at 40 K. Ferricyanide appears to be relaxing the P-700 + indirectly to the lattice by a cross-relaxation process. The possibility of dipolar-spin broadening of P-700 + due to either the iron-sulfur center A or plastocyanin was examined by determining the spin-packet linewidth for P-700 + when center A and plastocyanin were in either the reduced or oxidized states. Neither reduced center A nor oxidized plastocyanin was capable of broadening the spin-packet linewidth of the P-700 + signal. The absence of diplolar broadening indicates that both center A and plastocyanin are located at a distance at least 3.0 nm from the P-700 + reaction center chlorophyll. This evidence supports previous hypotheses that the electron donor and acceptor to P-700 are situated on opposite sides of the chloroplast membrane. It is also shown that the ratio of photo-oxidized P-700 to photoreduced centers A and B at low temperature is 2 : 1 if P-700 is monitored at a nonsaturating microwave power. 相似文献
5.
The kinetics of redox changes of P-700, plastocyanin and cytochrome f in chloroplasts suspended in a fluid medium at sub-zero temperatures have been studied following excitation of the chloroplasts with either a single-turnover flash, a series of flashes or continuous light. The results show that: (1) The kinetics of reduction of P-700 + and those of oxidation of plastocyanin are consistent with a bimolecular reaction between these two components as previously suggested (Olsen, L.F., Cox, R.P. and Barber, J. (1980) FEBS Lett. 122, 13–16). (2) Cytochrome f shows heterogeneity with respect to its kinetics of oxidation by Photosystem I. (3) In contrast to the situation when plastoquinol is the electron donor, reduction of cytochrome f by electrons derived from diaminodurene occurs with sigmoidal kinetics that shows a good fit to an apparent equilibrium constant of 12 between the cytochrome and P-700. (4) The rate of electron transfer from plastoquinol to Photosystem I depends on the redox state of the plastoquinone pool. (5) In relation to current ideas about the lateral heterogeneity of Photosystem I and Photosystem II in the thylakoid membrane, the results are consistent with the function of plastocyanin as a mobile carrier of electrons in the intrathylakoid space. 相似文献
6.
Salt- or pH-induced change of the rate of reduction of the phtooxidized membrane bound electron transfer components, P-700, by ionic and nonionic reductants added in the outer medium was studied in sonicated chloroplasts. The rate with the negatively charged reductants increased with the increase of salt concentration at a neutral pH or with the decrease of medium pH. Salts of divalent cations were much more effective than those of monovalent cations. A trivalent cation was even more effective. The rate with a nonionic reductant was little affected by salts. The change of the reduction rate was analyzed using the Gouy-Chapman theory, which explains the change of reduction rate by the changes of activities of ionic reductants at the charged membrane surface where the reaction takes place. This analysis gave more useful parameters and explained more satisfactorily the case with high-valence cation salts than the Brönsted type analysis. The values for the surface charge density and the surface potential of the membrane surface in the vicinity of P-700 estimated from the analysis were lower than those estimated for the surface in the vicinity of Photosystem II primary acceptor, suggesting the heterogeneity of the thylakoid surface. The salt-induced surface potential change was shown to affect the activation energy of the reaction between P-700 and the ionic reagent. 相似文献
7.
1. Changes in the fluorescence yield of aerobic Chlorella vulgaris have been measured in laser flashes of 15 ns, 30 ns and 350 ns half time. The kinetics after the first flash given after a 3 min dark period could be simulated on a computer using the hypothesis that the oxidized acceptor Q and primary donor P + are fluorescence quenchers, and Q − is a weak quencher, and that the reduction time for P + is 20–35 ns. 2. The P+ reduction time for at least an appreciable part of the reaction centers was found to be longer after the second and subsequent flashes. In the first 5 flashes an oscillation was observed. Under steady state conditions, with a pulse separation of 3 s, a reduction time for P+ of about 400 ns for all reaction centers gave the best correspondence between computed and experimental fluorescence kinetics. 相似文献
8.
PSI-G is an 11 kDa subunit of PSI in photosynthetic eukaryotes. Arabidopsis thaliana plants devoid of PSI-G have a decreased PSI content and an increased activity of NADP + photoreduction in vitro but otherwise no obvious phenotype [P.E. Jensen, L. Rosgaard, J. Knoetzel, H.V. Scheller, Photosystem I activity is increased in the absence of the PSI-G subunit. J. Biol. Chem. 277, (2002) 2798-2803.]. To investigate the biochemical basis for the increased activity, the kinetic parameters of the reaction between PSI and plastocyanin were determined. PSI-G clearly plays a role in the affinity for plastocyanin since the dissociation constant ( KD) is only 12 μM in the absence of PSI-G compared to 32 μM for the wild type. On the physiological level, plants devoid of PSI-G have a more reduced QA. This indicates that the decreased PSI content is due to unstable PSI rather than an adaptation to the increased activity. In agreement with this indication of decreased stability, plants devoid of PSI-G were found to be more photoinhibited both at low temperature and after high light treatment. The decreased PSI stability was confirmed in vitro by measuring PSI activity after illumination of a thylakoid suspension which clearly showed a faster decrease in PSI activity in the thylakoids lacking PSI-G. Light response of the P700 redox state in vivo showed that in the absence of PSI-G, P700 is more reduced at low light intensities. We conclude that PSI-G is involved in the binding dynamics of plastocyanin to PSI and that PSI-G is important for the stability of the PSI complex. 相似文献
9.
