The effect of o-phenanthroline on the midpoint potential of the primary electron acceptor of Photosystem II

The primary electron acceptor of Photosystem II has a midpoint oxidation-reduction potential of +95 mV at pH 7.0 in Photosystem II chloroplast fragments prepared by digitonin treatment. The midpoint potential of the acceptor has a pH dependence of −60 mV/pH unit. At concentrations that inhibit oxygen evolution, o-phenanthroline shifts the midpoint potential of the primary acceptor by +70 mV. The shifted potential retains the same dependence on pH. The effect of o-phenanthroline suggests that it interacts directly with the primary electron acceptor of Photosystem II in a manner similar to that reported previously for the primary electron acceptor in purple photosynthetic bacteria.     

The effect of o-phenanthroline on the midpoint potential of the primary electron acceptor of photosystem II.

The primary electron acceptor of Photosystem II has a midpoint oxidation-reduction potential of +95 mV at pH 7.0 in Photosystem II chloroplast fragments prepared by digitonin treatment. The midpoint potential of the acceptor has a pH dependence of -60 mV/pH unit. At concentrations that inhibit oxygen evolution, o-phenanthroline shifts the midpoint potential of the primary acceptor by +70 mV. The shifted potential retains the same dependence on pH. The effect of o-phenanthroline suggests that it interacts directly with the primary electron acceptor of photosystem II in a manner similar to that reported previously for the primary electron acceptor in purple photosynthetic bacteria.     

The effect of temperature on the primary reaction of chloroplast Photosystem II Evidence for a temperature-dependent back reaction

The primary reaction of Photosystem II has been studied over the temperature range from −196 to −20 °C. The photooxidation of the reaction-center chlorophyll (P680) was followed by the free-radical electron paramagnetic resonance signal of P680⁺, and the photoreduction of the Photosystem II primary electron acceptor was monitored by the C-550 absorbance change.

At temperatures below −100 °C, the primary reaction of Photosystem II is irreversible. However, at temperatures between −100 and −20 °C a back reaction that is insensitive to 3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) occurs between P680⁺ and the reduced acceptor.

The amount of reduced acceptor and P680⁺ present under steady-state illumination at temperatures between −100 and −20 °C is small unless high light intensity is used to overcome the competing back reaction. The amount of reduced acceptor present at low light intensity can be increased by adjusting the oxidation-reduction potential so that P680⁺ is reduced by a secondary electron donor (cytochrome b₅₅₉) before P680⁺ can reoxidize the reduced primary acceptor. The photooxidation of cytochrome b₅₅₉ and the accompanying photoreduction of C-550 are inhibited by DCMU. The inhibition of C-550 photoreduction by DCMU, the dependence of P680 photooxidation and C-550 photoreduction on light intensity, and the effect of the availability of reduced cytochrome b₅₅₉ on C-550 photoreduction are unique to the temperature range where the Photosystem II primary reaction is reversible and are not observed at lower temperatures.     

The oxidation-reduction potential of the reaction-centre chlorophyll (P700) in Photosystem I. Evidence for multiple components in electron-paramagnetic-resonance signal 1 at low temperature.

The oxidation-reduction potential of the reaction-centre chlorophyll of Photosystem I (P700) in spinach chloroplasts was determined by using the ability of the reaction centre to photoreduce the bound ferredoxin and to photo-oxidize P700 on illumination at 20K as an indicator of the oxidation state of P700. This procedure shows that P700 is oxidized with Em (pH8.0)(mid-point redox potential at pH8.0)congruent to +375mV. Further oxidation of the chloroplast preparations by high concentrations of K3Fe(CN)6(10mM) in the presence of mediating dyes leads to the appearance of a large radical signal with an apparent Em congruent to +470mVA second, light-inducible, radical also appears over the same potential range. We propose that these signals are due to bulk chlorophyll oxidation and not, as was previously thought [Knaff & Malkin (1973) Arch. Biochem. Biophys. 159, 555-562], to reaction-centre oxidation. A number of optical techniques were used to determine Em of P700. Dual-wavelength spectroscopy (697-720nm) indicates Em congruent to +460-+480mV. The spectrum of the sample during the titration showed a large contribution to the signal by bulk chlorophyll oxidation, in agreement with the electron-paramagnetic-resonance results and those of Ke, Sugahara & Shaw [(1975) Biochim. Biophys. Acta 408, 12-25]. The light-induced absorbance change at 435 nm, usually attributed to P700, showed a potential dependence similar to that of bulk chlorophyll oxidation. Determination of Em of P700 on the basis of the appearance of the P700 signal in oxidized-versus-reduced difference spectra showed Em (pH8.0) congruent to +360mV. Measurements of the effect of potential on the irreversible photo-oxidation of P700 at 77K showed that P700 became oxidized in this potential range. We conclude that the reaction-centre chlorophyll of Photosystem I has Em (pH8.0) congruent to +375mV.     

Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium.

We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic (t1/2 of less than 5 mus and approximately 50 mus). We belive that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll. We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below -450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD+ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae.     

Laser-flash-activated electron paramagnetic resonance studies of primary photochemical reactions in chloroplasts.

Electron paramagnetic resonance studies of the primary reactants of Photosystems I and II have been conducted at cryogenic temperatures after laser-flash activation with monochromatic light.P-700 photooxidation occurs irreversibly in chloroplasts and in Photosystem I fragments after activation with a 730 nm laser flash at a temperature of 35 degrees K. Flash activation of chloroplasts or Photosystem II chloroplast fragments with 660 nm light results in the production of a free-radical signal (g = 2.002, linewidth approximately 8 gauss) which decays with a half-time of 5.0 ms at 35 degrees K. The half-time of decay is independent of temperature in the range of 10-77 degrees K. This reversible signal can be eliminated by preillumination of the sample at 35 degrees K with 660 nm light (but not by 730 nm light), by preillumination with 660 nm light at room temperature in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) plus hydroxylamine, or by adjustment of the oxidation-reduction potential of the chloroplasts to - 150 mV prior to freezing. In the presence of ferricyanide (20-50 mM), two free-radical signals are photoinduced during a 660 nm flash at 35 degrees K. One signal decays with a half-time of 5 ms, whereas the second signal is formed irreversibly. These results are discussed in terms of a current model for the Photosystem II primary reaction at low temperature which postulates a back-reaction between P-680+ and the primary electron acceptor.     

Laser-flash-activated electron paramagnetic resonance studies of primary photochemical reactions in chloroplasts

Electron paramagnetic resonance studies of the primary reactants of Photosystems I and II have been conducted at cryogenic temperatures after laser-flash activation with monochromatic light.P-700 photooxidation occurs irreversibly in chloroplasts and in Photosystem I fragments after activation with a 730 nm laser flash at a temperature of 35 °;K. Flash activation of chloroplasts or Photosystem II chloroplast fragments with 660 nm light results in the production of a free-radical signal (g = 2.002, linewidth ～ 8 gauss) which decays with a half-time of 5.0 ms at 35 °;K. The half-time of decay is independent of temperature in the range of 10–77 °;K. This reversible signal can be eliminated by preillumination of the sample at 35 °;K with 660 nm light (but not by 730 nm light), by preillumination with 660 nm light at room temperature in the presence of 3-(3′, 4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) plus hydroxylamine, or by adjustment of the oxidation-reduction potential of the chloroplasts to — 150 mV prior to freezing. In the presence of ferricyanide (20–50 mM), two free-radical signals are photoinduced during a 660 nm flash at 35 °;K. One signal decays with a half-time of 5 ms, whereas the second signal is formed irreversibly. These results are discussed in terms of a current model for the Photosystem II primary reaction at low temperature which postulates a back-reaction between P-680⁺ and the primary electron acceptor.     

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.     

Cytochrome b and photosynthetic sulfur bacteria

Chromatophores isolated from the purple sulfur bacterium Chromatium and the green sulfur bacterium Chlorobium exhibit absorbance changes in the cytochrome -band region consistent with the presence of a b-type cytochrome. Cytochrome content determined by reduced minus oxidized difference spectra and by heme analysis suggests that each bacterium contains one cytochrome b per molecule of photochemically active bacteriochlorophyll (reaction-center bacteriochlorophyll).

The b-type cytochrome in Chromatium has an -band maximum at 560 nm and a midpoint oxidation-reduction potential of −5 mV at pH 8.0. The b-type cytochrome in Chlorobium has an -band maximum at 564 nm and an apparent midpoint oxidation-reduction potential near −90 mV.

Chromatophores isolated from both Chromatium and Chlorobium cells catalyze a photoreduction of cytochrome b that is enhanced in the presence of antimycin A. Antimycin A and 2-n-heptyl-4-hydroxyquinoline-N-oxide inhibit endogenous (but not phenazine methosulfate-mediated) cyclic photophosphorylation in Chromatium chromatophores and non-cyclic electron flow from Na₂S to NADP in Chlorobium chromatophores. These observations suggest that b-type cytochromes may function in electron transport reactions in photosynthetic sulfur bacteria.     

The influence of cytochrome b559 on the fluorescence yield of chloroplasts at low temperature

The maximum light-induced fluorescence yield, F_M, of spinach chloroplasts at − 196 °C was less when the chloroplasts were oxidized with ferricyanide prior to freezing; the minimum fluorescence yield, F₀, of the dark-adapted chloroplasts at − 196 °C was unaffected. The ratio of the fluorescence yields, F_M/F₀, measured at 695 nm at low temperature was 4.5–5.0 for normal chloroplasts and 2.0–2.5 in the presence of ferricyanide. The oxidative titration curve of F_M followed a 1 electron Nernst equation with a midpoint potential of 365 mV and followed closely to the oxidation of cytochrome b₅₅₉. The photoreduction of C−550 at low temperature was the same at all redox potentials over the range of 200–500 mV. It is suggested that a relatively strong oxidant associated with the water-splitting side of Photosystem II, possibly the primary electron donor, can chlorophyll fluorescence of Photosystem II as well as the primary electron acceptor.     

Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium

We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of < 5 μs and ≈ 50 μs). We believe that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll.We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below ?450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD⁺ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae.     

Photoinduced changes in adsorption at 800 nm related to photosystem I functioning]

The spectra and kinetics of light-induced absorbance changes in the near-infrared region of subchloroplast fragments enriched by P700 were studied. An increase in absorbancy within the region of 725--900 nm upon illumination was characterized by a maximum around 810 nm and by "shoulders" around 760 and 870 nm. Similar effects of thermal inactivation and low temperatures on the duration of dark recovery of light-induced absorbance changes at 700 nm and within the region of 725--900 nm suggest that the absorbance changes in the near-infrared region are due to photooxidation of P700. The values of P700 differential extinction coefficients at 810 nm are 8,2.10(3) M-1.cm-1 for digitonin fragments and 7,7.10(3) M-1.cm-1 for fragments prepared with the use of diethyl ester. It was shown that the value of midpoint oxidation-reduction potential measured for the absorbance changes at 810 nm (+492 mv) is higher than that measured at 700 nm (+475 mv).     

The oxidation-reduction potentials of electron carriers in chloroplast photosystem I fragments

Oxidation-reduction titrations of several electron carriers found in chloroplast Photosystem I fragments have been performed. The midpoint potential of P700 in these fragments and in chloroplasts has been found to be +520 mV by optical absorbance methods or electron paramagnetic resonance spectroscopy. The copper-containing protein plastocyanin is present in Photosystem I fragments and has a midpoint potential of +320 mV, significantly less positive than the midpoint potential of cytochrome f in the same fragments, which was measured to be +375 mV. Photo-system I fragments contain two b cytochromes, a low-potential form of cytochrome b₅₅₉ (E_m = +110 mV) and cytochrome b₅₆₃ (E_m = ?100 mV).     

