On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Coregulation of electron transport through PS I by Cyt b 6 f,excitation capture by P700 and acceptor side reduction. Time kinetics and electron transport requirement

Regulation of electron transport rate through Photosystem I (PS I) was investigated in intact sunflower leaves. The rate constant of electron donation via the cytochrome b ₆ f complex (k_q, s^–1) was obtained from the postillumination P700⁺ reduction rate, measured as the exponential decay of the light-dark difference (D830) of the 830 nm transmission signal. D830 corresponding to maximum oxidisable P700 (D830_m) was obtained by applying white light flashes of different intensity and extrapolating the plot of the quantum yield Y vs. D830 to the axis of abscissae (Y->0). Maximum quantum yield of PS I at completely reduced P700 (Y_m) was obtained by extrapolating the same plot to the axis of ordinates (D830->0). Regulation of k_q, D830_m and Y_m under rate-limiting CO₂ and O₂ concentrations applied after air (21% O₂, 310 ppm CO₂) was investigated. The amplitude of the downregulation of k_q (photosynthetic control) was maximal when electron transport rate (ETR) was limited to about 3 nmol cm^–2 s^–1 and decreased when ETR was higher or lower. Downregulation did not occur in the absence of CO₂ and O₂. These gases acted only as substrates of ribulosebisphosphate carboxylase-oxygenase, no high-affinity reaction of O₂ leading to enhanced photosynthetic control (e.g. Mehler reaction) was detected. After the transition, D830_m at first decreased and then increased again, showing that the reduction of the PS I acceptor side disappeared as a result of the downregulation of k_q. The variation of Y_m had two reasons, PS I acceptor side reduction and variable excitation capture efficiency by P700. It is concluded that electron transport through PS I is coregulated by the rate of plastoquinol oxidation at Cyt b ₆ f, excitation capture efficiency by P700, and by acceptor side reduction.Abbreviations Cyt b ₆ f cytochrome b ₆ f complex - D830 difference of the 830 nm signal from the dark level - ETR electron transport rate - PAD photon absorption density nmol cm^–2 s^–1 - PFD incident photon flux density, nmol cm^–2 s^–1 - PS I Photosystem I - PS II Photosystem II - PQH₂ plastoquinol - P700 Photosystem I donor pigment - Y quantum yield of PS I electron transport, rel. un.     

Electron transport pathways in isolated chromoplasts from Narcissus pseudonarcissus L.

During daffodil flower development, chloroplasts differentiate into photosynthetically inactive chromoplasts having lost functional photosynthetic reaction centers. Chromoplasts exhibit a respiratory activity reducing oxygen to water and generating ATP. Immunoblots revealed the presence of the plastid terminal oxidase (PTOX), the NAD(P)H dehydrogenase (NDH) complex, the cytochrome b₆f complex, ATP synthase and several isoforms of ferredoxin‐NADP⁺ oxidoreductase (FNR), and ferredoxin (Fd). Fluorescence spectroscopy allowed the detection of chlorophyll a in the cytochrome b₆f complex. Here we characterize the electron transport pathway of chromorespiration by using specific inhibitors for the NDH complex, the cytochrome b₆f complex, FNR and redox‐inactive Fd in which the iron was replaced by gallium. Our data suggest an electron flow via two separate pathways, both reducing plastoquinone (PQ) and using PTOX as oxidase. The first oxidizes NADPH via FNR, Fd and cytochrome b_h of the cytochrome b₆f complex, and does not result in the pumping of protons across the membrane. In the second, electron transport takes place via the NDH complex using both NADH and NADPH as electron donor. FNR and Fd are not involved in this pathway. The NDH complex is responsible for the generation of the proton gradient. We propose a model for chromorespiration that may also be relevant for the understanding of chlororespiration and for the characterization of the electron input from Fd to the cytochrome b₆f complex during cyclic electron transport in chloroplasts.     

