Photosynthetic and respiratory electron transport in a cyanobacterium

In the cyanobacterium Agmenellum quadruplicatum steady-state redox conditions were monitored in vivo for cytochrome (+c553) and P700 versus intensities of an actinic light 1 or light 2 (mainly absorbed by photosystems, and 2, respectively). Parallel measurements of O₂ evolution were used to calibrate intensities for rates of electron transfer. Results show that the quality of actinic light (as light 1 or light 2) depends on intensity as well as wavelength. The contribution of electron flow from respiration is confirmed by observations of relative rate of photoreaction 1 estimated from Ip (intensity × fraction of P700 reduced). With 3,- (3,4-dichlorophenyl-1, 1-dimethylurea) (DCMU) the rate of photoreaction 1 depends upon, and is sensitive to small changes in, the rate of dark respiration. Very slow transient dark reductions of Cyt (f+c553) and P700 following any low intensity actinic light 1 are attributed to respiratory electron flow. Cyclic electron flow around photoreaction 1 cannot be large compared to dark respiration and cannot vary significantly with light intensity.This paper is contributed in honor of my longtime friend, L.N.M. Duysens, who has carried still further the eminence of the Dutch tradition in biophysics.     

Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments.

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP+). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, I1/2, is defined as the actinic light intensity that, at a given concentration of electron donro, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, I1/2 is compared with another parameter, equivalent to I1/2, that is obtained independently of the reaciton-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow. The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP+ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.     

Light-induced redox changes of cytochrome b-559

Dark incubation of spinach or pea chloroplasts with 10 μm carbonylcyanide m-chlorophenylhydrazone (CCCP) had a negligible effect either on the redox state or the redox potential of the high potential form of cytochrome b-559 (cytochrome b-559_hp). A similar result was obtained with spinach chloroplasts on incubation with 3.3 μm carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), but pea chloroplasts showed a decrease of 10–20% in the amount of reduced cytochrome b-559.Light-induced redox changes of cytochrome b-559 were not observed in untreated spinach chloroplasts. In the presence of CCP or FCCP, cytochrome b-559 was photooxidized both in 655 nm actinic light and in far-red light. Addition of the plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) to CCCP- or FCCP-treated chloroplasts had only a small effect on the photooxidation of cytochrome b-559 in 655 light, but it completely inhibited the oxidation in far-red light.Electron flow from water to 2,3′,6-trichlorophenolindophenol was partly inhibited by CCCP or FCCP, but the degree of inhibition does not appear to be sufficient to account for the photooxidation of cytochrome b-559.The photooxidation of cytochrome b-559 by 655 nm light at liquid nitrogen temperature was not influenced by prior treatment of the chloroplasts at room temperature with CCCP, DBMIB, or CCCP + DBMIB.The results cannot be explained by the presence of two independent pools of cytochrome b-559 in CCCP-treated chloroplasts, one photooxidized by Photosystem II and the other photooxidized by Photosystem I and photoreduced by Photosystem II.     

Salts and chloroplast fluorescence

Chloroplast fluorescence was excited by a weak measuring beam. A time-separated actinic light was used to modify the redox states of Q which in turn induced a change in the fluorescence yield. In salt-depleted chloroplasts, fluorescence saturated at a low actinic light intensity. CaCl₂ increased the “variable” fluorescence as well as the rate of ferricyanide-Hill reaction. With Tris-washed chloroplasts, Photosystem II donor couple, phenylenediamine and ascorbate, did not increase the fluorescence to a large extent without the presence of CaCl₂. It is suggested that salt-depletion inactivates the Photosystem II reaction center of chloroplasts.     

Photooxidation of cytochrome b-559 in leaves and chloroplasts at room temperature

1. Light-induced absorbance changes of cytochrome b-559 and cytochrome f in the -band region were examined in leaves and in isolated chloroplasts.

2. Absorbance changes of cytochrome b-559 were not detected in untreated leaves or in most preparations of isolated chloroplasts. After treatment of leaves or chloroplasts with carbonyl cyanide m-chlorophenylhydrazone, high rates of photooxidation of cytochrome b-559 were obtained, both in far-red (>700 nm) and red actinic light. Cytochrome f was photooxidized in far-red light, but in red light it remained mainly in the reduced state. The initial rates of photooxidation of cytochrome b-559 in leaves or chloroplasts treated with carbonyl cyanide m-chlorophenylhydrazone were considerably decreased by 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea.

