Interactions between thylakoid electron transfer complexes. II. Modification studies with glutaraldehyde

Photosystem I (PSI) and photosystem II (PSII) complexes have been isolated from stacked spinach thylakoid membranes that had been treated with varying amounts of glutaraldehyde. The concentrations of cytochrome f, Q, and P700 have been determined by spectrophotometric methods. It was found that at low concentrations of glutaraldehyde, the amount of cytochrome f associated with either PSII or PSI increased significantly while the amounts of Q and P700 stayed relatively constant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analyses indicated the presence of cytochrome f and other components of the cytochrome b6-f complex in the PSII and PSI preparations after glutaraldehyde treatment, but no intermolecular cross-linked polypeptides could be detected. Solubilization of the cytochrome b6-f complex was also inhibited after thylakoid membranes were treated with low concentrations of glutaraldehyde. These results are discussed in relation to current models for the organization of the membrane complexes, and relate to the location of the cytochrome b6-f complex in appressed and nonappressed membrane regions of thylakoids.     

Differential detergent-solubilization of integral thylakoid membrane complexes in spinach chloroplasts. Localization of photosystem II, cytochrome b6-f complex and photosystem I

Progressive solubilization of spinach chloroplast thylakoids by Triton X-100 was employed to investigate the domain organization of the electron transport complexes in the thylakoid membrane. Triton/chlorophyll ratios of 1:1 were sufficient to disrupt fully the continuity of the thylakoid membrane network, but not sufficient to solubilize either photosystem I (PSI), photosystem II (PSII) or the cytochrome b6-f(Cyt b6-f) complex. Progressive with the Triton concentration increase (Triton/Chl greater than 1:1), a differential solubilization of the three electron transport complexes was observed. Solubilization of the Cyt b6-f complex from the thylakoid membrane preceded that of PSI and apparently occurred early in the solubilization of stroma-exposed segments of the chloroplast lamellae. The initial removal of chlorophyll (up to 40% of the total) occurred upon solubilization of PSI from the stroma-exposed lamella regions in which PSI is localized. The tightly appressed membrane of the grana partition regions was markedly resistant to solubilization by Triton X-100. Thus, solubilization of PSII from this membrane region was initiated only after all Cyt b6-f and PSI complexes were removed from the chloroplast lamellae. The results support the notion of extreme lateral heterogeneity in the organization of the electron transport complexes in higher plant chloroplasts and suggest a Cyt b6-f localization in the membrane of the narrow fret regions which serve as a continuum between the grana and stroma lamellae.     

Topographical studies of the polypeptide subunits of the thylakoid cytochrome b6-f complex

The orientation of specific polypeptides of the cytochrome b6-f complex with respect to the chloroplast stromal phase has been studied using trinitrobenzenesulfonate (TNBS) and pronase E as impermeant modifying reagents. Of the four polypeptides of the complex (33,23,20 and 17 kDa), only cytochrome f was labeled by 14C-TNBS in unfractionated membranes. However, to a varying degree, all of the constituent polypeptides were sensitive to pronase digestion and, in the case of cytochrome f, it was possible, by immunoblotting techniques to identify several degradation products. These results are discussed in relation to the organization of the cytochrome complex in thylakoid membranes and argue for an exposure to the stromal phase of all of the polypeptides, while functional considerations indicate that at least cytochrome f and the Rieske iron-sulfur protein have a possible transmembrane organization.     

Reevaluation of the stoichiometry of cytochrome b559 in photosystem II and thylakoid membranes.

The stoichiometry of cytochrome b559 (one or two copies) per reaction center of photosystem II (PSII) has been the subject of considerable debate. The molar ratio of cytochrome b559 has a number of significant implications on our understanding of the functional role of cytochrome b559, the mechanism of electron donation in PSII, and the stoichiometry of the other redox-active, reaction center components. We have reinvestigated the stoichiometry of cytochrome b559 in PSII-enriched and thylakoid membranes, using differential absorbance and electron paramagnetic resonance spectroscopies. The data from both quantitation procedures strongly indicate only one copy of cytochrome b559 per reaction center in PSII-enriched membranes and also suggest one copy of cytochrome b559 per reaction center in thylakoid membranes.     

Energy transfer between photosystem II and photosystem I in chloroplasts.

