Kinetics of the photoreduction of cytochrome b-559 by photosystem II in chloroplasts.

The kinetics of the photoreduction of cytochrome b-559 and plastoquinone were measured using well-coupled spinach chloroplasts. High potential (i.e, hydroquinone reducible) cytochrome b-559 was oxidized with low intensity far-red light in the presence of N-methyl phenazonium methosulfate or after preillumination with high intensity light. Using long flashes of red light, the half-reduction time of cytochrome b-559 was found to be 100 +/- 10 ms, compared to 6-10 ms for the photoreduction of the plastoquinone pool. Light saturation of the photoreduction of cytochrome b-559 occurred at a light intensity less than one-third of the intensity necessary for the saturation of ferricyanide reduction under identical illumination conditions. The photoreduction of cytochrome b-559 was accelerated in the presence of dibromothymoquinone with a t 1/2 = 25-35 ms. The addition of uncouplers, which caused stimulatory effect on ferricyanide reduction under the same experimental conditions resulted in a decrease in the rate of cytochrome b-559 reduction. The relatively slow photoreduction rate of cytochrome b-559 compared to the plastoquinone pool implies that electrons can be transferred efficiently from Photosystem II to plastoquinone without the involvement of cytochrome b-559 as an intermediate. These results indicate that it is unlikely that high potential cytochrome b-559 functions as an obligatory redox component in the main electron transport chain joining the two photosystems.

Critical analysis of the extinction coefficient of chloroplast cytochrome f.

In oxygenic photosynthesis the cytochrome bf complex links electron transport between photosystem II and photosystem I. The largest subunit of the complex is cytochrome f, a 32-kDa polypeptide that is anchored in the membrane by a transmembrane alpha helix located near the carboxyl end. The three-dimensional structure of the soluble domain of cytochrome f isolated from turnip has been determined by X-ray crystallography to 1.96 A resolution. The structure revealed several novel features compared to previously solved soluble c-type cytochrome structures, including a predominant beta-strand motif, the N-terminal alpha-amino group of a tyrosyl residue as an orthogonal ligand, and a bound internal water chain. Here we report a novel and unprecedented extinction coefficient for cytochrome f. Using the pyridine hemochrome assay, the reduced minus oxidized extinction coefficient for the soluble domain of turnip cytochrome f was 26 +/- 1 mM-1 cm-1 for the alpha-band wavelength peak at 554 nm relative to the isosbestic wavelengths (534, 543.5 and 560.5 nm), and 25 +/- 1 mM-1 cm-1 for spinach cytochrome f relative to the isosbestic wavelengths (533.5, 543.3 and 560.2). The extinction coefficients reported here are significantly higher than previously published values for cytochrome f. We believe earlier determinations underestimated the cytochrome f extinction coefficient and that the same is likely true for commonly used extinction coefficients of cytochrome b6. The cytochrome f extinction coefficient is large compared to most other c-type cytochromes, which could be due to the unique axial ligand of the cytochrome f heme. Polarographic measurements show the midpoint potential of soluble turnip cytochrome f to be 362 +/- 5 mV at pH 7.5. The midpoint potential was pH-independent from 5.0 to 8.5, and pH-dependent from pH 8.5 to 10.5 (-58 mV/pH unit) with a pK on the oxidized from near 9. Storage of some samples of purified turnip and spinach cytochrome f at -20 degrees C modified the heme environment in a fraction of the protein, shifting the midpoint potential to near -165 mV (pH 7.5) and the peak of the alpha-band absorption spectrum from 554 nm to 552 nm.

Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b(6) in Synechococcus sp. PCC 7002.

The cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b(6) subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b(6) at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc(1) complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc(1) complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc(1) complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure-function relationships in the cytochrome bf complex.

Extinction coefficients and midpoint potentials of cytochrome c(6) from the cyanobacteria Arthrospira maxima, Microcystis aeruginosa, and Synechocystis 6803.

Cytochrome c(6) is a soluble heme protein that serves as a photosynthetic electron transport component in cyanobacteria and algae, carrying electrons from the cytochrome bf complex to photosystem I. The rapid accumulation of cytochrome c(6) sequence data from a wide range of species, combined with significant advances in determining high resolution three-dimensional structures, provides a powerful database for investigating the relationship between structure and function. The fact that the gene encoding cytochrome c(6) can be readily modified in a number of species adds to the usefulness of cytochrome c(6) as a tool for comparative analysis. Efforts to relate cytochrome c(6) sequence information to structure, and structural information to function depend on knowledge of the physical and thermodynamic properties of the cytochrome from different species. To this end we have determined the optical extinction coefficient, the oxidation/reduction midpoint potential, and the pH dependence of the midpoint potential of cytochrome c(6) isolated from three cyanobacteria, Arthrospira maxima, Microcystis aeruginosa, and Synechocystis 6803.