Range of photosynthetic control of postillumination P700+ reduction rate in sunflower leaves

The kinetics of the postillumination reduction of P700⁺ which reflects the rate constant for plastoquinol (PQH₂) oxidation was recorded in sunflower leaves at different photon absorption densities (PAD), CO₂ and O₂ concentrations. The P700 oxidation state was calculated from the leaf transmittance at 830 nm logged at 50 s intervals. The P700⁺ dark reduction kinetics were fitted with two exponents with time constants of 6.5 and about 45 ms at atmospheric CO₂ and O₂ concentrations. The time constant of the fast component, which is the major contributor to the linear electron transport rate (ETR), did not change over the range of PADs of 14.5 to 134 nmol cm^-2 s^-1 in 21% O₂, but it increased up to 40 ms under severe limitation of ETR at low O₂ and CO₂. The acceptor side of Photosystem I (PS I) became reduced in correlation with the downregulation of the PQH₂ oxidation rate constant. It is concluded that thylakoid pH-related downregulation of the PQH₂ oxidation rate constant (photosynthetic control) is not present under normal atmospheric conditions but appears under severe limitation of the availability of electron acceptors. The measured range of photosynthetic control fits with the maximum variation of ETR under natural stress in C₃ plants. Increasing the carboxylase/oxygenase specificity would lead to higher reduction of the PS I acceptor side under stress.Abbreviations Cyt b ₆ f cytochrome b ₆ f complex - C_w cell-wall CO₂ concentration, M - ETR electron transport rate - Fd ferredoxin - FNR ferredoxin-NADP reductase - FRL far-red light - PC plastocyanin - PAD photon absorption density nmol cm^-2 s^-1 - PFD photon flux density nmol cm^-2 s^-1 - PS I Photosystem I complex - PQ plastoquinon - PQH₂ plastoquinol - PS II Photosystem II complex - P700 Photosystem I donor pigment, reduced - S830 830 nm signal (D830, difference of S830 from the dark level) - WL white light - Y_l maximum quantum yield of PS I electron transport, rel. un     

Electron transport and photophosphorylation by Photosystem I in vivo in plants and cyanobacteria

Recently, a number of techniques, some of them relatively new and many often used in combination, have given a clearer picture of the dynamic role of electron transport in Photosystem I of photosynthesis and of coupled cyclic photophosphorylation. For example, the photoacoustic technique has detected cyclic electron transport in vivo in all the major algal groups and in leaves of higher plants. Spectroscopic measurements of the Photosystem I reaction center and of the changes in light scattering associated with thylakoid membrane energization also indicate that cyclic photophosphorylation occurs in living plants and cyanobacteria, particularly under stressful conditions.In cyanobacteria, the path of cyclic electron transport has recently been proposed to include an NAD(P)H dehydrogenase, a complex that may also participate in respiratory electron transport. Photosynthesis and respiration may share common electron carriers in eukaryotes also. Chlororespiration, the uptake of O₂ in the dark by chloroplasts, is inhibited by excitation of Photosystem I, which diverts electrons away from the chlororespiratory chain into the photosynthetic electron transport chain. Chlororespiration in N-starved Chlamydomonas increases ten fold over that of the control, perhaps because carbohydrates and NAD(P)H are oxidized and ATP produced by this process.The regulation of energy distribution to the photosystems and of cyclic and non-cyclic phosphorylation via state 1 to state 2 transitions may involve the cytochrome b ₆-f complex. An increased demand for ATP lowers the transthylakoid pH gradient, activates the b ₆-f complex, stimulates phosphorylation of the light-harvesting chlorophyll-protein complex of Photosystem II and decreases energy input to Photosystem II upon induction of state 2. The resulting increase in the absorption by Photosystem I favors cyclic electron flow and ATP production over linear electron flow to NADP and poises the system by slowing down the flow of electrons originating in Photosystem II.Cyclic electron transport may function to prevent photoinhibition to the photosynthetic apparatus as well as to provide ATP. Thus, under high light intensities where CO₂ can limit photosynthesis, especially when stomates are closed as a result of water stress, the proton gradient established by coupled cyclic electron transport can prevent over-reduction of the electron transport system by increasing thermal de-excitation in Photosystem II (Weis and Berry 1987). Increased cyclic photophosphorylation may also serve to drive ion uptake in nutrient-deprived cells or ion export in salt-stressed cells.There is evidence in some plants for a specialization of Photosystem I. For example, in the red alga Porphyra about one third of the total Photosystem I units are engaged in linear electron transfer from Photosystem II and the remaining two thirds of the Photosystem I units are specialized for cyclic electron flow. Other organisms show evidence of similar specialization.Improved understanding of the biological role of cyclic photophosphorylation will depend on experiments made on living cells and measurements of cyclic photophosphorylation in vivo.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DCHC dicyclohexyl-18-crown-6 - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone - LHC light harvesting chlorophyll - LHCP II light harvesting chlorophyll protein of Photosystem II - PQ plastoquinone - PS I, II Photosystem I, II - SHAM salicyl hydroxamic acid - TBT Tri-n-butyltin CIW/DPB Publication No. 1146     

Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a     

Effect of NADP+ on light-induced cytochrome changes in membrane fragments from a blue-green alga

The effect of NADP⁺ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragments prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP⁺ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH₂) to NADP⁺. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH₂ as the electron donor, NADP⁺ had no effect on the light-induced redox changes of cytochromes: with or without NADP⁺, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an α peak at 549 nm. With 664 nm illumination and water as the electron donor, NADP⁺ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b₅₅₉. Cytochrome b₅₅₉ appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP⁺) for the electron flow from water.     

Effects of magnesium and chloride ions on light-induced electron transport in membrane fragments from a blue-green alga

The effects of magnesium and chloride ions on photosynthetic electron transport were investigated in membrane fragments of a blue-green alga, Nostoc muscorum (Strain 7119), noted for their stability and high rates of electron transport from water or reduced dichlorophenolindophenol to NADP⁺. Magnesium ions were required not only for light-induced electron transport from water to NADP⁺ but also for protection in the dark of the integrity of the water-photooxidizing system (Photosystem II). Membrane fragments suspended in the dark in a medium lacking Mg²⁺ lost the capacity to photoreduce NADP⁺ with water on subsequent illumination. Chloride ions could substitute, but less effectively, for each of these two effects of magnesium ions. By contrast, the photoreduction of NADP⁺ by DCIPH₂ was independent of Mg²⁺ (or Cl^?) for the protection of the electron transport system in the dark or during the light reaction proper. Furthermore, high concentrations of MgCl₂ produced a strong inhibition of NADP⁺ photoreduction with DCIPH₂ without significantly affecting the rate of NADP⁺ photoreduction with water. The implications of these findings for the differential involvement of Photosystem I and Photosystem II in the photoreduction of NADP⁺ with different electron donors are discussed.     

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

A 300 μs decay component of ESR Signal I (P-700⁺) in chloroplasts is observed following a 10 μs 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 E_m = 375±10 mV (pH = 7.5). These data suggest the assignment of the 300-μs 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)).     

Light-induced oxidation-reduction reactions in a cell-free preparation from the blue-green alga Nostoc muscorum: The role of cytochrome f, cytochrome b558, C550, and P700 in noncyclic electron transport

1. Cytochrome f (λ_max = 554 nm, E_m = +0.35 V) and cytochrome b₅₅₈ (λ_max = 558 nm, E_m = +0.35 V) were photooxidized by Photosystem I and photoreduced by Photosystem II in a cell-free preparation from the blue-green alga Nostoc muscorum. The steady-state oxidation levels of both cytochromes were affected by noncyclic electron acceptors and by inhibitors of noncyclic electron transport. These results are consistent with the hypothesis that the mechanism of NADP reduction by water involves a Photosystem II and a Photosystem I light reaction operating in series and linked by a chain of electron carriers that includes cytochrome f and cytochrome b₅₅₈.2. Phosphorylation cofactors shifted the steady-state of cytochrome f to a more reduced level under conditions of noncyclic electron transport but had no effect on cytochrome b₅₅₈. These observations suggest that the noncyclic phosphorylation site lies before cytochrome f (on the Photosystem II side) and that cytochrome f is closer to this site than is cytochrome b₅₅₈.3. A Photosystem II photoreduction of C550 at 77 °K was observed, suggesting that in blue-green algae, as in other plants, C550 is closely associated with the primary electron acceptor for Photosystem II. A Photosystem I photooxidation of P700 at 77 °K was observed, consistent with P700 serving as the primary electron donor of Photosystem I.     