A model for the photochemical apparatus of photosynthesis is presented which accounts for the fluorescence properties of Photosystem II and Photosystem I as well as energy transfer between the two photosystems. The model was tested by measuring at - 196 degrees C fluorescence induction curves at 690 and 730 nm in the absence and presence of 5mMMgCl2 which presumably changes the distrubution of excitation energy between the two photosystems. The equations describing the fluorescence properties involve terms for the distribution of absorbed quanta, alpha, being the fraction distributed to Photosystem I, and beta, the fraction to Photosystem II to Photosystem I, KT(II yields I). The data, analyzed within the context of the model, permit a direct comparison of alpha and kt(II yields I) in the absence (minus) and presence (+) of Mg-2+ :alpha minus/alpha-+ equals 1.2 and k-minus t)II yields I)/K-+T(II yields I) equal to 1.9. If the criterion that alpha + beta equal to 1 is applied absolute values can be calculated: in the presence of Mg-2+, alpha-+ equal to 0.27 and the yield of energy transfer, phi-+ t(II yields I) varied the presence of Mg-2+, alpha-+ equal to 0.27 and the yield of energy transfer, phi-+ t(II yields I) varied from 0.065 when the Photosystem II reaction centers were all open to 0.23 when they were closed. In the absence of Mg-2+, alpha-minus equal to 0.32 and phi t(II yields I) varied from 0.12 to 0.28. The data were also analyzed assuming that two types of energy transfer could be distinguished; a transfer from the light-harvesting chlorophyll of Photosystem II to Photosystem I, kt(II yields I), and a transfer from the reaction centers of Photosystem II to Photosystem I, kt(II yields I). In that case alpha-minus/alpha+ equal to 1.3, k-minus t(II yields I)/k+ t(II yields I)equal to 1.3 and k-minus t(II yields I) equal to 3.0. It was concluded, however, that both of these types of energy transfer are different manifestations of a single energy transfer process.     

The thermodynamics and kinetics of electron transfer between cytochrome b6f and photosystem I in the chlorophyll d-dominated cyanobacterium, Acaryochloris marina

We have investigated the photosynthetic properties of Acaryochloris marina, a cyanobacterium distinguished by having a high level of chlorophyll d, which has its absorption bands shifted to the red when compared with chlorophyll a. Despite this unusual pigment content, the overall rate and thermodynamics of the photosynthetic electron flow are similar to those of chlorophyll a-containing species. The midpoint potential of both cytochrome f and the primary electron donor of photosystem I (P(740)) were found to be unchanged with respect to those prevailing in organisms having chlorophyll a, being 345 and 425 mV, respectively. Thus, contrary to previous reports (Hu, Q., Miyashita, H., Iwasaki, I. I., Kurano, N., Miyachi, S., Iwaki, M., and Itoh, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13319-13323), the midpoint potential of the electron donor P(740) has not been tuned to compensate for the decrease in excitonic energy in A. marina and to maintain the reducing power of photosystem I. We argue that this is a weaker constraint on the engineering of the oxygenic photosynthetic electron transfer chain than preserving the driving force for plastoquinol oxidation by P(740), via the cytochrome b(6)f complex. We further show that there is no restriction in the diffusion of the soluble electron carrier between cytochrome b(6)f and photosystem I in A. marina, at variance with plants. This difference probably reflects the simplified ultrastructure of the thylakoids of this organism, where no segregation into grana and stroma lamellae is observed. Nevertheless, chlorophyll fluorescence measurements suggest that there is energy transfer between adjacent photosystem II complexes but not from photosystem II to photosystem I, indicating spatial separation between the two photosystems.     

Supercomplexes of plant photosystem I with cytochrome b6f,light-harvesting complex II and NDH