Dissipation of the Proton Electrochemical Potential in Intact Chloroplasts (II. The pH Gradient Monitored by Cytochrome f Reduction Kinetics).

The potency of various uncouplers for collapsing the light-induced pH gradient across thylakoid membranes in intact chloroplasts was investigated by time-resolved optical spectroscopy. The thylakoid transmembrane pH gradient ([delta]pH) was monitored indirectly by measuring the rate of cytochrome (Cyt) f reduction following a light flash of sufficient duration to create a sizable [delta]pH. The results show that the rate of Cyt f reduction is controlled in part by the internal pH of the thylakoid inner aqueous space. At pH values from 6.5 to 8.0, the Cyt f reduction rate was maximal, whereas at lower pH values from 6.5 to 5.5 the reduction rate decreased to 25% of the maximal rate. The ability of three uncouplers, nigericin, carbonylcyanide m-chlorophenylhydrazone, and gramicidin, to accelerate the rate of Cyt f reduction was determined for intact chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of the uncouplers for collapsing the [delta]pH was determined using the empirical relationship between the [delta]pH and the Cyt f reduction rate. For intact chloroplasts, nigericin was the most effective uncoupler, followed by carbonylcyanide m-chlorophenylhydrazone, which interacted strongly with bovine serum albumin. Gramicidin D, even at high gramicidin:chlorophyll ratios, did not completely collapse the pH gradient, probably because it partitions in the envelope membranes and does not enter the intact chloroplast.

Characterization of photosystem II activity and heterogeneity during the cell cycle of the green alga scenedesmus quadricauda

The photosynthetic activity of the green alga Scenedesmus quadricauda was investigated during synchronous growth in light/dark cycles. The rate of O2 evolution increased 2-fold during the first 3 to 4 h of the light period, remained high for the next 3 to 4 h, and then declined during the last half of the light period. During cell division, which occurred at the beginning of the dark period, the ability of the cells to evolve O2 was at a minimum. To determine if photosystem II (PSII) controls the photosynthetic capacity of the cells during the cell cycle we measured PSII activity and heterogeneity. Measurements of electron-transport activity revealed two populations of PSII, active centers that contribute to carbon reduction and inactive centers that do not. Measurements of PSII antenna sizes also revealed two populations, PSIIalpha and PSIIbeta, which differ from one another by their antenna size. During the early light period the photosynthetic capacity of the cells doubled, the O2-evolving capacity of PSII was nearly constant, the proportion of PSIIbeta centers decreased to nearly zero, and the proportion of inactive PSII centers remained constant. During the period of minimum photosynthetic activity 30% of the PSII centers were insensitive to the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which may be related to reorganization of the thylakoid membrane. We conclude from these results that PSII does not limit the photosynthetic activity of the cells during the first half of the light period. However, the decline in photosynthetic activity observed during the last half of the light period can be accounted for by limited PSII activity.

Kinetic imaging of chlorophyll fluorescence using modulated light.

Fluorometers that measure the kinetics of chlorophyll fluorescence have become invaluable tools for determining the photosynthetic performance of plants. Many of these instruments use high frequency modulated light to measure the rate, efficiency and regulation of photosynthesis. The technique is non-invasive and is effective under diverse environmental conditions. Recently, imaging fluorometers have been introduced that reveal variability in photosynthesis over the surface of a leaf or between individual plants. Most imaging instruments depend on continuous light or low frequency modulated light for fluorescence excitation, which imposes serious limitations on measurements of the fluorescence parameters, especially the minimum fluorescence (F(0)) and variable fluorescence (F(V)). Here, we describe a new instrument that combines the advantage of high frequency modulated light with two-dimensional imaging of chlorophyll fluorescence. The fluorometer produces dynamic images of chlorophyll fluorescence from leaves or plants, providing accurate mapping of F(0) and F(V), and non-photochemical quenching. A significant feature of the instrument is that it can record fluorescence images of leaves in daylight under field conditions.

African-American women who smoke crack cocaine: sexual trauma and the mother-daughter relationship.

This study evolved from previous writings that linked sexual abuse to the quality of the mother-daughter relationship. The purpose of this investigation was to examine the mother-daughter relationship among African-American women who abuse crack cocaine by comparing three different groups: women who have not experienced incest but have experienced sexual abuse; women who have experienced incest; and women who have not experienced sexual abuse. A convenience sample of 64 African-American women was used, 35 of whom were in drug abuse treatment and 29 of whom were abusing crack cocaine. Using analysis of variance studies, significant differences were found between the three groups in terms of the mother-daughter relationship. Differences were also found between the group that experienced incest and the group that experienced nonincestuous sexual abuse in terms of the amount of abuse and first age of abuse. This preliminary investigation indicates the need for further study of specific populations in this area--most notably, African-American women who abuse crack cocaine.

School nurses' skills in sexual health education.

School nurses are often called on teach sexual health without being given any formal teacher training or support. This article describes a study of the experiences of 50 school nurses. The author suggests ways in which schools and their nurses could create supportive and complementary teaching partnerships for effective sex education.

Proton decay kinetics for vesicles containing buffers-an analytical solution.

The problem of predicting the kinetics of proton efflux and the decay of the internal proton concentration for vesicles containing one or more buffers for which the internal proton concentration is initially higher than that of the surrounding medium is examined. An analytical solution is derived that describes the time course of the proton efflux from vesicles and the decay of the internal proton concentration under conditions of zero transmembrane electric potential. The effect of the internal buffers is to increase the time required for the proton concentration gradient to equilibrate across the membrane. To simplify the analysis we assume that the equilibration of the internal and external proton activity is due primarily to proton diffusion through the membrane, and not to hydroxyl ion flux. For a vesicle containing a single buffer the solution requires six independent physical parameters: the initial internal proton concentration, the external proton concentration, the ratio of the vesicle surface area to the internal volume, the permeability coefficient of the membrane for protons, the total concentration of the internal buffer, and the equilibrium constant for the dissociation of the internal buffer. Determination of these physical values is sufficient to predict the time dependence of the internal proton concentration and of the proton efflux. Over a pH range that is below or near the pK of the internal buffer the solution is complex. However, if the initial pH is one unit or more higher than the pK of the internal buffer the kinetics of the internal proton concentration and proton efflux can be described by a pseudo first order reaction. In this case the apparent rate constant depends linearly on the permeability coefficient and is dominated by the total internal buffer concentration and its pK. For example, increasing the internal buffer concentration inside a vesicle by 10-fold results in an approximately 10-fold increase in the half-time of the proton efflux kinetics. The theoretical analysis is applied to thylakiod vesicles using experimentally determined values for the physical parameters. The predictions of the analysis are compared to experimentally observed kinetics.

Dissipation of the proton electrochemical potential in intact and lysed chloroplasts : I. The electrical potential.

Effective ionophore:chlorophyll ratios were determined for various ionophores that decrease the electrical potential across thylakoid membranes in intact and hypo-osmotically lysed chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of gramicidin D, valinomycin, carbonylcyanide m-chlorophenylhydrazone, and dicyclohexano-18-crown-6 in collapsing the electrical potential was determined spectrophotometrically by the decay half-time of the absorbance change at 518 nanometers induced by a saturating, single turnover flash. The results show that the effectiveness of the ionophores in collapsing the electrical potential in intact and lysed chloroplasts depends on the amount of ionophore-accessible membrane in the assay medium. Only gramicidin exhibited a significant difference in efficacy between intact and lysed chloroplasts. The ratio of gramicidin to chlorophyll required to collapse the electrical potential was more than 50 times higher in intact chloroplasts than in lysed chloroplasts. The efficacy of carbonylcyanide m-chlorophenylhydrazone was significantly reduced in the presence of bovine serum albumin. The other ionophores tested maintained their potency in the presence of bovine serum albumin. Valinomycin was the most effective ionophore tested for collapsing the electrical potential in intact chloroplasts, whereas gramicidin was the most potent ionophore in lysed chloroplasts. The significance of the ionophore:chlorophyll ratios required to collapse the electrical potential is discussed with regard to bioenergetic studies, especially those that examine the contribution of the transmembrane electrochemical potential to protein transport into chloroplasts.

Light saturation curves show competence of the water splitting complex in inactive Photosystem II reaction centers.

