Photosynthetic electron transport and electrochromic effects at sub-zero temperatures

Spinach chloroplasts, suspended in a liquid medium containing ethyleneglycol, showed reversible absorbance changes near 700 and 518 nm due to P-700 and “P-518” in the region from ?35 to ?50 °C upon illumination. The kinetics were the same at both wavelengths, provided absorbance changes due to Photosystem II were suppressed. At both wavelengths, the decay was slowed down considerably, not only by the System I electron acceptor methyl viologen, but also by silicomolybdate. The effect of the latter compound is probably not due to the oxidation of the reduced acceptor of Photosystem I by silicomolybdate, but to the enhanced accessibility of the acceptor to some other oxidant.In the presence of both an electron donor and acceptor for System I, a strong stimulation of the extent of the light-induced absorbance increase at 518 nm was observed. The most effective donor tested was reduced N-methylphenazonium methosulphate (PMS). The light-induced difference spectrum was similar to spectra obtained earlier at room temperature, and indicated electrochromic band shifts of chlorophylls a and b and carotenoid, due to a large potential over the thylakoid membrane, caused by sustained electron transport. It was estimated that steady-state potentials of up to nearly 500 mV were obtained in this way; the potentials reversed only slowly in the dark, indicating a low conductance of the membrane. This decay was accelerated by gramicidin D. The absorbance changes were linearly proportional to the membrane potential.     

Electrochemical and spectro-kinetic evidence for an intermediate electron acceptor in photosystem I

Absorption changes accompanying light-induced P-700 formation and its decay in the dark at 15 K in Photosystem-I particles poised at various redox potentials have been examined. In unpoised samples, the light-induced absorption change is practically irreversible. At increasingly negative potentials, an increasing fraction of the absorption change, proportional to the fraction of bound iron-sulfur protein chemically reduced, becomes reversible, and the titration curve has a midpoint potential of --530 mV (vs. normal hydrogen electrode). At --66 mV, the P-700 absorption change is 97% reversible. The total P-700-signal amplitude decreases over the same potential span and levels off at about 43% (to slightly over 50% at a substantially higher excitation intensity). These results provide additional support to previous suggestions of an existence of an intermediate electron acceptor located between the primary donor, P-700, and the more stable primary electron acceptor (P-430 or bound iron-sulfur protein).     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment.

A comparative study is made, at 15 degrees C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O2 evolution was inhibited by low pH or by Tris-treatment. At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 mus. When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays (t 1/2 about 180 mus) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II (t 1/2 about 120 mus). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible. In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 mus.     

Light-induced absorbance changes due to Photosystems 1 and 2 in spinach chloroplasts at −50 °C

Absorbance changes in the region 500–565 nm and at 702 nm, brought about by excitation of Photosystems 1 and 2, respectively, were measured in spinach chloroplasts at ?50 °C. Either dark-adapted chloroplasts were used or chloroplasts preilluminated with a number of short saturating flashes just before cooling.Both photosystems were found to cause a light-induced increase of absorbance at 518 nm (due to “P518”). The System 1-induced change was not affected by preillumination. It decayed within 1 s in the dark and showed similar kinetics as P700. Experiments in the presence of external electron acceptors (methylviologen or Fe(CN)₆^3?) suggested that P518 was not affected by the redox state of the primary electron acceptor of System 1. The absorbance increase at 518 nm due to System 2 decayed in the dark with a half-time of several min. The kinetics were similar to those of C-550, the presumed indicator of the primary electron acceptor of System 2. After two flashes preillumination the changes due to P518 and C-550 were reduced by about 40%, and a relatively slow, System 2-induced oxidation of cytochrome b₅₅₉ occurred which proceeded at a similar rate as the increase in yield of chlorophyll a fluorescence. The results indicate that at ?50°C two different photoreactions of System 2 occur. One consists of a photoreduction of the primary electron acceptor associated with C-550, accompanied by the oxidation of an unknown electron donor; the other is less efficient and results in the photooxidation of cytochrome b₅₅₉.     

Evidence for two types of P-700 in membrane fragments from a blue-green alga.