3. A slow photoreduction of cytochrome b-559 was observed in aged mutant pea chloroplasts in red light.

4. The results do not support the view that cytochrome b-559 is a component of the electron transport chain between the light reactions. It is suggested that cytochrome b-559 is located on a side path from Photosystem II, but with a possible additional link to Photosystem I.     

Fluorescence decline as a function of redox potential and actinic light intensity in spinach thylakoids

Excitation of isolated thylakoids with sufficiently strong actinic light increases the fluorescence quantum yield up to a maximum level, F_max, followed by a slower decline under certain experimental conditions. In this study the latter effect was analyzed as a function of the ambient redox potential and the actinic light intensity. Two different types of fluorescence decrease were found. (a) In the presence of specific quinones widely used as redox mediators a fast and comparatively small decrease (30% of F_max), referred to as ΔF_SQ, was observed at moderate redox potentials (−300 <E_m < + 200 mV). ΔF_SQ disappears at positive values with E_{m, 7.5} = + 110 mV, whereas the decrease at negative redox potential depends on the midpoint potential of the quinone. (b) A more pronounced fluorescence decline was observed at redox potentials below −300 mV, which comprises 65–70% of the maximum fluorescence. The full expression of this effect, referred to as ΔF^max_LP, requires markedly higher actinic light intensities than ΔF^max_SQ. The extent of ΔF^max_LP as a function of the redox potential is dependent on the presence of redox mediators. In their absence the full expression of ΔF^max_LP can be only observed below −400 mV. Based on the hypothesis of Pheo⁻ photoaccumulation being responsible for the fluorescence decline at low redox potentials (Klimov, V.V., Klevanik, A.V. and Shuvalov, V.A. (1977) FEBS Lett. 82, 182–186), a reaction scheme is presented that qualitatively describes the time course of ΔF_LP at different actinic light intensities and redox potentials. Based on this analysis, the rate of Pheo^⨪ reoxidation is inferred to be limited by the reaction center apoprotein acting as a barrier to redox equilibration. The implications for the interpretations of redox titration curves are briefly discussed.     

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.     

质体醌库和细胞色素b6f参与调控蓝细菌 Synechocystis sp. PCC 6803的状态转换

用调制叶绿素荧光研究了对苯醌(1,4-benzoquinone,BQ)和二溴百里香醌(2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone,DBMIB)对蓝细菌Synechocystissp.PCC6803状态转换的作用。BQ和DBMIB是质体醌(PQ)的类似物,两者均可充当PQ的电子受体。其中,DBMIB能够和细胞色素b6f的Qo位点特异结合。在没有作用光的情况下,BQ诱导暗适应的蓝细菌进入状态Ⅰ;相反,DBMIB诱导Syne鄄chocystis6803向状态Ⅱ转换。据此提出,在生理状态下蓝细菌根据PQ库的氧化还原状态调节状态转换;细胞色素b6f参与此调控过程。     

The light-intensity-dependence of the efficacy of 2-(3-chloro-4-trifluoromethyl)-anilino-3,5-dinitrothiophene (Ant2p) to inhibit the photosystem 2 reactions of chloroplasts.

The mechanism by which Ant2p [2-(3-chloro-4-trifluoromethyl)anilino-3, 5-dinitrothiophene] inhibits the oxygen evolution capacity of chloroplasts is thought to be due to a rapid reduction of the S2 and S3 oxidation states of the oxygen-evolving complex mediated by the oxidation of endogenous donors such as cytochrome b559. The results presented in this paper show that the degree of inhibition by Ant2p of the photosystem 2-supported electron transfer reactions, registered by the light-dependent rate of dichlorophenolindophenol reduction, varies according to the actinic light intensity. Moreover, a similar intensity-dependence can be detected in the extent of the Ant2p-induced cytochrome b559HP photo-oxidation. We show, however, that the dependence of the cytochrome oxidation is not due to the oxidation per se, but reflects changes in the high light-driven re-reduction reaction. The close correlation between the two Ant2p reactions is interpreted as indicating that the effect of Ant2p might be due to an inhibition of the S-state turnovers and not necessarily due to a deactivation process.     