Cytochrome b 6 f complex: Dynamic molecular organization,function and acclimation

The cytochrome b ₆ f complex occupies a central position in photosynthetic electron transport and proton translocation by linking PS II to PS I in linear electron flow from water to NADP⁺, and around PS I for cyclic electron flow. Cytochrome b ₆ f complexes are uniquely located in three membrane domains: the appressed granal membranes, the non-appressed stroma thylakoids and end grana membranes, and also the non-appressed grana margins, in contrast to the marked lateral heterogeneity of the localization of all other thylakoid multiprotein complexes. In addition to its vital role in vectorial electron transfer and proton translocation across the membrane, cytochrome b ₆ f complex is also involved in the regulation of balanced light excitation energy distribution between the photosystems, since its redox state governs the activation of LHC II kinase (the kinase that phosphorylates the mobile peripheral fraction of the chlorophyll a/b-proteins of LHC II of PS II). Hence, cytochrome b ₆ f complex is the molecular link in the interactive co-regulation of light-harvesting and electron transfer.The importance of a highly dynamic, yet flexible organization of the thylakoid membranes of plants and green algae has been highlighted by the exciting discovery that a lateral reorganization of some cytochrome b ₆ f complexes occurs in the state transition mechanism both in vivo and in vitro (Vallon et al. 1991). The lateral redistribution of phosphorylated LHC II from stacked granal membrane regions is accompanied by a concomitant movement of some cytochrome b ₆ f complexes from the granal membranes out to the PS I-containing stroma thylakoids. Thus, the dynamic movement of cytochrome b ₆ f complex as a multiprotein complex is a molecular mechanism for short-term adaptation to changing light conditions. With the concept of different membrane domains for linear and cyclic electron flow gaining credence, it is thought that linear electron flow occurs in the granal compartments and cyclic electron flow is localised in the stroma thylakoids at non-limiting irradiances. It is postulated that dynamic lateral reversible redistribution of some cytochrome b ₆ f complexes are part of the molecular mechanism involved in the regulation of linear electron transfer (ATP and NADPH) and cyclic electron flow (ATP only). Finally, the molecular significance of the marked regulation of cytochrome b ₆ f complexes for long-term regulation and optimization of photosynthetic function under varying environmental conditions, particularly light acclimation, is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - PS Photosystem     

Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b6f Complex in Thermosynechococcus elongatus

The cyanobacterial cytochrome b₆f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b₆f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700⁺ reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b₆f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b₆f complexes with different functions that might be correlated with supercomplex formation.     

Inhibition of energy-transducing functions of chloroplast membranes by lipophilic iron chelators