The mathematical analysis described in the preceding paper (Biochim. Biophys. Acta (1977) 460, 65-75), in which the steady-state photooxidation of P-700 was compared with overall electron flux in Photosystem I chloroplast fragments, was applied to membrane fragments from the blue-gree alga Nostoc muscorum (Strain 7119) noted for their high activity of both Photosystem I and Photosystem II. The same analysis, which gave good agreement between the photooxidation of P-700 and the overall light-induced electron flux (measured as NADP+ reduction) in Photosystem I chloroplast fragments, revealed in the algal membrane fragments two P-700 components: one responding to high light intensity (P-700 HI), the photooxidation of which was in good agreement with the overall electron flux (measured as NADP+ reduction by reduced 2,6-dichlorophenolindophenol), and the other component responding to low light intensity (P-700 LI), the photooxidation of which was not correlated with the reduction of NADP+ by reduced 2,6-dichlorophenolindophenol.     

Reaction center triplet states in photosystem I and photosystem II

A photosystem I (PS I) particle has been prepared by lithium dodecyl sulfate digestion which lacks the acceptor X, and iron-sulfur centers B and A. Illumination of these particles at liquid helium temperature results in the appearance of a light-induced spin-polarized triplet signal observed by EPR. This signal is attributed to the triplet state of P-700, the primary donor, formed by recombination of the light induced radical pair P-700+ A1- (where A1 is the intermediate acceptor). Formation of the triplet does not occur if P-700 is oxidized or if A1 is reduced, prior to the illumination. A comparison of the P-700 triplet with that of P-680, the primary donor of Photosystem II, shows several differences. (1) The P-680 triplet is 1.5 mT (15 G) wider than the P-700 triplet. This is reflected by the zero-field splitting parameters, which indicate that P-700 is a slightly larger species than P-680. The zero-field splitting parameters do not indicate that either P-700 or P-680 are dimeric. (2) The P-700 triplet is induced by red and far-red light, while the P-680 triplet is induced only by red light. (3) The temperature dependences of the P-700 triplet and the P-680 triplet are different.     

Light-induced absorption changes in Photosystem I at low temperatures

Light-induced absorption changes associated with the primary photochemical reaction and dark relaxation in Photosystem I were measured at various low temperatures. A possible temperature-dependent long-range electron tunneling process was suggested to account for the unique temperature dependence of the dark decay process. The kinetics of the light-induced absorption changes are in good agreement with the light-induced EPR changes reported earlier (Ke, B., Sugahara, K., Shaw, E.R., Hansen, R. E., Hamilton, W. D. and Beinert, H. (1974) Biochim. Biophys. Acta 368, 401–408) for the same Photosystem I subchloroplast fragments at comparable temperatures.All absorption changes between 400 and 725 nm at 86 °K have identical kinetics. The light-minus-dark difference spectrum is very similar to that of P-700 at room temperature, with an additional prominent positive change at 690 nm. Possible contributions by P-430 to the blue and red spectral changes were discussed.It was demonstrated that the intensity of the measuring beam has a drastic effect on the light-induced absorption changes of Photosystem I at low temperatures. Various pretreatments of the Photosystem I fragments such as those that photochemically (or chemically) oxidize the primary donor or photoreduce the primary acceptor abolish the subsequent photochemical reaction. Continuous illumination of the Photosystem I fragments before and during freezing has the same effect.In the temperature range of ?20 to ?60 °C, an unusual counter absorption change as well as a counter EPR change were observed.     

Light-induced absorbance changes in chloroplasts mediated by Photosystem I and Photosystem II at low temperature

Stable light-induced absorbance changes in chloroplasts at −196 °C were measured across the visible spectrum from 370 to 730 nm in an effort to find previously undiscovered absorbance changes that could be related to the primary photochemical activity of Photosystem I or Photosystem II. A Photosystem I mediated absorbance increase of a band at 690 nm and a Photosystem II mediated absorbance increase of a band at 683 nm were found. The 690-nm change accompanied the oxidation of P₇₀₀ and the 683-nm increase accompanied the reduction of C-550. No Soret band was detected for P₇₀₀.