It has been shown that efficient functioning of photosynthesis and respiration in the cyanobacterium Synechocystis PCC 6803 requires the presence of either cytochrome c6 or plastocyanin. In order to check whether the blue copper protein plastocyanin can act as electron donor to cytochrome c oxidase, we investigated the intermolecular electron transfer kinetics between plastocyanin and the soluble CuA domain (i.e. the donor binding and electron entry site) of subunit II of the aa3-type cytochrome c oxidase from Synechocystis. Both copper proteins were expressed heterologously in Escherichia coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (5.1+/-0.2) x 10(4) M(-1) s(-1) and (8.5+/-0.4) x 10(5) M(-1) s(-1), respectively (20 mM phosphate buffer, pH 7). This corresponds to an apparent equilibrium constant of 0.06 in the physiological direction (reduction of CuA), which is similar to Keq values calculated for the reaction between c-type cytochromes and the soluble fragments of other CuA domains. The potential physiological role of plastocyanin in cyanobacterial respiration is discussed. 相似文献
10.
Three groups of isomeric nitrogen heterocycles, phenylpyridines, phenylimidazoles and pyridylimidazoles were studied in relation to the effect of steric factors on type II binding to cytochrome P-450 and inhibition of aryl hydrocarbon (benzo[ a]pyrene) hydroxylase (AHH) activity in hepatic microsomes from phenobarbital(PB)- and β-naphthoflavone(βNF)-induced rats. Type II binding affinity was lower (higher Ks) in compounds with substituents on the carbon adjacent to the nitrogen undergoing ligand interaction than in those where steric hindrance near the nitrogen was minimal. Binding affinities of the compounds as measured by their Ks values, were quite similar in both PB- and βNF-induced microsomes. In PB-induced microsomes, type II binding affinity was generally reflected by the ability of the compounds to inhibit AHH activity. In contrast, most of the compounds evaluated were inactive as AHH inhibitors in βNF-induced microsomes. 相似文献
11.
Absorption changes accompanying light-induced P-700 oxidation and the decay of P-700+ in the dark were measured in the temperature range 294-5 K over a broad time scale (three to four orders of magnitude). Two qualitatively different types of kinetics for the dark decay of P-700 + were observed. In the 294-240K region, a usual exponential kinetics is observed with the rate constant κ = 1 · 10 10 · exp(-16 000/RT) s ?1, with R in cal/mol per degree. Below 220 K, a rather unusual logarithmic or near-logarithmic kinetics are observed. These kinetics can be explained quantitatively if one assumes for the various ( P-700+ ··· X -) pairs a broad rectangular or near-rectangular distribution over the values of the rate constant. The following kinetic equation corresponding to this model was obtained: nt/ no = [In( κmax/ κmin)]-1 - [In(1/ κmin)? In t] where no and nt are respectively the initial concentration of P-700 + and its concentration at time t, and kmax and kmin the maximum and minimum values of the rate constant, respectively. The decay processes observed can be ascribed to electron tunneling. Distribution over the values of k can be accounted for by different environments or different mutual orientations of P-700 + and X ?, or by different distances between them in the various reacting pairs.The corresponding distribution function was reconstructed from the experimentally measured P-700+-decay curves. The rate of tunneling was found to be temperature dependent. In the 160-80-K region, the temperature dependence corresponds to an activation energy of 2.9 kcal/mol. Below 80 K, new modes of P-700+ decay with lower activation energy become operative. The tunneling distance for the majority of the ( P-700+ ··· X ?) pairs was estimated from the EPR linewidth of P-700+ to exceed 13.2 A. 相似文献
12.
The primary donor of Photosystem II (PS II), P-680, was photo-oxidized by a short flash and its rate of reduction was measured at different pH values by following the recovery of the absorption change at 820 nm in chloroplasts pretreated with a high concentration of Tris. The re-reduction is biphasic with a fast phase (dominant after the first flash) attributed to the donation by a donor, D 1, and a slow phase (usually dominant after the second flash) attributed to a back-reaction with the primary acceptor. It is found that pH has a strong influence on the donation from D1 (τ = 2 μs at pH 9, 44 μs at pH 4), but no influence on the back reaction (τ ≈ 200 μs). pH also influences the stability of the charge separation since the contribution of donation from D1 at the second flash increases at lower pH, getting close to 100% at pH 4. 相似文献
13.