Lipophilic metal chelators inhibit various energy-transducing functions of chloroplasts. The following observations were made.1. Photophosphorylation coupled to any known mode of electron transfer, i.e. whole-chain noncyclic, the partial noncyclic Photosystem I or Photosystem II reactions, or cyclic, is inhibited by several lipophilic chelators, but not by hydrophilic chelators.2. The light- and dithioerythritol-dependent Mg²⁺-ATPase was also inhibited by the lipophilic chelators.3. Electron transport through either partial reaction, Photosystem I or Photosystem II was not inhibited by lipophilic chelators. Whole-chain coupled electron transport was inhibited by bathophenanthroline, and the inhibition was not reversed by uncouplers. The diketone chelators diphenyl propanedione and nonanedione inhibited the coupled, whole-chain electron transport and the inhibition was reversed by uncouplers, a pattern typical of energy transfer inhibitors.The electron transport inhibition site is localized in the region of plastoquinone → cytochrome f. This inhibition site is consistent with other recent work (Prince et al. (1975) FEBS Lett. 51, 108 and Malkin and Aparicio (1975) Biochem. Biophys. Res. Commun. 63, 1157) showing that a non-heme iron protein is present in chloroplasts having a redox potential near +290 mV. A likely position for such a component to function in electron transport would be between plastoquinone and cytochrome f, just where our data suggests there to be a functional metalloprotein.4. Some of the lipophilic chelators induce H⁺ leakiness in the chloroplast membrane, making interpretation of their phosphorylation inhibition difficult. However, 1–3 mM nonanedione does not induce significant H⁺ leakiness, while inhibiting ATP formation and the Mg²⁺-ATPase. Nonanedione, at those concentrations, causes a two- to four-fold increase in the extent of H⁺ uptake.5. These results are consistent with, but do not prove, the involvement of a non-heme iron or a metalloprotein in chloroplast energy transduction.     

Interaction of flavodoxin with cyanobacterial thylakoids

Flavodoxin from the cyanobacterium Anabaena PCC 7119 has been shown to mediate, under illumination, the transfer of electrons from the thylakoidal membranes that were isolated from the same organism, to both the enzyme ferredoxin-NADP⁺ reductase and cytochrome c. Chemical cross-linking of ferredoxin or flavodoxin to the photosynthetic membranes provides a preparation that is active in cytochrome c photoreduction without the addition of external protein carrier. NADP⁺ photoreduction, albeit diminished, was observed only after addition of exogenous electron carrier protein. Immunoblotting analysis of the chemical adduct reveals that flavodoxin binds to a 10 kDa polypeptide subunit in the cyanobacterial Photosystem I which appears to act as its physiological partner in the electron transfer process.Abbreviations Fd ferredoxin - Fld flavodoxin - cyt c cytochrome c - EDC 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide - PS I Photosystem I     

Electron-transport chain and coupled oxidative phosphorylation in methanol-grown Paracoccus denitrificans

Methanol dehydrogenase of Paracoccus denitrificans was shown to be very similar to the enzyme of Pseudomonas sp, M. 27. The K _m value for methanol with excess activator (ammonium ions) is 35 M. The pH optimum for enzyme activity with 2,6-dichlorophe-nolindophenol as electronacceptor was at 9.0 A CO-binding type of cytochrome c was present only in cells grown with methanol as carbon and energy source.It has been shown that methanol-oxidation involves electron-transport via cytochrome c and an a-type cytochrome to oxygen. Antimycin A did not inhibit this electron transport and 90% inhibition was obtained by 375 M potassium cyanide. Electron transport from endogenous substrates is possible via cytochrome b and possibly cytochrome o to oxygen. Potassium cyanide inhibited 90% of the electron transport via this pathway at a concentration of 1.42 mM. Measurement of respiration-driven proton translocation proved that during oxidation of methanol to formaldehyde by oxygen one mole of adenosine triphosphate is synthesized in the site 3 region of the electron transport chain. The H⁺/O value found confirmed the H⁺/site ratio of 3–4 found in heterotrophic grown cells. During electron transport from endogenous substrates to oxygen there is a possible synthesis of 3 moles of adenosine triphosphate.In heterotrophically grown cells electron transfer to oxygen follows almost only the branch of the respiratory chain containing cytochrome o. In methanol-grown cells the pathway via the a-type cytochrome seems more important.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - EPR electron paramagnetic resonance - S.D. standard deviation - ATP adenosine triphosphate     