A specific effort was made to measure the difference spectrum for the photooxidation of P₆₈₀ under conditions (chloroplasts frozen to −196 °C in the presence of ferricyanide) where a stable, Photosystem II mediated EPR signal, attributed to P₆₈₀⁺ has been reported. The difference spectra, however, did not show that P₆₈₀⁺ was stable at −196 °C under any conditions tested. Absorbance measurements induced by saturating flashes at −196 °C (in the presence or absence of ferricyanide) indicated that all of the P₆₈₀⁺ formed by the flash was reduced in the dark either by a secondary electron donor or by a backreaction with the primary electron acceptor. We conclude that P₆₈₀⁺ is not stable in the dark at −196 °C: if the normal secondary donor at −196 °C is oxidized by ferricyanide prior to freezing, P₆₈₀⁺ will oxidize other substances.     

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.     

Flash photolysis-electron spin resonance studies of photosystem I. A fast reduction of component of P-700+.

A 300 mus decay component of ESR Signal I (P-700+) in chloroplasts is observed following a 10 mus actinic xenon flash. This transient is inhibited by treatments which block electron transfer from Photosystem II to Photosystem I (e.g. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), KCN and HgCl2). The fast transient reduction of P-700+ can be restored in the case of DCMU or DBMIB inhibition by addition of an electron donor couple (2,6-dichlorophenol indophenol (Cl2Ind)/ascorbate) which supplies electrons to cytochrome f. However, this donor couple is inefficient in restoring electron transport in chloroplasts which have been inhibited with the plastocyanin inactivators, KCN and HgCl2. Oxidation-reduction measurements reveal that the fast P-700+ reduction component reflects electron transfer from a component with Em = 375 +/- 10 mV (pH = 7.5). These data suggest the assignment of the 300-mus decay kinetics to electron transfer from cytochrome f (Fe2+) to P-700+, thus confirming the recent observations of Haehnel et al. (Z. Naturforsch. 26b, 1171-1174 (1971)).     

A partial reaction in photosystem II: reduction of silicomolybdate prior to the site of dichlorophenyldimethylurea inhibition.

Silicomolybdate functions as an electron acceptor in a Photosystem II water oxidation (measured as O2 evolution) partial reaction that is 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) insensitive, that is, reduction os silicomolybdate occurs at or before the level of Q, the primary electron acceptor for Photosystem II. This report characterizes the partial reaction with the principal findings being as follows: 1. Electron transport to silicomolybdate significantly decreased room temperature Photosystem I side of the DCMU had no effect on the fluorescence level, consistent with silicomolybdate accepting electrons at or before Q. In the absence of DCMU, silicomolybdate is also reduced at a site on the Photosystem I side of the DCMU block, prior to or at plastoquinone, since the plastoquinone antagonist dibromothymoquinone (DBMIB) did not affect the electron transport rate. 3. Electron transport from water to silicomolybdate (+ DCMU) is not coupled to ATP formation, nor is there a measurable accumulation of protons within the membrane (measured by amine uptake). Silicomolybdate is not inhibitory to phosphorylation per se since neither cyclic nor post-illumination (XE) phosphorylation were inhibited. 4. Uncouplers stimulated electron transport from water to silicomolybdate in the pH range of 6 to 7, but inhibited at pH values near 8. These data are consistent with the view that when electron flow is through the abbreviated sequence of water to Photosystem II to silicomolybdate (+ DCMU), conditions are not established for the water protons to be deposited within the membrane. Experiments reported elsewhere (Fiaquinta, R.T., Dilley, R.A. and Horton, P.(19741 J. Bioenerg. 6, 167-177) and these data, are consistent with the hypothesis that electron transport between Q and plastoquinone energizes a membrane conformational change that is required to interact with the water oxication system so as to result in the deposition of water protons either within the membrane itself or within the inner oxmotic space.     

Primary photochemistry of Photosystem I in chloroplasts. Dynamics of reversible charge separation in open reaction centers at 25 K

The reduction rate of oxidized reaction center chlorophyll of Photosystem I after laser-flash excitation at 25 K has been determined for D-144 subchloroplast fragments and chloroplasts. A maximum of 40% of Photosystem I reaction centers undergo irreversible charge separation (P-700, Cluster A: P-700⁺, Cluster A^?) at 25 K, a percentage which is independent of laser-flash intensity. The remaining reaction centers in chloroplasts and D-144 fragments undergo reversible charge separation with biphasic recombination. Similar amplitudes and time constants (chloroplasts, 49 μs (61%); D-144 fragments, 90 μs (67%)) were obtained for the fast component, while the slower component differed considerably in time (chloroplasts, 2.9 ms; D-144 fragments, 170 ms). It is known that Fe-S Cluster A is photoreduced in less than 1 ms at 25 K. Data obtained support a model for Photosystem I involving a single intermediate in the decay path between the reduced primary electron acceptor (A^?₁) and P-700⁺ and a second intermediate in the decay path between a reduced secondary electron acceptor and P-700⁺. Dual laser-flash experiments to determine rate constants for these processes are included.     