Fifteen ancestral genotypes of United States soybean cultivars were screened for differences in photosynthetic electron transport capacity using isolated thylakoid membranes. Plants were grown in controlled environment chambers under high or low irradiance conditions. Thylakoid membranes were isolated from mature leaves. Photosynthetic electron transport was assayed as uncoupled Hill activity using 2,6-dichlorophenolindophenol (DCIP). Soybean electron transport activity was dependent on genotype and growth irradiance and ranged from 6 to 91 mmol DCIP reduced [mol chlorophyll] –1 s –1. Soybean plastocyanin pool size ranged from 0.1 to 1.3 mol plastocyanin [mol Photosystem I] –1. In contrast, barley and spinach electron transport activities were 140 and 170 mmol DCIP reduced [mol chlorophyll] –1 s –1, respectively, with plastocyanin pool sizes of 3 to 4 mol plastocyanin [mol Photosystem I] –1. No significant differences in the concentrations of Photosystem II, plastoquinone, cytochrome b 6f complexes, or Photosystem I were observed. Thus, genetic differences in electron transport activity were correlated with plastocyanin pool size. The results suggested that plastocyanin pool size can vary significantly and may limit photosynthetic electron transport capacity in certain species such as soybean. Soybean plastocyanin consisted of two isoforms with apparent molecular masses of 14 and 11 kDa, whereas barley and spinach plastocyanins each consisted of single polypeptides of 8 and 12 kDa, respectively.Abbreviations DAP
days after planting
- DCIP
2,6-dichlorophenolindophenol
- LiDS
lithium dodecyl sulfate
- PPFD
photosynthetic photon flux density (mol photons m –2 s –1)
- PS I
Photosystem I
- PS II
Photosystem II
- P700
reaction center of Photosystem I
The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged. 相似文献
14.
Cytochrome c 6 and plastocyanin are soluble metalloproteins that act as mobile carriers transferring electrons between the two membrane-embedded photosynthetic complexes cytochrome b 6 f and photosystem I (PSI). First, an account of recent data on structural and functional features of these two membrane complexes is presented. Afterwards, attention is focused on the mobile heme and copper proteins – and, in particular, on the structural factors that allow recognition and confer molecular specificity and control the rates of electron transfer from and to the membrane complexes. The interesting question of why plastocyanin has been chosen over the ancient heme protein is discussed to place emphasis on the evolutionary aspects. In fact, cytochrome c 6 and plastocyanin are presented herein as an excellent case study of biological evolution, which is not only convergent (two different structures but the same physiological function), but also parallel (two proteins adapting themselves to vary accordingly to each other within the same organism). Received: 4 July 1996 / Accepted: 16 September 1996 相似文献
15.
Electron transport has been studied by flash absorption and EPR spectroscopies at 10–30 K in Photosystem I particles prepared with digitonin under different redox conditions. In the presence of ascorbate, an irreversible charge separation is progressively induced at 10 K between P-700 and iron-sulfur center A by successive laser flashes, up to a maximum which corresponds to about two-thirds of the reaction centers. In these centers, heterogeneity of the rate for center A reduction is also shown. In the other third of reaction centers, the charge separation is reversible and relaxes with a t1/2 ≈ 120 μs. When the iron-sulfur centers A and B are prereduced, the 120 μs relaxation becomes the dominant process (70–80% of the reaction centers), while a slow component ( t1/2 = 50–400 ms) reflecting the recombination between P-700 + and center X − occurs in a minority of reaction centers (10–15%). Flash absorption and EPR experiments show that the partner of P-700 + in the 120 μs recombination is neither X nor a chlorophyll but more probably the acceptor A −1 as defined by Bonnerjea and Evans (Bonnerjea, J. and Evans, M.C.W. (1982) FEBS Lett. 148, 313–316). The role of center X in low-temperature electron flow is also discussed. 相似文献
16.
Plastocyanin is specifically cross-linked by incubation with N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) to a subunit of photosystem I in stroma lamellae and in isolated photosystem I complex. SDS-PAGE shows the disappearance of a 18.5 kDa subunit and the appearance of a new 31.5 kDa protein which was recognized by anti-plastocyanin antibodies. The isolated subunit was identified by its N-terminal amino acid sequence as the mature peptide coded by the nuclear gene psaF [Steppuhn et al. (1988) FEBS Lett. 237, 218–224]. P700 + was reduced by cross-linked plastocyanin with the same halftime of 13 μs as found in the native complex. This is evidence that cross-linking conserved the orientation of the complex and that the 18.5 kDa subunit provides the conformation of photosystem I necessary for the extremely rapid electron transfer from plastocyanin to P700 +. 相似文献
17.
