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.     

The effect of quinolines on electron transport and proton gradients associated with photophosphorylation in spinach chloroplasts

The effect of several different quinolines and related compounds was investigated on electron transport and proton translocation associated with photophosphorylation in spinach chloroplasts. It was found that many quinolines, regardless of their chelating ability, strongly inhibited dimethylbenzoquinone reduction by photosystem II (PS II) or the ascorbate + diaminodurene (DAD) → methylviologen (MV) pathway in photosystem I (PS I). Ascorbate + N,N,N¹,N¹-tetramethyl-p-phenylene diamine (TMPD) oxidation by PS I could also be inhibited by some quinolines and related compounds. Three modes of action presumably explain the effects observed: (1) chelation of non-heme irons found in PS II between the plastoquinone pool and cytochrome f, (2) non-specific lipid effect on chloroplast membranes by quinoline-like nitrogen bases and (3) sulfhydryl effects on chloroplast proteins by mercaptoquinoline type substances. The inhibition of proton gradients associated with cyclic and non-cyclic photophosphorylation resulted from removal of protons by the quinolines.     

p-nitroacetophenoxime N-methylcarbamate,a new electron acceptor in isolated thylakoids

p-Nitroacetophenoxime N-methylcarbamate (MCPNA) is a rather potent inhibitor of the electron transfer in spinach class A chloroplasts. In isolated thylakoids, MCPNA is an electron acceptor at the level of photosystem I (PS I). It inhibits O₂ evolution in the presence of NADP and ferredoxin but not the reduction of ferricyanide. MCPNA is active as an acceptor between 3 μM and 100 μM. At concentrations higher than 300 μM, inhibition of photosystem II (PS II) occurs. MCPNA has no uncoupling effect on photophosphorylation. Reduction of MCPNA by thylakoids in the presence of light is in accordance with the E′_o of this compound (??0.57 V) and is followed by an electron transfer to O₂. This reaction probably explains the inhibitory effect of MCPNA on class A chloroplasts.     

Vanadium in photosynthesis of Chlorella fusca and higher plants

The influence of vanadium compounds (vanadate, vanadyl citrate) on photosynthesis in Chlorella fusca and in algal and spinach chloroplasts has been investigated. It was found that: 1. At moderately high concentrations (at least 0.1 mM) both vanadate and vanadyl citrate enhance photosynthetic O₂ production in intact C. fusca cells. At lower V concentration (about 2 μM) only vanadate stimulates photosynthesis. The increase is dependent on culture conditions and on light intensity. 2. Up to 1 mM V, neither vanadium compound influences PS II activity, either in intact cells or in algal or spinach chloroplasts. 3. The PS I reaction in algal and spinach chloroplasts is maximally enhanced (3-fold) in presence of vanadium (20 μM). The increase is independent of light intensity. 4. Cr(VI), Mo(VI), and W(VI) (1 mM) stimulate photosynthesis in intact C. fusca cells, but do not influence the photosystems of isolated chloroplasts. Vanadium is suggested to act as a redox catalyst in the electron transport from PS II to PS I.     

Correlated influence of cation concentration and excitation intensity on PS II activity-II. Comparative study between green plant and brown-alga chloroplasts

A preparation of photochemically active chloroplasts of Fucus was added to a low-salt medium with high osmolarity (HEPES AMPD buffer, 1M sorbitol). The rate of DCIP reduction (DCIPr) and the variable fluorescence (Fv) of these phaeoplasts were measured and compared with the same activities in spinach chloroplasts. A study of the influence of mono- and divalent-cations showed that salt effects on PS II activity also exist in Fucus.

1. Mg⁺⁺ action on Fv is similar, although noticeably weaker in Fucus than in spinach chloroplasts.
2. Na⁺ has no effect on Fv of Fucus chloroplasts, but its influence on DCIPr is more pronounced than in spinach.
3. Mg⁺⁺ influence on DCIPr is largely dependent upon excitation energy. In subsaturating light (100\2-1000 W m^\t-2), Mg⁺⁺ stimulation increases up to 100 mM, almost doubling the level. In very low wight conditions (3Wm^\t02), however, this stimulation saturates at about 10 mM; higher concentrations are no longer effective but do not quench DCIPr noticeably, unlike the case in spinach.