Discrete catalytic sites for quinone in the ubiquinol-cytochrome c2 oxidoreductase of Rhodopseudomonas capsulata. Evidence from a mutant defective in ubiquinol oxidation

A non-photosynthetic mutant (Ps-) of Rhodopseudomonas capsulata, designated R126, was analyzed for a defect in the cyclic electron transfer system. Compared to a Ps+ strain MR126, the mutant was shown to have a full complement of electron transfer components (reaction centers, ubiquinone-10, cytochromes b, c1, and c2, the Rieske 2-iron, 2-sulfur (Rieske FeS) center, and the antimycin-sensitive semiquinone). Functionally, mutant R126 failed to catalyze complete cytochrome c1 + c2 re-reduction or cytochrome b reduction following a short (10 microseconds) flash of actinic light. Evidence (from flash-induced carotenoid band shift) was characteristic of inhibition of electron transfer proximal to cytochrome c1 of the ubiquinol-cytochrome c2 oxidoreductase. Three lines of evidence indicate that the lesion of R126 disrupts electron transfer from quinol to Rieske FeS: 1) the degree of cytochrome c1 + c2 re-reduction following a flash is indicative of electron transfer from Rieske FeS to cytochrome c1 + c2 without redox equilibration with an additional electron from a quinol; 2) inhibitors that act at the Qz site and raise the Rieske FeS midpoint redox potential (Em), namely 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole or 3-alkyl-2-hydroxy-1,4-napthoquinone, have no effect on cytochrome c1 + c2 oxidation in R126; 3) the Rieske FeS center, although it exhibits normal redox behavior, is unable to report the redox state of the quinone pool, as metered by its EPR line shape properties. Flash-induced proton binding in R126 is indicative of normal functional primary (QA) and secondary (QB) electron acceptor activity of the photosynthetic reaction center. The Qc functional site of cytochrome bc1 is intact in R126 as measured by the existence of antimycin-sensitive, flash-induced cytochrome b reduction.     

Cyclin E and cyclin A are likely targets of Src for PDGF-induced DNA synthesis in fibroblasts

The effects of benzoquinone analogues, phenyl-1,4-benzoquinone (PBQ) and 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB), on state transitions in Synechocystis sp. PCC 6803 were investigated. PBQ induced a transition from state 2 to state 1 in the absence of actinic light whereas DBMIB caused a state 2 transition. 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea could not eliminate the effects of PBQ and DBMIB. These results imply that the redox state of the plastoquinone pool controls the state transitions in vivo and cytochrome b6f complex is involved in this process. As a working hypothesis, we propose that the occupancy of the quinol oxidation site and the movement of the Rieske protein may be pivotal in this regulation.     

Evidence for the operation of alternative electron transport routes through photosystem I in intact barley leaves under weak and moderate white light

The functioning of alternative routes of photosynthetic electron transport was analyzed from the kinetics of dark reduction of P700⁺ , an oxidized primary donor of PSI, in barley (Hordeum vulgare L.) leaves irradiated by white light of various intensities. Redox changes of P700 were monitored as absorbance changes at 830 nm using PAM 101 specialized device. Irradiation of dark-adapted leaves caused a gradual P700⁺ accumulation, and the steady-state level of oxidized P700 increased with intensity of actinic light. The kinetics of P700⁺ dark reduction after a pulse of strong actinic light, assayed from the absorbance changes at 830 nm, was fitted by a single exponential term with a halftime of 10–12 ms. Two slower components were observed in the kinetics of P700⁺ dark reduction after leaf irradiation by attenuated actinic light. The contribution of slow components to P700⁺ reduction increased with the decrease in actinic light intensity. Two slow components characterized by halftimes similar to those observed after leaf irradiation by weak white light were found in the kinetics of dark reduction of P700⁺ oxidized in leaves with far-red light specifically absorbed by PSI. The treatment of leaves with methyl viologen, an artificial PSI electron acceptor, significantly accelerated the accumulation of P700⁺ under light. At the same time, the presence of methyl viologen, which inhibits ferredoxin-dependent electron transport around PSI, did not affect three components of the kinetics of P700⁺ dark reduction obtained after irradiations with various actinic light intensities. It was concluded that some part of PSI reaction centers was not reduced by electron transfer from PSII under weak or moderate intensities of actinic light. In this population of PSI centers, P700⁺ was reduced via alternative electron transport routes. Insensitivity of the kinetics of P700⁺ dark reduction to methyl viologen evidences that the input of electrons to PSI from the reductants (NADPH or NADH) localized in the chloroplast stroma was effective under those light conditions.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 5–11.Original Russian Text Copyright © 2005 by Bukhov, Egorova.     