Site of salicylaldoxime interaction with photosystem II

The reversible inhibition of Photosystem II by salicylaldoxime was studied in spinach D-10 particles by fluorescence, optical absorption, and electron spin resonance spectroscopy. In the presence of 15 mM salicylaldoxime, the initial fluorescence yield was raised to the level of the maximum fluorescence, indicating efficient charge recombination between reduced pheophytin (Ph) and P680⁺. In agreement with the rapid (ns) backreaction expected between Ph^– and P680⁺, the optical absorption transient at 820 mm was not observed. When the particles were washed free of salicylaldoxime, the optical absorption transient resulting from the rereduction of P680⁺ was restored to the µs timescale. These results, along with the previously observed inhibition of electron transport reactions and diminution of the 515-nm absorption change in chloroplasts [Golbeck, J.H. (1980) Arch Biochem Biophys 202, 458–466], are consistent with a site of inhibition between Ph and QA in Photosystem II. ESR Signal IIf and Signal Its were abolished in the presence of 25 mM salicylaldoxime, but both signals could be recovered by washing the D-10 particles free of the inhibitor. The loss of Signal Ilf is most likely a consequence of the inhibition between Ph and Q_A; the rapid charge recombination between Ph^– and P680⁺ would preclude electron transfer from an electron donor on the oxidizing side of Photosystem II. The loss of Signal Its may be due to a change in the environment of the donor complex such that the semiquinone radical giving rise to Signal Its interacts with a nearby reductant.Abbreviations D₁ electron donor to P680⁺ in oxygen-inhibited chloroplasts - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F₀ prompt chlorophyll a fluorescence yield - F_i initial chlorophyll a fluorescence yield - F_max maximum chlorophyll a fluorescence yield - F_var variable chlorophyll a fluorescence yield - FWHM full width at half maximum - Mes 2-(N-morpholino) ethanesulfonic acid - P680 reaction center chlorophyll a of photosystem II - Ph pheophytin intermediate electron acceptor - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - Tris tris(hydroxymethyl)aminomethane - Z electron donor to P680⁺     

Changes in the mode of electron flow to photosystem I following chilling-induced photoinhibition in a C3 plant, Cucumis sativus L.

This study provides evidence for enhanced electron flow from the stromal compartment of the photosynthetic membranes to P700⁺ via the cytochrome b₆/f complex (Cyt b₆/f) in leaves of Cucumis sativus L. submitted to chilling-induced photoinhibition. The above is deduced from the P700 oxidation–reduction kinetics studied in the absence of linear electron transport from water to NADP⁺, cyclic electron transfer mediated through the Q-cycle of Cyt b₆/f and charge recombination in photosystem I (PSI). The segregation of these pathways for P700⁺ rereduction were achieved by the use of a 50-ms multiple turnover white flash or a strong pulse of white or far-red illumination together with inhibitors. In cucumber leaves, chilling-induced photoinhibition resulted in ∼20% loss of photo-oxidizible P700. The measurement of P700⁺ was greatly limited by the turnover of cyclic processes in the absence of the linear mode of electron transport as electrons were rapidly transferred to the smaller pool of P700⁺. The above is explained by integrating the recent model of the cyclic electron flow in C₃ plants based on the Cyt b₆/f structural data [Joliot and Joliot (2006) Biochim Biophys Acta 1757:362–368] and a photoprotective function elicited by a low NADP⁺/NAD(P)H ratio [Rajagopal et al. (2003) Biochemistry 42:11839–11845]. Over-reduction of the photosynthetic apparatus results in the accumulation of NAD(P)H in vivo to prevent NADP⁺-induced reversible conformational changes in PSI and its extensive damage. As the ferredoxin:NADP reductase is fully reduced under these conditions, even in the absence of PSII electron transport, the reduced ferredoxin generated during illumination binds at the stromal openings in the Cyt b₆/f complex and activates cyclic electron flow. On the other hand, the excess electrons from the NAD(P)H pool are routed via the Ndh complex in a slow process to maintain moderate reduction of the plastoquinone pool and redox poise required for the operation of ferredoxin:plastoquinone reductase mediated cyclic flow.     