Photosystem I (PSI) is a pigment-protein complex required for the light-dependent reactions of photosynthesis and participates in light-harvesting and redox-driven chloroplast metabolism. Assembly of PSI into supercomplexes with light harvesting complex (LHC) II, cytochrome b₆f (Cytb₆f) or NAD(P)H dehydrogenase complex (NDH) has been proposed as a means for regulating photosynthesis. However, structural details about the binding positions in plant PSI are lacking. We analyzed large data sets of electron microscopy single particle projections of supercomplexes obtained from the stroma membrane of Arabidopsis thaliana. By single particle analysis, we established the binding position of Cytb₆f at the antenna side of PSI. The rectangular-shaped Cytb₆f dimer binds at the side where Lhca1 is located. The complex binds with its short side rather than its long side to PSI, which may explain why these supercomplexes are difficult to purify and easily disrupted. Refined analysis of the interaction between PSI and the NDH complex indicates that in total up to 6 copies of PSI can arrange with one NDH complex. Most PSI-NDH supercomplexes appeared to have 1–3 PSI copies associated. Finally, the PSI-LHCII supercomplex was found to bind an additional LHCII trimer at two positions on the LHCI side in Arabidopsis. The organization of PSI, either in a complex with NDH or with Cytb₆f, may improve regulation of electron transport by the control of binding partners and distances in small domains.     

Heat-induced changes in photosystem I activity as measured with different electron donors in isolated spinach thylakoid membranes.

Heat-induced changes in photosystem I (PSI) have been studied in terms of rates of oxygen consumption using various donors (DCPIPH2, TMPDred and DADred), formation of photo-oxidized P700 and changes in Chl a fluorescence emission at 77 K. Linear heating of thylakoid membranes from 35 degrees C to 70 degrees C caused an enhancement in PSI-mediated electron transfer rates (DCPIPH2-->MV) up to 55 degrees C. However, no change was observed in PSI rates when other electron donors were used (TMPDred and DADred). Similarly, Chl a fluorescence emission spectra at 77 K of heat-treated thylakoid membranes did not show any increase in peak at 735 nm, however, a significant decrease was observed as a function of temperature in the peaks at 685 and 694 nm. In DCMU-treated control thylakoid membranes maximum photo-oxidized P700 was generated at g = 2.0025. In heat-treated thylakoid membranes maximum intensity of photo-oxidized P700 signal was observed at approximately 50-55 degrees C without DCMU treatment. The steady-state signal of the photo-oxidized P700 was studied in the presence of DCPIPH2 and TMPDred as electron donors in DCMU-treated control and in 50 degrees C treated thylakoid membranes. We present here the first of such comparative study of PSI activity in terms of the rates of oxygen consumption and re-reduction kinetics of photo-oxidized P700 in the presence of different electron donors. It appears that the formation of the P700+ signal in heat-treated thylakoid membranes is due to an inhibited electron supply from PSII and not due to spillover or antenna migration.     

Mapping the electron transfer interface between cytochrome b5 and cytochrome c

To characterize the cytochrome b(5) (Cyt b(5))-cytochrome c (Cyt c) interactions during electron transfer, variants of Cyt b(5) have been employed to assess the contributions of electrostatic interactions (substitution of surface charged residues Glu44, Glu48, Glu56, and Asp60 and heme propionate), hydrophobic interactions, and the thermodynamic driving forces (substitutions for hydrophobic residues in heme pocket residues Phe35, Pro40, Val45, Phe58, and Val61). The electrostatic interactions play an important role in maintaining the stability and specificity of the Cyt b(5)-Cyt c complex that is formed. There is no essential effect on the intraprotein complex electron transfer even if most of the involved negatively charged residues on the surface of Cyt b(5) have been removed. The results support a dynamic docking paradigm for Cyt b(5)-Cyt c interactions. The orientation that is optimal for binding may not be optimal form for electron transfer. Substitution of hydrophobic residues does not have a significant effect on the binding between Cyt b(5) and Cyt c; rather, it regulates the electron transfer rates via changes in the driving force. Combining the electron transfer studies of the Cyt b(5)-Cyt c system and the Cyt b(5)-Zn-Cyt c system, we obtain the reorganization energy (0.6 eV) at an ionic strength of 150 mM.     

Inhibition of electron transport at the cytochrome b(6)f complex protects photosystem II from photoinhibition

Photoinhibition of photosystem II (PS II) activity was studied in thylakoid membranes illuminated in the presence of the inhibitor of the cytochrome b(6)f complex 2'iodo-6-isopropyl-3-methyl-2',4, 4'-trinitrodiphenylether (DNP-INT). DNP-INT was found to decrease photoinhibition. In the absence of DNP-INT, anaerobosis, superoxide dismutase and catalase protected against photoinhibition. No effect of these treatments was observed in the presence of DNP-INT. These data demonstrate that photoinhibition under these conditions is caused by reactive oxygen species which are formed most probably by the reduction of oxygen at photosystem I. The results are discussed in terms of the importance of photosynthetic control in protection against photoinhibition in vivo.     