Shifts of bacteriochlorophyll and carotenoid absorption bands linked to cytochrome c-555 photooxidation in Chromatium

Shifts in the absorption bands of bacteriochlorophyll and carotenoids in Chromatium vinosum chromatophores were measured after short actinic flashes, under various conditions. The amplitude of the bacteriochlorophyll band shift correlated well with the amount of cytochrome c-555 that was oxidized by P₈₇₀⁺ after a flash. No bacteriochlorophyll band shift appeared to accompany the photooxidation of P₈₇₀ itself, nor the oxidation of cytochrome c-552 by P₈₇₀⁺. The carotenoid band shift also correlated with cytochrome c-555 photooxidation, although a comparatively small carotenoid shift did occur at high redox potentials that permitted only P₈₇₀ oxidation.

The results explain earlier observations on infrared absorbance changes that had suggested the existence of two different photochemical systems in Chromatium. A single photochemical system accounts for all of the absorbance changes.

Previous work has shown that the photooxidations of P₈₇₀ and cytochrome c-555 cause similar changes in the electrical charge on the chromatophore membrane. The specific association of the band shifts with cytochrome c-555 photooxidation therefore argues against interpretations of the band shifts based on a light-induced membrane potential.     

Cytochrome b-563 redox changes in intact CO-fixing spinach chloroplasts and in developing pea chloroplasts.

Intact spinach chloroplasts, capable of high rates of photochemical oxygen evolution with CO2 as electron acceptor (120-350 mumol O2 mg chlorophyll-1 h-1) were examined for cytochrome redox changes. The response of the cytochromes in intact chloroplasts to oxidants and reductants appears to be governed by the permeability of the chloroplast envelope. The low potential cytochromes (b-559LP and b-563) were more slowly reduced at 25 degrees C by dithionite than is the case with broken chloroplasts. At 0 degrees C, the reduction of the low potential cytochromes in intactchloroplasts was extremely slow. The chloroplast envelope is impermeable to ferricyanide, slowly permeable to ascorbate and rapidly permeable to reduced dichlorophenolindophenol. Light-induced redox changes of cytochrome b-563 in intact chloroplasts were examined both at 0 degrees and 25 degrees C. A red/far-red antagonism on the redox changes of cytochrome b-563 was observed at 0 degrees C under anaerobic conditions. 3-(3,4-dichlorophenyl)-1, 1-dimethlyurea (DCMU) inhibited the photoreduction of cytochrome b-563 in red light following far-red illumination. The photooxidation of cytochrome b-563 under anaerobic conditions was not influenced by DCMU or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The photoreduction of cytochrome b-563 under aerobic conditions was much less efficient than its photooxidation under anaerobic conditions. Developing pea chloroplasts showed much greater light-induced redox changes of cytochrome b-563 than did intact spinach chloroplasts. Our data are consistent with the view that cytochrome b-563 functions on a cyclic pathway around Photosystem I, but it appears that cyclic flow is sensitive to the relative poising of the redox levels of cytochrome b-563 and the components of the non-cylic pathway.     

Fluorescence induction of photosynthetic bacteria

The kinetics of bacteriochlorophyll fluorescence in intact cells of the purple nonsulfur bacterium Rhodobacter sphaeroides were measured under continuous and pulsed actinic laser diode (808 nm wavelength and maximum 2 W light power) illumination on the micro- and millisecond timescale. The fluorescence induction curve was interpreted in terms of a combination of photochemical and triplet fluorescence quenchers and was demonstrated to be a reflection of redox changes and electron carrier dynamics. By adjustment of the conditions of single and multiple turnovers of the reaction center, we obtained 11 ms^–1 and 120 μs^–1 for the rate constants of cytochrome c₂³⁺ detachment and cyclic electron flow, respectively. The effects of cytochrome c₂ deletion and chemical treatments of the bacteria and the advantages of the fluorescence induction study on the operation of the electron transport chain in vivo were discussed.     