Reconstitution of electron transport by cytochrome c-553 in a cell-free system of Nostoc muscorum

Treatment of spheroplasts of Nostoc museorum with hypotonic buffer results in membranes depleted of cytochrome c-553, but still active in photosynthetic and respiratory electron transport. These membranes retain full photosystem II activity (H₂ODAD_ox). Complete linear electron transport (H₂ONADP⁺), however, is decreased as compared with untreated spheroplasts. Addition of basic Nostoc cytochrome c-553 to depleted membranes reconstitutes NADP⁺ reduction and redox reactions of the photosystem I region as well.Using NADPH as electron donor, respiration of depleted membranes is also stimulated by adding cytochrome c-553, indicative of its function in respiratory electron transport.Cytochrome c-553 from Bumilleriopsis filiformis, Spirulina platensis (acidic types), Phormidium foveolarum (basic type), and mitochondrial horse-heart cytochrome c-550 are not effective in reconstituting both photosynthetic and respiratory electron transport, which points to a specific role of Nostoc cytochrome c-553.Abbreviations BSA bovine serum albumin - DAD 3,6-diaminodurene - DAD_ox 3,6-diaminodurene oxidized by potassium ferricyanide - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - Fd ferredoxin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2(-N-morpholino)-ethanesulfonic acid - MV methylviologen (1,1-dimethyl-4,4-bipyridylium dichloride) - PS I photosystem I - PS II photosystem II - Tris tris-(hydroxymethyl)-aminomethane     

Dissection of respiratory and cyclic electron transport in Synechocystis sp. PCC 6803

Cyclic electron transport (CET) is an attractive hypothesis for regulating photosynthetic electron transport and producing the additional ATP in oxygenic phototrophs. The concept of CET has been established in the last decades, and it is proposed to function in the progenitor of oxygenic photosynthesis, cyanobacteria. The in vivo activity of CET is frequently evaluated either from the redox state of the reaction center chlorophyll in photosystem (PS) I, P700, in the absence of PSII activity or by comparing PSI and PSII activities through the P700 redox state and chlorophyll fluorescence, respectively. The evaluation of CET activity, however, is complicated especially in cyanobacteria, where CET shares the intersystem chain, including plastoquinone, cytochrome b₆/f complex, plastocyanin, and cytochrome c₆, with photosynthetic linear electron transport (LET) and respiratory electron transport (RET). Here we sought to distinguish the in vivo electron transport rates in RET and CET in the cyanobacterium Synechocystis sp. PCC 6803. The reduction rate of oxidized P700 (P700⁺) decreased to less than 10% when PSII was inhibited, indicating that PSII is the dominant electron source to PSI but P700⁺ is also reduced by electrons derived from other sources. The oxidative pentose phosphate (OPP) pathway functions as the dominant electron source for RET, which was found to be inhibited by glycolaldehyde (GA). In the condition where the OPP pathway and respiratory terminal oxidases were inhibited by GA and KCN, the P700⁺ reduction rate was less than 1% of that without any inhibitors. This study indicate that the electron transport to PSI when PSII is inhibited is dominantly derived from the OPP pathway in Synechocystis sp. PCC 6803.

     

Respiration in energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus: I. Relation of the respiratory and photosynthetic electron transport

The thermophilic cyanobacterium Mastigocladus laminosus was grown at different CO₂ concentrations and temperatures. Respiratory and photosynthetic electron transport in isolated membranes were measured and their activities were compared. Cells grown at low CO₂ concentration showed respiratory electron transport, whereas Photosystem-II-dependent transport was optimal in cells grown at high CO₂ concentrations. The respiratory electron transport from NADH and succinate were KCN-sensitive, whereas NADPH-dependent O₂ uptake was not. It could be shown that NADH and succinate donate electrons in the photosynthetic electron pathway via Photosystem I. In cytochrome-c-553-depleted membranes added cytochrome c-553 could stimulate photosynthetic and respiratory electron transport. A common electron transport pathway between the quinone and cytochrome c is postulated.