Therefore, cations act through similar pathways on Fucus as on spinach isolated chloroplasts but the effects on PS II centers are preponderant in Fucus whereas the modifications in non-radiative decay or pigment array size are weaker.     

Structural and functional organization of the photosystems in spinach chloroplasts. Antenna size,relative electron-transport capacity,and chlorophyll composition

The structural and functional organization of the spinach chloroplast photosystems (PS) I, II_α and II_β was investigated. Sensitive absorbance difference spectrophotometry in the ultraviolet (?A₃₂₀) and red (?A₇₀₀) regions of the spectrum provided information on the relative concentration of PS II and PS I reaction centers. The kinetic analysis of PS II and PS I photochemistry under continuous weak excitation provided information on the number (N) of chlorophyll (Chl) molecules transferring excitation energy to PS II_α, PS II_β and PS I. Spinach chloroplasts contained almost twice as many PS II reaction centers compared to PS I reaction centers. The number N_α of chlorophyll (Chl) molecules associated with PS II_α was 234, while N_β = 100 and N_{PS I} = 210. Thus, the functional photosynthetic unit size of PS II reaction centers was different from that of PS I reaction centers. The relative electron-transport capacity of PS II was significantly greater than that of PS I. Hence, under light-limiting green excitation when both Chl a and Chl b molecules are excited equally, the limiting factor in the overall electron-transfer reaction was the turnover of PS I. The Chl composition of PS I, PS II_α and PS II_β was analyzed on the basis of a core Chl a reaction center complex component and a Chl $\text{a}\text{b-}\text{LHC}$ component. There is a dissimilar Chl $\text{a}\text{b-}\text{LHC}$ composition in the three photosystems with 77% of total Chl b associated with PS II_α only. The results indicate that PS II_α, located in the membrane of the grana partition region, is poised to receive excitation from a wider spectral window than PS II_β and PS I.     

Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of Mg2+

Single-photon timing with picosecond resolution is used to investigate the effect of Mg²⁺ on the room-temperature fluorescence decay kinetics in broken spinach chloroplasts. In agreement with an earlier paper (Haehnel, W., Nairn, J.A., Reisberg, P. and Sauer, K. (1982) Biochim. Biophys. Acta 680, 161–173), we find three components in the fluorescence decay both in the presence and in the absence of Mg²⁺. The behavior of these components is examined as a function of Mg²⁺ concentration at both the F₀ and the F_max fluorescence levels, and as a function of the excitation intensity for thylakoids from spinach chloroplasts isolated in the absence of added Mg²⁺. Analysis of the results indicates that the subsequent addition of Mg²⁺ has effects which occur at different levels of added cation. At low levels of Mg²⁺ (less than 0.75 mM), there appears to be a decrease in communication between Photosystem (PS) II and PS I, which amounts to a decrease in the spillover rate between PS II and PS I. At higher levels of Mg²⁺ (about 2 mM), there appears to be an increase in communication between PS II units and an increase in the effective absorption cross-section of PS II, probably both of these involving the chlorophyll $\text{a}\text{b}$ light-harvesting antenna.     

Evidence for alpha-Tocopherol Function in the Electron Transport Chain of Chloroplasts

The effect of three different stable radicals-2,2-diphenyl-1-picrylhydrazyl, 1,3,5-triphenyl-verdazyl, and galvinoxyl-was studied in photosystem II of spinach (Spinacia oleracea) chloroplasts. Inhibition by the three was noted on dimethylbenzoquinone reduction in presence of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and on silicomolybdate reduction in presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in photosystem II and on the H₂O → methylviologen reaction encompassing both photosystems. Inhibition of all photosystem II reactions except silicomolybdate reduction could be partially restored by α-tocopherol or by 9-ethoxy-α-tocopherone but not by other quinones or radical chasers. On this basis, a functional role for α-tocopherol in the electron transport chain of spinach chloroplasts between the DCMU and DBMIB inhibition sites is postulated.     