Evidence that the intermediate electron acceptor,A2, in Photosystem I is a bound iron-sulfur protein

Absorption changes accompanying the formation of light-induced P-700⁺ were investigated in a highly enriched Photosystem I preparation where an intermediate electron acceptor preceding P-430 could be detected. In an enriched Photosystem I particle, light-induced reversible absorption changes observed at 700 nm in the presence of dithionite resembled those previously seen at 703 nm and 820 nm [9], thus indicating the presence of a backreaction between P-700⁺ and A^?₂. After this same Photosystem I particle was treated to denature the bound iron-sulfur centers, the photochemical changes that could be attributed to P-700 A₂ were completely lost. These results provide evidence that the intermediate electron acceptor, A₂, is a bound iron-sulfur protein. Additional studies in the 400–500 nm region with Photosystem I particles prepared by sonication indicate that the spectrum of A₂ is different from that of P-430.     

PRoperties of the low-temperature photosystem I primary reaction in the P-700-chlorophyll alpha-protein.

The Photosystem I primary reaction, as measured by electron paramagnetic resonance changes of P-700 and a bound iron-sulfur center, has been studied at 15 degrees K in P-700-chlorophyll alpha-protein complexes isolated from a blue-green alga. One complex, prepared with sodium dodecyl sulfate shows P-700 photooxidation only at 300 degrees K, whereas a second complex, prepared with Triton X-100, is photochemically active at 15 degrees K as well as at 300 degrees K. Analysis of these two preparations shows that the absence of low-temperature photoactivity in the sodium dodecyl sulfate complex reflects a lack of bound iron-sulfur centers in this preparation and supports the assignment of an iron-sulfur center as the primary electron acceptor of Photosystem I.     

Light-induced changes of absorbance and electron spin resonance in small Photosystem II particles

Photosystem II reaction center components have been studied in small system II particles prepared with digitonin. Upon illumination the reduction of the primary acceptor was indicated by absorbance changes due to the reduction of a plastoquinone to the semiquinone anion and by a small blue shift of absorption bands near 545 nm (C550) and 685 nm. The semiquinone to chlorophyll ratio was between 1/20 and 1/70 in various preparations. The terminal electron donor in this reaction did not cause large absorbance changes but its oxidized form was revealed by a hitherto unknown electron spin resonance (ESR) signal, which had some properties of the well-known signal II but a linewidth and g-value much nearer to those of signal I. Upon darkening absorbance and ESR changes decayed together in a cyclic or back reaction which was stimulated by 3-(3,4 dichlorophenyl)-1,1-dimethylurea. The donor could be oxidized by ferricyanide in the dark.

Illumination in the presence of ferricyanide induced absorbance and ESR changes, rapidly reversed upon darkening, which may be ascribed to the oxidation of a chlorophyll a dimer, possibly the primary electron donor of photosystem II. In addition an ESR signal with 15 to 20 gauss linewidth and a slower dark decay was observed, which may have been caused by a secondary donor.     

Light-induced changes of absorbance and electron spin resonance in small photosystem II particles.