Dynamic docking and electron transfer between myoglobin and cytochrome b 5

The interaction of trypsin-digested bovine cytochrome b(5) (cyt b(5)) with horse heart myoglobin (Mb) and the interprotein electron transfer (ET) between these redox partners have been studied to gain better understanding of ET processes between weakly bound protein partners. The bimolecular rate constant ( k(2)) for photo-induced ET between zinc-substituted Mb (ZnMb) and cyt b(5) decreases with increasing ionic strength, consistent with the predominantly electrostatic character of this complex. The formation of a protein-protein complex has been confirmed and the binding affinities of metMb and ZnMb for cyt b(5) have been measured by two techniques: (1)H NMR titrations at pH 6.0 give binding constants of K(a) approximately (1.0+/-0.1)x10(3) M(-1) for metMb and K(a) approximately (0.75+/-0.1)x10(3) M(-1) for ZnMb; isothermal calorimetry gives K(a) approximately (0.35+/-0.1)x10(3) M(-1) for ZnMb. Brownian dynamic (BD) simulations show that cyt b(5) binds over a broad surface of Mb that includes its heme edge. The experimental results are described in terms of a dynamic docking model which proposes that Mb binds cyt b(5) in a large ensemble of protein binding conformations, not one or a few dominant ones, but that only a small subset are ET reactive. Aided by the BD simulations, this model explains why k(2) decreases with increasing pH: increasing pH not only weakens the binding affinity but also reduces the number of binding conformations with high ET reactivity.     

Correlation between thylakoid stacking and cation-induced decrease in photosystem I electron transport at saturating light intensity

A Mg²⁺-induced decrease of the rate of photosystem I (PS I) electron transport (DCIPH₂ → methyl viologen) in thylakoids under saturated light intensities has been reported earlier (S. Bose, J. E. Mullet, G. E. Hoch, and C. J. Arntzen, 1981, Photobiochem. Photobiophys.2, 45–52). A similar effect is observed with Na⁺, although the concentration required for half-maximal inhibition was higher by about two orders of magnitude. The cation effect was gradually abolished as the thylakoids were aged by incubation at 30 °C for 6 h. The loss of cation effect on PS I electron transport rate during aging was parallel to the corresponding loss of cation effect on thylakoid stacking. The cation concentration required for thylakoid stacking and the degree of inhibition as a function of cation concentration correlated strongly with the degree of thylakoid stacking. These observations indicated that the inhibition of the rate of PS I electron transport by cations is a consequence of cation-induced stacking of thylakoid membranes. The observed inhibition of the rate of PS I electron transport is discussed in terms of two hypotheses: (i) a fraction (20–30%) of the PS I complexes is trapped in the appressed region of grana and becomes unavailable to the electron donor (DCIPH₂) and (ii) the membrane structure is altered by the cations in such a manner that the rate constant of electron donation by the donor to the electron transport chain in the thylakoid is decreased.     

Light-induced alteration of low-temperature interprotein electron transfer between photosystem I and flavodoxin

Electron paramagnetic resonance (EPR) was used to study light-induced electron transfer in Photosystem I-flavodoxin complexes. Deuteration of flavodoxin enables the signals of the reduced flavin acceptor and oxidized primary donor, P(700)(+), to be well-resolved at X- and D-band EPR. In dark-adapted samples, photoinitiated interprotein electron transfer does not occur at 5 K. However, for samples prepared in dim light, significant interprotein electron transfer occurs at 5 K and a concomitant loss of the spin-correlated radical pair P(+)A(1A)(-) signal is observed. These results indicate a light-induced reorientation of flavodoxin in the PSI docking site that allows a high quantum yield efficiency for the interprotein electron transfer reaction.     

Light-induced voltage changes associated with electron and proton transfer in photosystem II core complexes reconstituted in phospholipid monolayers.