Photosystem II complexes of higher plants are structurally and functionally heterogeneous. While the only clearly defined structural difference is that Photosystem II reaction centers are served by two distinct antenna sizes, several types of functional heterogeneity have been demonstrated. Among these is the observation that in dark-adapted leaves of spinach and pea, over 30% of the Photosystem II reaction centers are unable to reduce plastoquinone to plastoquinol at physiologically meaningful rates. Several lines of evidence show that the impaired reaction centers are effectively inactive, because the rate of oxidation of the primary quinone acceptor, QA, is 1000 times slower than in normally active reaction centers. However, there are conflicting opinions and data over whether inactive Photosystem II complexes are capable of oxidizing water in the presence of certain artificial electron acceptors. In the present study we investigated whether inactive Photosystem II complexes have a functional water oxidizing system in spinach thylakoid membranes by measuring the flash yield of water oxidation products as a function of flash intensity. At low flash energies (less that 10% saturation), selected to minimize double turnovers of reaction centers, we found that in the presence of the artificial quinone acceptor, dichlorobenzoquinone (DCBQ), the yield of proton release was enhanced 20±2% over that observed in the presence of dimethylbenzoquinone (DMBQ). We argue that the extra proton release is from the normally inactive Photosystem II reaction centers that have been activated in the presence of DCBQ, demonstrating their capacity to oxidize water in repetitive flashes, as concluded by Graan and Ort (Biochim Biophys Acta (1986) 852: 320-330). The light saturation curves indicate that the effective antenna size of inactive reaction centers is 55±12% the size of active Photosystem II centers. Comparison of the light saturation dependence of steady state oxygen evolution in the presence of DCBQ or DMBQ support the conclusion that inactive Photosystem II complexes have a functional water oxidation system.

Evidence that cytochrome b559 protects photosystem II against photoinhibition.

Light that exceeds the photosynthetic capacity of a plant can impair the ability of photosystem II to oxidize water. The light-induced inhibition is initiated by inopportune electron transport reactions that create damaging redox states. There is evidence that secondary electron transport pathways within the photosystem II reaction center can protect against potentially damaging redox states. Experiments using thylakoid membranes poised at different ambient redox potentials demonstrate that light-induced damage to photosystem II can be controlled by a redox component within the reaction center [Nedbal, L., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7929-7933]. The rate of photoinhibition is slow when the redox component is oxidized, but increases by more than 10-fold when the redox component is reduced. Here, using spinach thylakoid membranes, we provide evidence that the redox component is cytochrome b559, an intrinsic heme protein of the photosystem II reaction center. The results support a model in which the low-potential (LP) form of cytochrome b559 protects photosystem II by deactivating a rarely formed, but hazardous redox state of photosystem II, namely, P680/Pheo-/ QA-. Cytochrome b559LP is proposed to deactivate this potentially lethal redox state by accepting electrons from reduced pheophytin. The key observations supporting this proposal are as follows: (1) The oxidation-reduction potential of cytochrome b559LP is in the range predicted by redox titrations of photoinhibition. (2) If cytochrome b559LP is reduced prior to illumination, the rate of photoinhibition is fast, whereas if the cytochrome is oxidized prior to illumination, the rate of photoinhibition is slow.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome f function in photosynthetic electron transport.

The questions of whether the stoichiometry of the turnover of cytochrome f, and the time-course of its reduction subsequent to a light flash, are consistent with efficient function in noncyclic electron transport have been investigated. Measurements were made of the absorbance change at the 553-nm alpha-band maximum relative to a reference wavelength. In the dark cytochrome f is initially fully reduced, oxidized by a 0.3-s flash, and reduced again in the dark period after the flash. In the presence of gramicidin at 18 degrees C, the dark reduction was characterized by a half-time of 25-30 ms, stoichiometries of cytochrome f:chlorophyll and P700:chlorophyll of 1:670 and 1:640, respectively, and a short time delay. The time delay in the dark reduction of cytochrome f, which is expected for a component in an intermediate position in the chain, becomes more apparent in the presence of valinomycin and K+. Under these conditions the half-time for cytochrome f dark reduction is 130-150 ms, and the delay is approximately equal to 20 ms. The measured value for the activation energy of the dark reduction of cytochrome f (11 +/- 1 kcal/mol) is the same as that for noncyclic electron transport in steady-state light. A sigmoidal time-course for the reduction of cytochrome f has been calculated for a simple linear electron transport chain. The kinetics for reduction of cytochrome f predicted by the calculation, in the presence of valinomycin and K+, are in reasonably good agreement with the experimental data. There is an appreciable amount of data in the literature to document complex properties of cytochrome f after illumination with short flashes, and evidence for complexity in a light-minus-dark transition. The data presented here, obtained after a long flash that should establish steady-state conditions, either fulfill or are consistent with the basic criteria for efficient function of cytochrome f in noncyclic electron transport.

