An analysis of proton fluxes coupled to electron transport and ATP synthesis in chloroplast thylakoids

Electron transport, phosphorylation and internal proton concentration were measured in illuminated spinach chloroplast thylakoid membranes under a number of conditions. Regardless of the procedure used to vary these parameters, the data fit a simple chemiosmotic model. Protons from Photosystem II did not appear to be utilized differently from those derived from Photosystem I. The maximal phosphorylation efficiency ($\text{P}\text{e}{}_2$) for photophosphorylation in washed thylakoids under oxidizing conditions is likely to be $\text{4}\text{3}$. This value is consistent with a proton-to-electron-pair ratio of 4 for electron flow through both photosystems and a proton-to-ATP ratio of 3 for the chloroplast proton-ATPase.     

EPR detection of a cryogenically photogenerated intermediate in photosynthetic oxygen evolution

In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S₁ and S₂ states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S₂ state. Low-temperature illumination of samples prepared in the S₂ state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH₂OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl^? with F^? exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a $\text{S =}\text{5}\text{2}$ spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.     

The chloride requirement for photosynthetic oxygen evolution. Analysis of the effects of chloride and other anions on amine inhibition of the oxygen-evolving complex

In the presence of Cl^?, the severity of ammonia-induced inhibition of photosynthetic oxygen evolution is attenuated in spinach thylakoid membranes (Sandusky, P.O. and Yocum, C.F. (1983) FEBS Lett. 162, 339–343). A further examination of this phenomenon using steady-state kinetic analysis suggests that there are two sites of ammonia attack, only one of which is protected by the presence of Cl^?. In the case of Tris-induced inhibition of oxygen evolution only the Cl^? protected site is evident. In both cases the mechanism of Cl^? protection involves the binding of Cl^? in competition with the inhibitory amine. Anions (Br^? and NO^?₃) known to reactive oxygen evolution in Cl^?-depleted membranes also protect against Tris-induced inhibition, and reactivation of Cl^?-depleted membranes by Cl^? is competitively inhibited by ammonia. Inactivation of the oxygen-evolving complex by NH₂OH is impeded by Cl^?, whereas Cl^? does not affect the inhibition induced by so-called ADRY reagents. We propose that Cl^? functions in the oxygen-evolving complex as a ligand bridging manganese atoms to mediate electron transfer. This model accounts both for the well known Cl^? requirement of oxygen evolution, and for the inhibitory effects of amines on this reaction.     

Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes

(1) Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. (2) The midpoint redox potential of the cytochrome is 100–130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5–7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. (3) The Nernst plots of titration data have slopes of 75–115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. (4) Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. (5) Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4–6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.     

Measurement of the relative electron-transport capacity of Photosystem I and Photosystem II in spinach chloroplasts

The ratio of Photosystem (PS) II to PS I electron-transport capacity in spinach chloroplasts was compared from reaction-center and steady-state rate measurements. The reaction-center electron-transport capacity was based upon both the relative concentrations of the PS II_α, PS II_β and PS I centers, and the number of chlorophyll molecules associated with each type of center. The reaction-center ratio of total PS II to PS I electron-transport capacity was about 1.8:1. Steady-state electron-transport capacity data were obtained from the rate of light-induced absorbance-change measurements in the presence of ferredoxin-NADP⁺, potassium ferricyanide and 2,5-dimethylbenzoquinone (DMQ). A new method was developed for determining the partition of reduced DMQ between the thylakoid membrane and the surrounding aqueous phase. The ratio of membrane-bound to aqueous DMQH₂ was experimentally determined to be 1.3:1. When used at low concentrations (200 μM), potassium ferricyanide is shown to be strictly a PS I electron acceptor. At concentrations higher than 200 μM, ferricyanide intercepted electrons from the reducing side of PS II as well. The experimental rates of electron flow through PS II and PS I defined a PS II/PS I electron-transport capacity ratio of 1.6:1.     

Role of uptake hydrogenase in providing reductant for nitrogenase in Rhizobium leguminosarum bacteroids

The role of uptake hydrogenase in providing reducing power to nitrogenase was investigated in Rhizobium leguminosarum bacteroids from nodules of Pisum sativum L. (cv. Homesteader). H₂ increased the rate of C₂H₂ reduction in the absence of added substrates. Malate also increased nitrogenase (C₂H₂) activity while decreasing the effect of H₂. At exogenous malate concentrations above 0.05 mM no effect of H₂ was seen. Malate appeared to be more important as a source of reductant than of ATP. When iodoacetate was used to minimize the contribution of endogenous substrates to nitrogenase activity in an isolate in which H₂ uptake was not coupled to ATP formation, H₂ increased the rate of C₂H₂ reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C₂H₂ reduction, but when H₂ was also included, the rate of C₂H₂ reduction was increased by 280% over that with the ATP-generating system alone. The data suggest that, under conditions of substrate starvation, the uptake hydrogenase in R. leguminosarum could provide reductant as well as ATP in an isolate in which the H₂ uptake is coupled to ATP formation, to the nitrogenase complex.     

Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition

Lipid peroxidation in rat brain mitochondria was induced by NADH in the presence of ADP and FeCl3. CV-2619 inhibited the lipid peroxidation in a concentration-dependent manner; the concentration giving 50% inhibition (IC50) was 84 microM. In addition, the inhibitory effect of CV-2619 was strongly enhanced by adding substrates of mitochondrial respiration; when succinate, glutamate, or succinate plus glutamate was added, the IC50 of CV-2619 was changed to 1.1, 10, or 0.5 microM, respectively. Metabolites of CV-2619 also inhibited the lipid peroxidation. The inhibitory effect of CV-2619 on mitochondrial lipid peroxidation disappeared when TTFA, an inhibitor of complex II in mitochondrial respiratory chain, was added. The results indicate that in mitochondria CV-2619 is changed to its reduced form which inhibits lipid peroxidation.     

Proton-induced membrane fusion role of phospholipid composition and protein-mediated intermembrane contact

Glycolipid-phospholipid vesicles containing phosphatidate and phosphatidylethanolamine were found to undergo proton-induced fusion upon acidification of the suspending medium from pH 7.4 to pH 6.5 or lower, as determined by an assay for lipid intermixing based on fluorescence resonance energy transfer. Lectinmediated contact between the vesicles was required for fusion. Incorporation of phosphatidylcholine in the vesicles inhibited proton-induced fusion. Vesicles in which phosphatidate was replaced by phosphatidylserine underwent fusion only when pH was reduced below 4.5, while no significant fusion occured (pH ? 3.5) when the anionic phospholipid was phosphatidylinositol. It is suggested that partial protonation of the polar headgroup of phosphatidate and phosphatidylserine, respectively, causes a sufficient reduction in the polarity and hydration of the vesicle surface to trigger fusion at sites of intermembrane contact.     

Effect of dibromothymoquinone (DBMIB) on reduction rates of Photosystem I donors in intact chloroplasts

Dual effect of dibromothymoquinone ( DBMIB ), inhibitor and reducing agent at the donor side of Photosystem I, was investigated in isolated intact chloroplasts by flash-induced absorbance changes at 820 and 515 nm. We show that in the absence of other electron donors, rereduction of P700+ by DBMIB proceeds at a very low rate (half-time of approximately 10 s) Dual effect of DBMIB explains that the initial rise of electrochromic absorbance change induced by repetitive flashes is usually not diminished while the slow rise is fully inhibited by this compound.     

A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D

We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells.