Differential sensitivity of bicarbonate-reversible formate effects on herbicide-resistant mutants of Synechocystis 6714.

Herbicide-resistant mutants of the cyanobacterium Synechocystis 6714, that are altered in specific amino acids in their D-1 protein, shows differential sensitivity to formate treatment. Measurements on oxygen yield in a sequence of flashes, chlorophyll (Chl) a fluorescence transients and Chl a fluorescence yield decay after a flash reveal that the resistance of cells to formate treatment is in the following (highest to lowest) order: [double mutant] A251V/F211S (Az V) greater than [single mutant] F211S (Az I) congruent to wild type greater than [single mutant] S264A (DCMU II-A). Significance of these results in terms of overlapping between the herbicide and bicarbonate binding niches on the D-1 protein is discussed.

Intrinsic uncoupling in proton-pumping cytochrome c oxidase: pH dependence of cytochrome c oxidation in coupled and uncoupled phospholipid vesicles.

The pH dependence of the transient aerobic kinetics of cytochromes c and a has been investigated with cytochrome oxidase reconstituted in phospholipid vesicles in the absence and presence of an uncoupler and an ionophore. The cytochrome a reduction level immediately after the burst phase was 60-80% and was not significantly changed by the addition of uncoupler and/or ionophore. The coupled rate of ferro-cytochrome c oxidation increases linearly with decreasing pH in the range 8.4-5.4. The increase in rate on uncoupling becomes less with decreasing pH and low cytochrome c concentration, being almost zero at pH 5.4. The coupled rate is increased by a lowering of the outside pH when the inside pH is constant. Varying the inside pH with a constant outside pH of 7.4 has little effect on the rate. It is suggested that the electrochemical potential has two separate effects on the coupled rate: the pH gradient mainly slows down the intramolecular electron transfer, but the membrane potential also lowers the second-order rate constant for the reaction with cytochrome c. The results are interpreted in terms of a model in which protonation of an acid-base group with a pKa of 6.4 from the inside increases the catalytic constant. Protonation from the outside, on the other hand, leads to an intrinsic uncoupling, because the protonated enzyme in the output state can return to the input state. This has no adverse physiological effect, since it becomes significant only at pH values well below 7.

Acid-induced double hits in glutaraldehyde-treated Chlorella.

Glutaraldehyde-treated Chlorella cells show pH-dependent oxygen evolution characteristics. 1. The oxygen yield on the second flash (Y2) of a sequence of 2 mus flashes is pH-dependent, the rest of the sequence is unchanged. 2. This yield on the second flash increases as the dark-time between sequences is increased. 3. The deactivation times for the S2 and S3 states are not significantly changed at different pH values. 4. Using a short flash (0.5 mus) for the first flash, no oxygen was detected on the second flash, indicating that there is no S2 remaining in the dark. 5. When the intensity of the first flash is varied, a sigmoidal curve is obtained for Y2 as a function of flash intensity of the first flash is varied, a sigmoidal curve is obtained for Y2 as a function of flash intensity at pH 6.0 but not at pH 7.5. This is indicative of a double hit process. 6. At pH 6.0 the turnover of the Photosystem II centers after the first flash has a fast rise (t 1/2 < 25 mus) followed by a slow phase. This behaviour is not seen at pH 7.5, nor after two flashes. 7. We propose a scheme in which an auxiliary acceptor is in series with Q1 and in competition with B. The redox potential of the auxiliary acceptor is pH dependent.

Effects of sodium azide on photosystem II of Chlorella pyrenoidosa.

The action of sodium azide on the electron transport chain was investigated by means of oxygen evolution, fluorescence and luminescence measurements. (1) The damping of the oxygen oscillations is progressively reduced with increasing azide concentration in the range of 10(-5) - 10(-1) M. (2) The rate of the dark decay of the S2 and S3 states is considerably slowed. The degree of slowing is dependent on concentration. (3) Luminescence is inhibited by azide both in the presence and absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (4) The fluorescence induction curve in strong light is modified in the presence of azide and its shape depends on azide concentration and on incubation time. (5) At a given time after a saturating flash, the fluorescence yield in the presence of azide is much higher than that of the control. It seems to be due to a general fluorescence increase rather than to a slower Q- reduction. (6) We tentatively propose an accelerated reduction of the primary donor P+ in state S2 and S3, by the intermediate donor Z in the presence of azide. Additionally, we have to assume that in the S2 and S3 states, some centers are blocked in an inactive low fluorescent form and that azide decreases their concentration.