Cobalt ions inhibit electron-transport activity of Photosystem II without affecting Photosystem I

Investigations on photosynthesis have greatly benefited by the use of specific inhibitors that affect a specific site of inhibition on the electron-transport chain. We show here for the first time that cobalt (Co²⁺) ions can be used specifically to inactivate electron donation to the reaction centre of Photosystem (PS) II without affecting PS I reactions. This conclusion is based on the following observations: (1) addition of exogenous electron donors such as NH₂OH does not relieve Co²⁺-induced inactivation of photoelectron transport or the lowering of steady-state chlorophyll a fluorescence yield; this suggests that the inhibition is beyond the NH₂OH donation site and before the fluorescence quencher Q, i.e., on the reaction centre complex itself. (2) Washing of Co²⁺-pretreated chloroplasts with isolation buffer to remove Co²⁺ does not relieve Co²⁺-induced inhibition of Hill activity, suggesting that the Co²⁺ effect is irreversible. (3) Co²⁺ did not alter the PS I reactions. Thus, Co²⁺-treated chloroplasts can be used to study PS I functions free from PS II reactions in isolated chloroplasts.     

Uptake of l-Ascorbate by Intact Spinach Chloroplasts

Uptake of l-[1-¹⁴C]ascorbate by intact ascorbate-free spinach (Spinacia oleracea L. cv Vital^r) chloroplasts has been investigated using the technique of silicone oil filtering. Rates greater than 100 micromoles per milligram chlorophyll per hour (external concentration, 10 millimolar) of ascorbate transport were observed. Ascorbate uptake into the sorbitol-impermeable space (stroma) followed the Michaelis-Menten-type characteristic for substrate saturation. A K_m of 18 to 40 millimolar was determined. Transport of ascorbate across the chloroplast envelope resulted in an equilibrium of the ascorbate concentrations between stroma and medium. A pH optimum of 7.0 to 7.5 and the lack of alkalization of the medium upon ascorbate uptake suggest that only the monovalent ascorbate anion is able to cross the chloroplast envelope. The activation energy of ascorbate uptake was determined to be 65.8 kilojoules (16 kilocalories) per mole (8 to 20°C). Interference of ascorbate transport with substrates of the phosphate or dicarboxylate translocator could not be detected, but didehydroascorbate was a competitive inhibitor. Preloading of chloroplasts with didehydroascorbate resulted in an increase of V_max but did not change the K_m for ascorbate. Millimolar concentrations of the sulfhydryl reagent p-chloromercuriphenyl sulfonate inhibited ascorbate uptake. The data are interpreted in terms of ascorbate uptake into chloroplasts by the mechanism of facilitated diffusion mediated by a specific translocator.     

Evidence for multiple sites of ferricyanide reduction in chloroplasts

Various sites of ferricyanide reduction were studied in spinach chloroplasts. It was found that in the presence of dibromothymoquinone a fraction of ferricyanide reduction was dibromothymoquinone sensitive, implying that ferricyanide can be reduced by photosystem I as well as photosystem II. To separate ferricyanide reduction sites in photosystem II, orthophenanthroline and dichlorophenyl dimethylurea inhibitions were compared at various pH's. It was noted that at low pH ferricyanide reduction was not completely inhibited by orthophenanthroline. At high pH's, however, inhibition of ferricyanide reduction by orthophenanthroline was complete. It was found that varying concentration of orthophenanthroline at a constant pH showed different degrees of inhibition. In the study of ferricyanide reduction by photosystem II various treatments affecting plastocyanin were performed. It was found that Tween-20 or KCN treatments which inactivated plastocyanin did not completely inactivate ferricyanide reduction. These data support the conclusion that ferricyanide accepts electrons both before and after plastoquinone in photosystem II.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyurea - MV methyl viologen - DBMIB 2,5-dibromothymoquinone - DMBQ 2,6-dimethyl benzoquinone - OP 1,10-orthophenanthroline - TMPD tetramethyl-p-phenylenediamine - PS 1 photosystem I - PS II photosystem II - SN sucrose-sodium chloride chloroplasts Supported by NSF Grant BMS 74-19689.     

Inhibitory effect of p-nitrothiophenol in the light on the Photosystem II activity of spinach chloroplasts

The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH's caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiophenol to causing the specific inhibition of Photosystem II.     