Photosystem II reaction center components have been studied in small system II particles prepared with digitonin. Upon illumination the reduction of the primary acceptor was indicated by absorbance changes due to the reduction of a plastoquinone to the semiquinone anion and by a small blue shifts of absorption bands near 545 nm (C550) and 685 nm. The semiquinone to chlorophyll ratio was between 1/20 and 1/70 in various preparations. The terminal electron donor in this reaction did not cause large absorbance changes but its oxidized form was revealed by a hitherto unknown electron spin resonance (ESR) signal, which had some properties of the well-known signal II but a linewidth and g-value much nearer to those of signal I. Upon darkening absorbance and ESR changes decayed together in a cyclic or back reaction which was stimulated by 3-(3,4 dichlorophenyl)-1,1-dimethylurea. The donor could be oxidized by ferricyanide in the dark. Illumination in the presence of ferricyanide induced absorbance and ESR changes, rapidly reversed upon darkening, which may be ascribed to the oxidation of a chlorophyll a dimer, possibly the primary electron donor of photosystem II. In addition an ESR signal with 15 to 20 gauss linewidth and a slower dark decay was observed, which may have been caused by a secondary donor.     

The use of light-induced absorbance changes at 820 nm to monitor the oxidation state of P-700 in leaves

Abstract The use of the light-induced absorbance change at 820 nm (ΔA 820) to monitor the oxidation and reduction of P-700 in irradiated leaves is examined. Results obtained from leaves irradiated with a range of wavelengths of light, poisoned with DCMU, or lacking PS I, are consistent with the proposition that the light-induced ΔA 820 can be used to monitor P-700 oxidation in leaves.     

Electrochemical and spectro-kinetic evidence for an intermediate electron acceptor in Photosystem I

Absorption changes accompanying light-induced P-700⁺ formation and its decay in the dark at 15 K in Photosystem-I particles poised at various redox potentials have been examined. In unpoised samples, the light-induced absorption change is practically irreversible. At increasingly negative potentials, an increasing fraction of the absorption change, proportional to the fraction of bound iron-sulfur protein chemically reduced, becomes reversible, and the titration curve has a midpoint potential of −530 mV (vs. normal hydrogen electrode). At −666 mV, the P-700 absorption change is 97% reversible. The total P-700-signal amplitude decreases over the same potential span and levels off at about 43% (to slightly over 50% at a substantially higher excitation intensity). These results provide additional support to previous suggestions of an existence of an intermediate electron acceptor located between the primary donor, P-700, and the more stable primary electron acceptor (P-430 or bound iron-sulfur protein).     

Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — characteristics and reinterpretation of single-turnover cyclic electron transfer

The contributions of ferredoxin, P-700, plastocyanin and the cytochromes c-554, and b-563 to single-turnover electron transfer in Photosystem (PS) I-enriched subchloroplast vesicles were deconvoluted by fitting the literature-derived spectra of these components to the observed absorption data at a series of wavelengths, according to a linear least-squares method. The obtained corresponding residuals showed that the applied component spectra were satisfactory. The deconvoluted signals of cytochromes c-554 and b-563 differed in some cases significantly from the classical dual-wavelength signals recording at 554–545 nm and 563–575 (or −572) nm, due to interference from other electron-transferring components. KCN, DNP-INT (2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether), DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzo-quinone) and antimycin A all inhibited electron transfer, although antimycin and DBMIB inhibited only after a few turnovers of the cytochrome bf complex. Fast flash-induced reduction of cytochrome b-563 exclusively reflected oxidant-induced reduction. Fast electron flow from cytochrome c-554 to plastocyanin and P-700 resulted in an apparent rereduction of cytochrome c-554 that was slower than the reduction of cytochrome b-563. Model simulations indicate that under highly oxidizing conditions for the Rieske FeS centre and reducing conditions for cytochrome b-563, the semiquinone at the Q_z site cannot only reduce cytochrome b-563, but can also oxidize cytochrome b-563 and reduce the Rieske FeS centre. The effect of 10 μM gramicidin D was evaluated in order to determine the contributions by electrochromic absorption changes around 518 nm. Gramicidin left electron transfer, monitored in the 550–600 nm range, unchanged. The gramicidin-sensitive (membrane potential-associated) signal at 518 nm differed from the signals recorded in the absence of gramicidin at 518 nm or 518–545 nm, due to spectral interference from electron-transferring components in the latter signals. KCN, DBMIB and antimycin A affected both the fast and slow components of the electrochromic signal, but did not proportionally affect the initial electron transfer from P-700 to ferredoxin (charge separation in PS I). Not only the slow (10–100 ms) component of the 518 nm absorption change, but also part of the fast (less than 1 ms) component appears to minitor electrogenic events in the cytochrome bf complex.     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment

A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O₂ evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.