The pathways through which NADPH, NADH and H 2 provide electrons to nitrogenase were examined in anaerobically isolated heterocysts. Electron donation in freeze-thawed heterocysts and in heterocyst fractions was studied by measuring O 2 uptake, acetylene reduction and reduction of horse heart cytochrome c. In freeze-thawed heterocysts and membrane fractions, NADH and H 2 supported cyanide-sensitive, respiratory O 2 uptake and light-enhanced, cyanide-insensitive uptake of O 2 resulting from electron donation to O 2 at the reducing side of Photosystem I. Membrane fractions also catalyzed NADH-dependent reduction of cytochrome c. In freeze-thawed heterocysts and soluble fractions from heterocysts, NADPH donated electrons in dark reactions to O 2 or cytochrome c through a pathway involving ferredoxin:NADP reductase; these reactions were only slightly influenced by cyanide or illumination. In freeze-thawed heterocysts provided with an ATP-generating system, NADH or H 2 supported slow acetylene reduction in the dark through uncoupler-sensitive reverse electron flow. Upon illumination, enhanced rates of acetylene reduction requiring the participation of Photosystem I were observed with NADH and H 2 as electron donors. Rapid NADPH-dependent acetylene reduction occurred in the dark and this activity was not influenced by illumination or uncoupler. A scheme summarizing electron-transfer pathways between soluble and membrane components is presented. 相似文献
18.
The oxidation and reduction of cytochrome f and P-700 is measured spectrophotometrically in leaves of low-light and high-light plants. After illumination with red light, an induction phenomenon for cytochrome f oxidation is observed which indicates a regulation of photosystem I activity through energy distribution between the pigment systems by the energy state of the membrane. After far-red excitation the reduction of cytochrome f in the dark is much slower in low-light leaves. This shows that cyclic electron transport is not improved in low-light plants under these conditions. P-700 is oxidized on excitation with far-red light. However, with high intensities of far-red light, P-700 is partially reduced again which is due to a low extent of photosystem II excitation with the far-red used in the experiments. The low-light leaves show greater sensitivity of photosystem II to this excitation. The initial rate of the cytochrome f oxidation-rate is the same in low-light and high-light leaves. This shows that several P-700 are connected with only one electron transport chain. The consequences of these results concerning the tripartite concept and the photosynthetic unit are discussed. In the high-light plants the experimental data can be well explained by the tripartite organization of the photosynthetic unit. In low-light plants, however, a multipartite organization has to be postulated. In the partition regions of the grana, several antennae systems I, antennae systems II, and light-harvesting complexes can communicate with one electron transport chain.Abbreviations CP I
P-700-chlorophyll a-protein
- Cyt f
cytochrome f
- DCMU
3-(3,4 dichlorophenyl)-1,1-dimethylurea
- DBMIB
2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone
- LA
leaf-area
- PhAR
photosynthetically active radiation
- PS
photosystem 相似文献
19.
The transient complex between cytochrome f and plastocyanin from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by X-ray Absorption Spectroscopy in solution, using both proteins in their oxidized and reduced
states. Fe K-edge data mainly shows that the atypical metal coordination geometry of cytochrome f, in which the N-terminal amino acid acts as an axial ligand of the heme group, remains unaltered upon binding to its redox
partner, plastocyanin. This fact suggests that cytochrome f provides a stable binding site for plastocyanin and minimizes the reorganization energy required in the transient complex
formation, which could facilitate the electron transfer between the two redox partners. 相似文献
20.
The kinetics of the reaction of hydrated electron (e −aq) and carboxyl anion radical (CO 2) with Pseudomonas aeruginosa ferricytochrome c-551 were studied by pulse radiolysis. The rate of reaction of e −aq with the negatively charged ferricytochrome c-551 (17 nM −1 · s −1) is significantly slower than the larger positively charged horse heart ferricytochrome c (70 nM − · s −). This difference cannot be explained solely by electrostatic effects on the diffusion-controlled reactions. After the initial encounter of e −aq with the protein, ferricytochrome c-551 is less effective in transferring an electron to the heme which may be due to the negative charge on the protein. The charge on ferricytochrome c-551 is estimated to be −5 at pH 7 from the effect of ionic strength on the reaction rate. A slower relaxation (2 · 10 4 s −1) observed after fast e −aq reduction is attributed to a small conformational change. The rate of reaction of CO 2 with ferricytochrome c-551 (0.7 nM −1 · s −) is, after electrostatic correction, the same as ferricytochrome c, indicating that the steric requirements for reaction are similar. This reaction probably takes place through the exposed heme edge. 相似文献
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