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.     

Isolation and properties of particles containing the reactioncenter complex of Photosystem II from spinach chloroplasts

By density gradient centrifugation of the 80000 × g supernatant of digitonintreated spinach chloroplasts two main green bands and one minor green band were obtained. The purification and properties of the particles present in the main bands, which were shown to be derived from Photosystem I and Photosystem II, have been described previously; those of the particles in the minor fraction will be described in the present paper.

After purification, these particles show Photosystem II activity but are devoid of Photosystem I activity. They have a high chlorophyll a/chlorophyll b ratio and are enriched in β-carotene and cytochrome b₅₅₉. At liquid nitrogen temperature, photoreduction of C550 and photooxidation of cytochrome-b₅₅₉ can be observed. At room temperature, cytochrome b₅₅₉ undergoes slight photooxidation.

These properties indicate that this particle may be the reaction-center complex of Photosystem II. It is suggested that, in vivo, the Photosystem II unit is made up of a reaction-center complex and an accessory complex, the latter being found in one of the main green bands of the density gradient.     

Four different b-type cytochromes in the halophilic archaebacterium, Halobacterium halobium

The halophilic archaebacterium, Halobacterium halobium has been found to contain four different b-type cytochromes. The four components were recognized by their potentiometric characteristics in situ in their functional environment in the membrane of H. halobium. Oxidation-reduction midpoint potentials of these four b-type cytochromes were determined to be +261, +160, +30, and -153 mV, respectively. We also demonstrate that the pathway involved in the transport of reducing equivalents from succinate to oxygen proceeds through the b-type cytochromes with oxidation-reduction midpoint potentials of +261 and +161 mV. The cytochrome with oxidation-reduction midpoint potential of -153 mV was not substrate reducible by NADH but was chemically reducible by dithionite. Antimycin inhibits reduction of b-type cytochrome in the succinate pathway, but has no effect on b-type cytochrome reduction when reducing equivalents are provided by NADH. The carbon monoxide difference spectrum of H. halobium membranes shows at least one carbon monoxide-binding b-type cytochrome, indicating a terminal oxidase. A scheme for electron transport in H.halobium involving the b-type cytochromes and terminal oxidase is suggested.     

Redox-potentiometric titrations of the electrochromic absorption change in chloroplasts

Two phases of the electrochromic 515 nm absorption change in chloroplasts elicited by microsecond flashes can be resolved kinetically. Redox-potentiometric titrations indicate that the initial amplitude appearing within 0.5 ms, and designated as phase a, has three components in the low-potential region with E_m7.5 values of +60 mV, −195 mV and less than −400 mV. From the insensitivity to DCMU, we propose that the species with E_m7.5 values of −195 mV and less than −400 mV are both related to Photosystem I. This conclusion was supported by the loss of both components when the Photosystem I reaction centre (P-700) was chemically oxidised (E_m7.5 = +370 mV). The species having an E_m7.5 less than −400 mV is presumed to be the Photosystem I primary acceptor, while the E_m7.5 = −195 mV wave could be due to a secondary electron acceptor, such as cytochrome b-563_LP, whose photoreduction is possible owing to the long duration of the excitation flash. The DCMU-sensitive component with an E_m7.5 of +60 mV is assumed to be the primary quinone acceptor (Q_A) of Photosystem II. Unlike the Photosystem I redox components, the midpoint potential of this species is sensitive to the background ionic level: the E_m7.5 is shifted to −100 mV when the cation concentration is lowered to facilitate membrane unstacking. The slow phase of the electrochromic signal (phase b) has been estimated by measuring the 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-sensitive amplitude of the absorption change at 20 ms. The signal appears with an estimated E_m7.5 = +50 mV, becomes maximal at −50 mV and attenuates with an E_m7.5 of about −180 mV. These results suggest that phase b occurs when the plastoquinone pool is reduced and cytochrome b-563_LP is oxidised.     