We have measured light-induced voltage changes (electrogenic events) in photosystem II (PSII) core complexes oriented in phospholipid monolayers. These events are compared to those measured in the functionally and structurally closely related reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides. In both systems we observed a rapid (< 100 ns) light-induced increase in voltage associated with charge separation. In PSII reaction centers it was followed by a decrease (decay) of approximately 14% of the charge-separation voltage and a time constant of approximately 500 microseconds. In bacterial reaction centers this decay was approximately 9% of the charge-separation voltage, and the time constant was approximately 200 microseconds. The decay was presumably associated with a structural change. In bacterial reaction centers, in the presence of excess water-soluble cytochrome c2+, it was followed by a slower increase of approximately 30% of the charge-separation voltage, associated with electron transfer from the cytochrome to the oxidized donor, P+. In PSII reaction centers, after the decay the voltage remained on the same level for > or = 0.5 s. In PSII reaction centers the electron transfer Q-AQB-->QA Q-B contributed with an electrogenicity of < or = 5% of that of the charge separation. In bacterial reaction centers this electrogenicity was < or = 2% of the charge-separation electrogenicity. Proton transfer to Q2-B in PSII reaction centers contributed with approximately 5% of the charge-separation voltage, which is approximately a factor of three smaller than that observed in bacterial reaction centers.     

Electron transfer between soluble and immobilized mammalian cytochrome c. Equilibrium and kinetic studies on immobilized cytochrome c.

Horse heart cytochrome c was covalently bound to Sepharose 4B and its redox properties were measured under various experimental conditions. The equilibrium constant for the electron exchange between the oxidized and the reduced form of cytochrome c when one of the two forms was in the semi-solid state and the other one in solution was close to 1. Matrix-bound ferrocytochrome c is very stable to autoxidation and is not oxidized by O2 even in the presence of mammalian cytochrome oxidase. Oxidation occurs if catalytic amounts of soluble cytochrome c are added to the reaction mixture. The rate of oxidation of matrix-bound ferrocytochrome c in the presence of cytochrome oxidase and catalytic amounts of soluble cytochrome c may be correlated with the rate of electron transfer between soluble and matrix-bound cytochrome c. This rate is more than two orders of magnitude lower than that reported for the homonuclear (between identical species) electron transfer in solution.     

Studies on cytochromes in photosynthetic electron transport system I. photoreduction and photo-oxidation of cytochrome b559 by photosystem II in spinach chloroplasts

Light-induced redox-reactions of cytochrome b₅₅₉ in spinachchloroplasts were investigated. Illumination of chloroplastsinduced photoreduction of cytochrorne b₅₅₉ Red light (650 nm)was more effective than far-red light (725 nm), indicating thatthe photoreduction is a photosystem II-mediated reaction. Onaddition of DCMU, the photoreduction was eliminated and a photooxidationof cytochrome b₅₅₉ was observed. The rate of this photooxidationwas faster with photosystem II light than with photo-systemI light. On addition of Mn⁺⁺ the photooxidation was partly suppressed;far-red light became as effective as red light in inducing photooxidationof cytochrome b₅₉₉, in the presence of DCMU and Mn⁺⁺. Ascorbate completely suppressed photooxidation of cytochromeb⁵⁵⁹ In the presence of ascorbate, however, photooxidation wasobserved in the presence of inhibitors or after inhibitory treatmentsof chloroplasts which affected the oxidizing side of systemII. These inhibitors and inhibitory treatments, but not DCMU,decreased the redoxpotential of cytochrome b₅₅₉. Reactivationof Hill reaction in Tris-washed chloroplasts by indophenol-ascorbatetreatment was not accompanied by an abolishment of photooxidationof cytochrome b₅₅₉. A possible mechanism is proposed to account for these reactionsof cytochrome b₅₅₉ in the photosynthetic electron transportin chloroplasts. (Received April 4, 1972; )     

Flash-photolysis studies of the electron transfer from genetically modified spinach plastocyanin to photosystem I.

Plastocyanin (Pc) has been modified by site-directed mutagenesis at two separate electron-transfer (ET) sites: Leu-12-Glu at a hydrophobic patch, and Tyr-83-His at an acidic patch. The reduction potential at pH 7.5 is decreased by 26 mV in Pc(Leu-12-Glu) and increased by 35 mV in Pc(Tyr-83-His). The latter mutant shows a 2-fold slower intracomplex ET to photosystem I (PSI) as expected from the decreased driving force. The affinity for PSI is unaffected for this mutant but is drastically decreased for Pc(Leu-12-Glu). It is concluded that the hydrophobic patch is more important for the ET to PSI.