Demonstration of a highly-sensitive portable double-flash kinetic spectrophotometer for measurement of electron transfer reactions in intact plants

A highly sensitive, portable spectrophotometer for use in measuring flash-induced absorbance changes in intact leaves is demonstrated. The design of the instrument is modified for whole plant use from that suggested by Joliot and Joliot (Biochim. Biophys. Acta 765, 210–218). The spectrophotometer uses trifurcated light guides to deliver measuring and actinic beams to two comparable areas of the leaf. The measuring beam is provided by a series of short, relatively intense light pulses from a xenon flashlamp in place of the constant weak measuring beam used in conventional machines. The use of a flash measuring beam and differential detection allows for a high signal-to-noise ratio (noise levels of 10^-5A) without significant actinic effects. The time resolution of the instrument is 2 sec and the noise level is independent of the experimental time range. The instrument is battery or mains powered, computer operated, and has a liquid crystal display for computer-user interface and dialogue, and to show the kinetic traces graphically. Wavelength selection is provided by interchangeable interference filters. The instrument can communicate with a laboratory-based computer, receiving programming information and sending experimental data to be processed and plotted. The instrument is demonstrated by following the kinetics of the electrochromic shift, the change in redox states of cytochrome f and the b cytochromes in an intact cucumber leaf, and in the same leaf after infiltration with DCMU.     

A simple low-cost microcontroller-based photometric instrument for monitoring chloroplast movement

A new microcontroller-based photometric instrument for monitoring blue light dependent changes in leaf transmission (chloroplast movement) was developed based on a modification of the double-beam technique developed by Walzcak and Gabrys [(1980) Photosynthetica 14: 65–72]. A blue and red bicolor light emitting diode (LED) provided both a variable intensity blue actinic light and a low intensity red measuring beam. A phototransistor detected the intensity of the transmitted measuring light. An inexpensive microcontroller independently and precisely controlled the light emission of the bicolor LED. A typical measurement event involved turning off the blue actinic light for 100 μs to create a narrow temporal window for turning on and measuring the transmittance of the red light. The microcontroller was programmed using LogoChip Logo (http://www.wellesley.edu/Physics/Rberg/logochip/) to record fluence rate response curves. Laser scanning confocal microscopy was utilized to correlate the changes in leaf transmission with intercellular chloroplast position. In the dark, the chloroplasts in the spongy mesophyll exhibited no evident asymmetries in their distribution, however, in the palisade layer the cell surface in contact with the overlying epidermis was devoid of chloroplasts. The low light dependent decrease in leaf transmittance in dark acclimated leaves was correlated with the movement of chloroplasts within the palisade layer into the regions previously devoid of chloroplasts. Changes in leaf transmittance were evident within one minute following the onset of illumination. Minimal leaf transmittance was correlated with chloroplasts having retreated from cell surfaces perpendicular to the incident light (avoidance reaction) in both spongy and palisade layers.Electronic Supplementary Material Supplementary material is available for this article at     

Phototactic Responses of Cell Population to Repeated Pulses of Yellow Light in a Phytoflagellate Cryptomonas sp

Positive phototaxis in cell populations of a phytoflagellate Cryptomonas sp. was recorded photoelectrically when the duration and intensity of repeated pulses of monochromatic yellow light (570 nm) interspersed with darkness were varied. Irrespective of the duration of the light pulses, phototactic responses to repeated pulses were as great as those to continuous irradiation and were linearly dependent on the logarithm of total incident light energy when the dark interval was shorter than 60 milliseconds. Under these conditions, reciprocity between duration and intensity held well. In contrast, when the dark interval exceeded 250 milliseconds, the responses were remarkably reduced regardless of light duration and were not affected by increasing the intensity of actinic light pulses.

The present results clearly indicate that continuous stimulation with actinic light is not essential for the maximum effect, but that the length of dark interval is crucial in phototactic response.

     

Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP⁺). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, is defined as the actinic light intensity that, at a given concentration of electron donor, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ is compared with another parameter, equivalent to $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, that is obtained independently of the reaction-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow.The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP⁺ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.