A quantitative study of the slow decline of chlorophyll a fluorescence in isolated chloroplasts.

A detailed study of the photo-induced decline in chlorophyll a fluorescence intensity (Kautsky phenomenon) in coupled isolated chloroplasts from a high level (P) to a low stationary level (S) is presented. 1. A linear relationship between P leads to S quenching and intrathylakoid H+ concentration was found. When the light-induced proton gradient was abolished by uncoupling, the fluorescence emission at room temperature was lowered proportionally to increased H+ concentration in the medium. 2. Fluorescence spectra at -196 degrees C of samples frozen at the P and S states showed no significant differences in the Photosystem I/Photosystem II ratio of fluorescence emission. Furthermore, freezing to -196 degrees C reversed the P leads to S quenching. This indicates that the P leads to S quenching is not related to an increase of spillover of excitation energy from Photosystem II to Photosystem I. 3. When Mg2+ was added to thylakoids suspended in a medium free of divalent cations, the inhibition of spillover required lower Mg2+ concentrations (half saturation at 0.6 mM). Increased proton concentration in the medium also inhibited spillover. 4. The results are interpreted in terms of two sites of Mg2+ and H+ effects on excitation deactivation in Photosystem II. One site is located on the outer face of the thylakoid membrane; action of both Mg2+ and H+ at this side diminishes spillover. The second site is located on the inner face of the membrane; as Mg2+ is displaced there by protons, a non-photochemical quenching of Photosystem II fluorescence is induced, which is manifested by the P leads to S decline.     

Characterization of the iron-sulfur protein of the mitochondrial outer membrane partially purified from beef kidney cortex.

The iron-sulfur protein present in the mitochondrial outer membrane has been partially purified from beef kidney cortex mitochondria by means of selective solubilization followed by DEAE-cellulose chromatography. The EPR spectrum of the iron-sulfur protein with g-values at 2.01, 1.94 and 1.89 was well resolved up to 200 K which is unusual for an iron-sulfur protein. Analyses confirmed a center with two iron and two labile sulfur atoms in the protein. By measuring the effect of oxidation-reduction potential on the EPR signal amplitude, midpoint potentials at pH 7.2 were determined both for the purified iron-sulfur protein, +75 (+/- 5) mV, and in prepared mitochondrial outer membrane, +62 (+/- 6) mV. At pH 8.2 slightly lower values were indicated, +62 and 52 mV, respectively. The oxidation-reduction equilibrium involved a one electron transfer. A functional relationship to the rotenone-insensitive NADH-cytochrome c oxidoreductase in the mitochondrial outer membrane is suggested. Both this activity and the iron-sulfur center were sensitive to acidities slightly below pH 7 in contrast to the iron-sulfur centers of the inner membrane.     

Redox study of electron donation to P-680 in Photosystem II

Flash-induced absorption changes at 820 nm were studied as a function of redox potential in Tris-extracted Photosystem II oxygen-evolving particles and Triton subchloroplast fraction II particles. The rereduction kinetics of P-680+ in both preparations showed biphasic recovery phases with half-times of 42 and 625 microseconds at pH 4.5. The magnitude of the 42 microseconds phase of P-680+ rereduction was strongly dependent on the redox potential of the medium. This absorption transient, attributed to electron donation from D1 (the secondary electron donor in oxygen-inhibited chloroplasts), titrated as a single redox component with a midpoint potential of +240 +/- 35 mV. The experimentally determined midpoint potential was found to be independent of pH over the tested range 4.5-6.0. In contrast, the magnitude of the 625 microseconds phase of P-680+ rereduction was independent of redox potential between +350 and +100 mV. These results are interpreted in terms of a model in which an alternate electron donor with Em approximately equal to 240 mV, termed D0, serves as a rapid donor (t 1/2 less than or equal to 2 microseconds) to P-680+ in Tris-extracted and Triton-treated Photosystem-II preparations. According to this model, the slower electron donor, D1, is functional only when D0 becomes oxidized.