Photooxidation of the high-potential iron-sulfur center in chloroplasts.

The high-potential iron-sulfur center associated with the g = 1.89-1.90 electron paramagnetic resonance signal in spinach chloroplasts is fully reduced in the dark. It is photooxidized in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and reduced after illumination with red actinic light in the absence of inhibitors. The light-induced redox changes of the high-potential iron-sulfur center are shown to correlate with those of plastocyanin detected at g = 2.05. Demonstration of light-induced redox changes in the high-potential iron-sulfur center provides support for a functional role of this component in the chloroplast electron transport chain.

Effect of integral membrane proteins on the lateral mobility of plastoquinone in phosphatidylcholine proteoliposomes.

PYRENE FLUORESCENCE QUENCHING BY PLASTOQUINONE WAS USED TO ESTIMATE THE RATE OF PLASTOQUINONE LATERAL DIFFUSION IN SOYBEAN PHOSPHATIDYLCHOLINE PROTEOLIPOSOMES CONTAINING THE FOLLOWING INTEGRAL MEMBRANE PROTEINS: gramicidin D, spinach cytochrome bf complex, spinach cytochrome f, reaction centers from Rhodobacter sphaeroides, beef heart mitochondrial cytochrome bc(1), and beef heart mitochondrial cytochrome oxidase. The measured plastoquinone lateral diffusion coefficient varied between 1 and 3 . 10(-7) cm(2) s(-1) in control liposomes that lacked protein. When proteins were added, these values decreased: a 10-fold decrease was observed when 16-26% of the membrane surface area was occupied by protein for all the proteins but gramicidin. The larger protein complexes (cytochrome bf, Rhodobacter sphaeroides reaction centers, cytochrome bc(1), and cytochrome oxidase), whose hydrophobic volumes were 15-20 times as large as that of cytochrome f and the gramicidin transmembrane dimer, were 15-20 times as effective in decreasing the lateral-diffusion coefficient over the range of concentrations studied. These proteins had a much stronger effect than that observed for bacteriorhodopsin in fluorescence photobleaching recovery measurements. The effect of high-protein concentrations in gramicidin proteoliposomes was in close agreement with fluorescence photobleaching measurements. The results are compared with the predictions of several theoretical models of lateral mobility as a function of integral membrane concentration.

Light saturation response of inactive photosystem II reaction centers in spinach.

The effective absorption cross section of inactive photosystem II (PS II) centers, which is the product of the effective antenna size and the quantum yield for photochemistry, was investigated by comparing the light saturation curves of inactive PS II and active reaction centers in intact chloroplasts and thylakoid membranes of spinach (Spinacia oleracea). Inactive PS II centers are defined as the impaired PS II reaction centers that require greater than 50 ms for the reoxidation of QA (-) subsequent to a single turnover flash. Active reaction centers are defined as the rapidly turning over PS II centers (recovery time less than 50 ms) and all of the PS I centers. The electrochromic shift, measured by the flash-induced absorbance increase at 518 nm, was used to probe the activity of the reaction centers. Light saturation curves were generated for inactive PS II centers and active reaction centers by measuring the extent of the absorbance increase at 518 nm induced by red actinic flashes of variable energy. The light saturation curves show that inactive PS II centers required over twice as many photons as active reaction centers to achieve the same yield. The ratio of the flash energy required for 50% saturation for active reaction centers (PS II active + PS I) compared to inactive PS II centers was 0.45±0.04 in intact chloroplasts, and 0.54±0.11 in thylakoid membranes. Analysis of the light saturation curves using a Poisson statistical model in which the ratio of the antenna size of active PS II centers to that of PS I is considered to range from 1 to 1.5, indicates that the effective absorption cross section of inactive PS II centers was 0.54-0.37 times that of active PS II centers. If the quantum yield for photochemistry is assumed to be one, we estimate that the antenna system serving the inactive PS II centers contains approx. 110 chlorophyll molecules.