Stable Electron Acceptor Research Articles

Evidence for only one stable electron acceptor in the reaction center of Photosystem II in spinach chloroplasts

Evidence for only one stable electron acceptor in the reaction center of Photosystem II in spinach chloroplasts

Bicarbonate in vivo Requirement of Photosystem II in the Green Alga Chlamydobotrys stellata

Flash induced 685 nm fluorescence emission of preilluminated and dark kept Chlamydobotrys stellata has been measured under conditions of CO 2-deprivation. The difference in fluorescence intensity between dark kept and preilluminated cells is taken as a measure for the reduced state of the primary stable electron acceptor of photosystem II, Q, at the given intensity of preillumination. CO 2 removal from growing cultures of this alga for 15 min diminishes photosynthetic electron transport at the oxidizing side of this photosystem. Prolonged CO 2-absence influences also its reducing side. Measurements of flash induced oxygen yields support the conclusion that both sides of photosystem II are affected in the absence of bicarbonate.

Chlorophyll fluorescence and water relations of salt-stressed plants

The yield of chlorophyll a variable fluorescence from leaves of grapevine, bean, barley, spinach, citrus ( Citrus myrtifolia) and mangrove ( Avicennia marina) remained unaffected by salt treatment declining only when excessive ion accumulation led to losses of cell turgor pressure. Salt treatment was found to accelerate the rate of fluorescence quenching after maximal fluorescence yield was attained in barley, spinach, citrus and mangrove, species which accumulate both sodium and chloride into their leaves, but not in grapevine or bean, species which accumulate chloride but exclude sodium from their leaves. Stimulation of fluorescence quenching in intact chloroplasts and thylakoids isolated from spinach was induced upon addition of sodium chloride, but not sorbitol. Energy-dependent fluorescence quenching associated with establishment of a pH gradient appears to be stimulated by sodium ion in isolated chloroplasts and thylakoids. In intact leaves stimulation of fluorescence quenching is more likely a consequence of accelerated photochemical reoxidation of Q, the stable electron acceptor of photosystem II.

Herbicide resistance in Chlamydomonas reinhardtii results from a mutation in the chloroplast gene for the 32-kilodalton protein of photosystem II

We report the isolation and characterization of a uniparental mutant of Chlamydomonas reinhardtii that is resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine). Such herbicides inhibit photosynthesis by preventing transfer of electrons in photosystem II from the primary stable electron acceptor Q to the secondary stable electron acceptor complex B, which is thought to contain a protein of 32 kDa and a bound quinone. It has been proposed that herbicide binding to the 32-kDa protein alters the B complex so that electron transfer from Q is prohibited. Both whole and broken-cell preparations of the mutant alga show a resistance to the effects of herbicide on electron transfer from Q to B, as measured by fluorescence-induction kinetics. In the absence of herbicide, mutant cells exhibit a slower rate of Q to B electron transfer than do wild-type cells. The 32-kDa protein from wild-type cells, but not mutant cells, binds azido[(14)C]atrazine at 0.1 muM. We have isolated psbA, the chloroplast gene for the 32-kDa protein, from both wild-type and herbicide-resistant algae and sequenced the coding regions of the gene that are contained in five exons. The only difference between the exon nucleotide sequences of the wild-type and mutant psbA is a single T-A to G-C transversion. This mutation results in a predicted amino acid change of serine in the wild-type protein to alanine in the mutant. We suggest that this alteration in the 32-kDa protein is the molecular basis for herbicide resistance in the C. reinhardtii mutant.

Interactions of herbicides and azidoquinones at a Photosystem II binding site in the thylakoid membrane

6-Azido-5-decyl-2,3-dimethoxy- p-benzoquinone (6-azido-Q 0C 10) was found to replace the native plastoquinone at B (the second stable electron acceptor to Photosystem II (PS II)). The 6-azido-Q 10C 10 would accept electrons from the primary electron-accepting quinone, Q, thus allowing electron transport through PS II to the plastoquinone pool in thylakoids. The synthetic azidoquinone also competes with the PS II herbicides ioxynil and atrazine for binding. This observation strongly favors the hypothesis that PS II herbicides block electron transport by replacing the native quinone which acts as the second electron carrier on the reducing side of PS II (termed B). Covalent linkage of 6-azido-Q 0C 10 to its binding environment by ultraviolet irradiation greatly reduces herbicide-binding affinity but does not lead to a loss in herbicide-binding sites. We take this as evidence that covalent attachment of 6-azido-Q 0C 10 allows some freedom of quinone head-group movement such that the herbicides can enter the binding site. This indicates that the protein determinants which regulate quinone and herbicide binding are very closely related, but not identical. A compound somewhat related to 6-azido-Q 0C 10 is 2-azido-3-methoxy-5-geranyl-6-methyl- p-benzoquinone (2-azido-Q 2). This compound was found to be an ineffective competitor with respect to herbicide binding. Thus, interactions with protein-binding determinants are highly dependent on the molecular structure of quinones. The 2-azido-Q 2 was an inhibitor of electron flow in the intersystem portion of the chain.

Iron Nutrition-Mediated Chloroplast Development

Membrane development in chloroplasts was explored by resupplying iron to iron-deficient sugar beet (Beta vulgaris L. cv F58-554H1) and monitoring changes in lamellar components during regreening. The synthesis of chlorophyll a, chlorophyll b, and Q, the first stable electron acceptor of photosystem II, exhibited a lag phase during the first 24 to 48 hours of resupply. In contrast, the per area amounts of P(700) and cytochrome f increased linearly over the first 48 hours. During the early regreening period, the Q to P(700) ratio was 2.6 and decreased to 0.7 after 96 hours of regreening. The rate of photosynthesis (net CO(2) uptake) per chlorophyll increased during the first 48 hours of resupply, then by 96 hours decreased to values typical of control plants. The results suggest that there was preferential synthesis of the measured photosystem I components during the first 24 to 48 hours, while from 48 to 96 hours there was rapid synthesis of all components. The iron nutrition-mediated chloroplast development system provides a useful experimental approach for studying biomembrane synthesis and structural-functional relations of the photosynthetic apparatus.

Identification of the triazine receptor protein as a chloroplast gene product

The triazine herbicides inhibit photosynthesis by blocking electron transport at the second stable electron acceptor of photosystem II. This electron transport component of chloroplast thylakoid membranes is a protein-plastoquinone complex termed "B." The polypeptide that is believed to be a component of the B complex has recently been identified as a 32- to 34-kilo-dalton polypeptide by using a photoaffinity labeling probe, azido-[(14)C]atrazine. A 34-kilodalton polypeptide of pea chloroplasts rapidly incorporates [(35)S]methionine in vivo and is also a rapidly labeled product of chloroplast-directed protein synthesis. Trypsin treatment of membranes tagged with azido-[(14)C]atrazine, [(35)S]methionine in vivo, or [(35)S]methionine in isolated intact chloroplasts results in identical, sequential alterations of the 34-kilo-dalton polypeptide to species of 32, then 18 and 16 kilodaltons. From the identical pattern of susceptibility to trypsin we conclude that the rapidly synthesized 34-kilodalton polypeptide that is a product of chloroplast-directed protein synthesis is identical to the triazine herbicide-binding protein of photosystem II. Chloroplasts of both triazine-susceptible and triazine-resistant biotypes of Amaranthus hybridus synthesize the 34-kilodalton polypeptide, but that of the resistant biotype does not bind the herbicide.

Picosecond spectroscopy of photosystem I reaction centers

When iron-sulfur centers A and B [l] (or P430 [2]), the stable primary electron acceptors of photosystem I reaction centers, are in the reduced state, photochemical electron transfer occurs between P700 and the earlier acceptors A1 and AZ [3]. Acceptor AZ has been identified by EPR and optic spectroscopies as an iron-sulfur center [4]. Reduced AZ has an EPR signal with g-values at 1.78,1.88 and 2.08 [5,6], and it recombines with P700’ with a halftime of 130 ms at 5 K [4]. The absorbance change at 694 nm (U6w) measured 50 ps after excitation with a 694 nm excitation pulse is-2-times that ascribable solely to WOO photooxidation; it decays in % 200 ps to the level of WOO’ alone [7]. When iron-sulfur centers A and B are reduced, the aA has the same initial amplitude, but it decays in 2 phases with lifetimes of IO ns and 3 ps [7]. The optic and EPR spectra of the 3 ps component are consistent with the assumption that photochemical charge separation occurs between P700 and a chl a dimer (electron acceptor A,) [4,7]. This electron transfer apparently occurs in < 60 ps, followed by re-oxidation of the chl a dimer by P430 in % 200 ps. This work shows that the spectrum of the 200 ps decay component of the A,4 is consistent with that produced by the formation of an anion radical of a chl a dimer whose absorption-band maximum is at 695 nm.

EFFECTS OF 2,5‐DIBROMO‐3‐METHYL‐6‐ISOPROPYL BENZOQUINONE (DBMIB) ON PHOTOCHEMICAL EVENTS IN RHODOSPIRILLUM RUBRUM*

Abstract. The quinone antagonist dibromothymoquinone (2,5‐dibromo‐3‐methyl‐6‐isopropyl benzoquinone, DBMIB)‡ was used to inhibit early photochemical changes in chromatophores of the photo‐synthetic bacterium, Rhodospirillum rubrum. With continuous illumination with near infrared light, we observed an approximately threefold decrease in efficiency of P865+ formation at 100–200 μM DBMIB as measured by absorbance changes at 605 or 430 nm. However, with continuous illumination of several seconds duration, maximal absorbance changes were observed. At low concentrations (1–10 μM) of DBMIB, a decrease in the decay rate of the ΔA430 or ΔA605 was observed. Using a short (2–10 μs), low intensity light pulse for excitation, we observed that DBMIB inhibits P865+ formation. The concentration dependency of this inhibition corresponds to that seen with continuous illumination. We tested the possibility that DBMIB was displacing the first stable electron acceptor, which has been shown to be a tightly bound ubiquinone (UQ) in R. rubrum. Chromatophores prepared from cells grown with 14C‐p‐hydroxybenzoic acid in their media provided a sample in which the benzoquinones were labelled specifically with 14C. These labelled chromatophores were first extracted with petroleum ether to remove the loosely bound ubiquinone pool and then were treated with DBMIB. DBMIB displaced about 1/2 of the remaining tightly‐bound UQ present. The DBMIB inhibition was tested for reversibility by adding an excess of UQ to DBMIB‐treated samples. In these cases, restoration to 80% could be achieved from samples which were only 20% active relative to the untreated samples. A similar inhibitory effect by DBMIB was also demonstrated in whole cells of Rhodospirillum rubrum, the G‐9 mutant of Rhodospirillum rubrum, Rhodopseudomoms sphaeroides, Rhodopseudomonas capsulata, and Chromatium vinosum. The mechanism for inhibition is most consistent with displacement of, or interference with the first stable electron acceptor (ubiquinone) rather than by random quenching of excited states at the level of antenna bacteriochlorophyll.

Electrochemical and spectro-kinetic evidence for an intermediate electron acceptor in Photosystem I

Absorption changes accompanying light-induced P-700 + formation and its decay in the dark at 15 K in Photosystem-I particles poised at various redox potentials have been examined. In unpoised samples, the light-induced absorption change is practically irreversible. At increasingly negative potentials, an increasing fraction of the absorption change, proportional to the fraction of bound iron-sulfur protein chemically reduced, becomes reversible, and the titration curve has a midpoint potential of −530 mV (vs. normal hydrogen electrode). At −666 mV, the P-700 absorption change is 97% reversible. The total P-700-signal amplitude decreases over the same potential span and levels off at about 43% (to slightly over 50% at a substantially higher excitation intensity). These results provide additional support to previous suggestions of an existence of an intermediate electron acceptor located between the primary donor, P-700, and the more stable primary electron acceptor ( P-430 or bound iron-sulfur protein).