Flash-induced carotenoid radical cation formation in Photosystem II

Light excitation of chloroplasts at low temperature produces absorption changes (ΔA) with a large positive peak at 990 nm and a bleaching around 480 nm. ΔA at 990 nm rises with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.6}\text{ms}$ at 20–77 K and remains largely stable. This signal is not observed when Photosystem II (PS II) photochemistry is blocked by reduction of the primary plastoquinone. It is observed also in purified PS II particles, in which case it could be shown that during a sequence of short flashes, the absorption at 990 nm rises in parallel with plastoquinone reduction measured at 320 nm. In chloroplasts the light-induced 990-nm ΔA at 77 K is increased under oxidizing conditions (addition of ferricyanide) and upon addition of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT2p). At 21°C, flash excitation of chloroplasts or of PS II particles induces only a very small ΔA at 990 nm, even when this is measured with a 100-ns time resolution or when the material is preilluminated. In both materials, however, a large flash-induced ΔA takes place when various lipophilic anions are added. After a flash the signal rises in less than 100 μs and its decay varies with experimental conditions; the decay is strongly accelerated by benzidine. The difference spectrum measured in PS II particles includes a broad peak around 990 nm and a bleaching around 490 nm. These absorption changes are attributed to a carotenoid radical cation formed at the PS II reaction center. It is estimated that in the presence of lipophilic anions at room temperature, one cation can be formed by a single flash in 80% of the reaction centers. At cryogenic temperature approx. 8% of the PS II reaction centers can oxidize a carotenoid after a single flash.     

Measurement of degree of chlorophyll fluorescence polarization in relation to the regulation of excitation energy transfer between Photosystems I and II in pea chloroplasts

The degree of chlorophyll fluorescence polarization (p) at 740 nm was measured at room temperature for pea chloroplasts subjected to various conditions. (1) In agreement with previous published observations, p decreased when the Photosystem (PS) II traps were closed by illumination in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. (2) Under these conditions, the magnitude of p was also sensitive to the presence of salts. Under conditions when ‘spillover’ of the excitation energy from PS II to PS I was low, p was also low, being consistent with increased migration of energy between the PS II light-harvesting chlorophylls. In contrast, when spillover was at a maximum p increased. (3) The change in p in the presence of salts was dependent on the concentration and valency of the cations in such a way as to suggest the changes were mediated through electrostatic forces. The dependency of p on ionic composition of the experimental medium was closely related to the associated changes in fluorescence yield. (4) Membrane stacking, caused by lowering pH of the chloroplast suspension, did not induce a significant change in p, suggesting that this pH-induced process is different from the membrane stacking brought about by manipulating the salt levels. (5) Incubation of thylakoids with ATP induces light-dependent phosphorylation of the light-harvesting chlorophyll-protein complexes, and regulates excitation energy transfer between PS I and PS II (Bennett, J., Steinback, K.R. and Arntzen, C.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5253–5257). Under conditions which bring about this phosphorylation it was found that p increased to a value indicative of spillover.     

Light-induced redox changes of cytochrome b-559

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

Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77 K II. ATP-dependent quenching

In intact, uncoupled type B chloroplasts from spinach, added ATP causes a slow light-induced decline ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{min}$) of chlorophyll a fluorescence at room temperature. Fluorescence spectra were recorded after fast cooling to 77 K and normalized with fluorescein as an internal standard. Related to the fluorescence quenching at room temperature, an increase in Photosystem (PS) I fluorescence (F₇₃₅) and a decrease in PS II fluorescence (F₆₉₅) were observed in the low-temperature spectra. The change in the $\text{F}{}_{735}\text{F}{}_{695}$ ratio was abolished by the presence of methyl viologen. Fluorescence induction at 77 K of chloroplasts frozen in the quenched state showed lowered variable (F_v) and initial (F₀) fluorescence at 690 nm and an increase in F₀ at 735 nm. The results are interpreted as indicating an ATP-dependent change of the initial distribution of excitation energy in favor of PS I, which is controlled by the redox state of the electron-transport chain and, according to current theories, is caused by phosphorylation of the light-harvesting complex.     

Photoreactions of cytochrome b-559 and cyclic electron flow in Photosystem II of intact chloroplasts

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO₂ showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.     

Comparison of photosynthetic activities of spinach chloroplasts with those of corn mesophyll and corn bundle sheath tissue

Bundle sheath and mesophyll chloroplasts from Zea mays showed comparable rates of O₂ evolution, which amounted to about half of the rate observed in spinach (Spinacia oleracea) chloroplasts.

Ratios of 4.5, 4.6, and 6.2 Mn²⁺ atoms per 400 chlorophylls were observed in mesophyll, bundle sheath, and spinach chloroplasts, respectively. These ratios roughly correspond to the observed O₂ evolution rates.

Rates of electron transport from water to methylviologen (photosystem I and II) in both types of corn chloroplasts were about one-third that in spinach. Compared to spinach, transport rates from reduced diaminodurene to methylviologen (photosystem I) were about one-third and greater than one-half in mesophyll and bundle sheath material, respectively.

In both types of corn chloroplasts, electron flow from photosystem II to P700 was abnormal. This observation, together with the low rates of all activities, suggests that damage occurred during isolation. Such damage may limit the quantitative significance of observations made with these materials (including the following data).

Measurements of flash yields of O₂ evolution or O₂ uptake showed that the size of the photosynthetic unit was the same in photosystems I and II and in all three types of chloroplasts (about 400 chlorophylls per equivalent).

Similarity of the photochemical cross-section of the two photosystems in the three preparations was also found in optical experiments: that is the half-times of the fluorescence rise in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (photosystem II) and of the photooxidation of P700 (photosystem I).

The ratio of P700 to chlorophyll appeared to be about 2-fold higher in bundle sheath chloroplasts than in the other materials (1/200 versus 1/400).

     

Covalent modification of chloroplast Photosystem II polypeptides by p-nitrothiophenol

Illumination of the chlorophyll $\text{a}\text{b}$ light-harvesting complex in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused quenching of fluorescence emission at 685 nm (77 K) relative to 695 nm and covalent modification of light-harvesting complex polypeptides. Fluorescence quenching saturated with one p-nitrothiophenol bound per light-harvesting complex polypeptide (10–13 chlorophylls); $\text{1}\text{2}$ maximal quenching occurred with one p-nitrothiophenol bound per light-harvesting complex polypeptides (190–247 chlorophylls). This result provides direct evidence for excitation energy transfer between light-harvesting complex subunits which contain 4–6 polypeptides plus 40–78 chlorophylls per complex.Illumination of chloroplasts or Photosystem II (PS II) particles in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused inhibition of PS II activity and labeling of several polypeptides including those of 42–48 kilodaltons previously identified as PS II reaction center polypeptides. In chloroplasts, inhibition of oxygen evolution accelerated p-nitrothiophenol modification reactions; DCMU or donors to PS II decreased p-nitrothiophenol modification. These results are consistent with the hypothesis that accumulation of oxidizing equivalents on the donor side of PS II creates a ‘reactive state’ in which polypeptides of PS II are susceptible to p-nitrothiophenol modification.     

Organization of Electron Transport in Photosystem II of Spinach Chloroplasts According to Chelator Inhibition Sites

The organization of electron transport in photosystem II of spinach (Spinacia oleracea) chloroplasts was studied by means of various chelators and uncouplers. The partial reactions used included H₂O→methyl viologen, H₂O→silicomolybdic acid H₂O→ferricyanide, and H₂O→dimethylbenzoquinone. Three types of chelator inhibition were found (a) inhibition common to all pathways and presumably affecting the Mn or water oxidation site in photosystem II (salicylaldoxime, dithizone, acridine, 4,4,4-trifluoro-1-(2-thienyl)-1,1-butanedione, 4,4,4-trifluoro-0-(2-furyl)-1,3-butanedione; (b) strong inhibition of the H₂O→silicomolybdic acid pathway in presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea by lipophilic chelators (bathocuproine, tertoctylcatechol) but stimulation by orthophenanthroline; and (c) 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-insensitive dimethylbenzoquinone reduction inhibited by all phenanthrolines while ferricyanide reduction was remarkably stimulated by bathophenanthroline but inhibited by orthophenanthroline and bathocuproine. The action of lipophilic chelators on silicomolybdic acid reduction presumes the presence of a metallo protein in photosystem II. The differential action of bathophenanthroline on dimethylbenzoquinone and ferricyanide reduction indicated the possible existence of a metalloprotein in this pathway which is different from the site of orthophenanthroline inhibition.