Regulation of photosynthetic electron transport by bicarbonate formate and herbicides in isolated broken and intact chloroplasts

In this paper, we have presented a minireview on the interaction of bicarbonate, formate and herbicides with the thylakoid membranes.The regulation of photosynthetic electron transport by bicarbonate, formate and herbicides is described. Bicarbonate, formate, and many herbicides act between the primary quinone electron acceptor Q_A and the plastoquinone pool. Many herbicides like the ureas, triazines and the phenol-type herbicides act, probably, by the displacement of the secondary quinone electron acceptor Q_B from its binding site on a Q_B-binding protein located at the acceptor side of Photosystem II. Formate appears to be an inhibitor of electron transport; this inhibition can be removed by the addition of bicarbonate. There appears to be an interaction of the herbicides with bicarbonate and/or It has been suggested that both the binding of a herbicide and the absence of bicarbonate may cause a conformational alteration of the environment of the Q_B-binding site. The alteration brought about by a herbicide decreases the affinity for another herbicide or for bicarbonate; the change caused by the absence of bicarbonate decreases the affinity for herbicides. Moreover, this change in conformation causes an inhibition of electron transport. A bicarbonate-effect in isolated intact chloroplasts is demonstrated.Paper presented at the FESPP meeting (Strasbourg, 1984)     

Mechanism of bicarbonate action on photosynthetic electron transport in broken chloroplasts

In CO2-depleted chloroplasts electron transport between the Photosystem II electron acceptor Q and plastoquinone is largely suppressed. In the presence of a high concentration of sodium formate (greater than 10 mM), which probably binds to the bicarbonate site, addition of bicarbonate restores the ferricyanide Hill reaction only after incubation in the dark. With lower formate concentrations bicarbonate is able to restore electron transport in the light. The Hill reaction rate in CO2-depleted chloroplasts after bicarbonate addition, divided by the rate in CO2-depleted chloroplasts before bicarbonate addition, shows a sharp optimum at pH 6.5. Furthermore, the rate-limiting step in bicarbonate action is probably diffusion. The results are explained in terms of a hypothetical model: the bicarbonate-binding site is located at the outer side of the thylakoid membrane, but not directly accessible from the "bulk". To reach the site from the bulk, the molecule has to pass a channel with negatively charge groups on its side walls. In the light these groups are more negatively charged than in the dark. Therefore, the formate ion cannot exchange for bicarbonate in the light, and a dark period is necessary to enable exchange of formate for bicarbonate.     

Inhibition of O2 evolution by NADP in isolated potato tuber chloroplast and its identity with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea inhibition site.

Chloroplast from greening potato tuber showed good photosynthetic capacity. The evolution of O₂ was dependent upon the intensity of light. A light intensity of 30 lux gave maximum O₂ evolution. At higher intensities inhibition was observed. The presence of bicarbonate in the reaction mixture was essential for O₂ evolution. NADP was found to be a potent inhibitor of O₂ evolution in this system. NADP and 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) inhibited the O₂ evolution completely at a 3 μm concentration level, which was reversed by oxidized 2,6-dichlorophenol-indophenol (DCIP). Cyanide (CN)-treated chloroplasts showed full O₂ evolution capacity, when a lipophilic electron acceptor like N-tetramethyl-p-phenylenediamine (TMPD) or DCIP was used along with ferricyanide. Ferricyanide alone showed only 20% reduction. NADP or DCMU could inhibit O₂ evolution only when TMPD was the acceptor but not with DCIP. Photosystem II (PS II) isolated from these chloroplasts also showed inhibition by NADP or DCMU and its reversal by DCIP. Here also the evolution of O₂ with only TMPD as acceptor was sensitive to NADP or DCMU. In the presence of added silicotungstate in PS II NADP or DCMU did not affect ferricyanide reduction or oxygen evolution. The chloroplasts were able to bind exogenously added NADP to the extent of 120 nmol/mg chlorophyll. It is concluded that the site of inhibition of NADP is the same as in DCMU, and it is between the DCIP and TMPD acceptor site in the electron transport from the quencher (Q) to plastoquinone (PQ).     

Kinetics of the reactivation of the Hill reaction in CO2-depleted chloroplasts by addition of bicarbonate in the absence and in the presence of herbicides

In isolated broken chloroplasts photosynthetic electron transport requires the presence of CO₂ and/or bicarbonate. This bicarbonate effect on electron flow was measured in a medium containing 100 m M sodium formate. In this medium a dark incubation time with bicarbonate is required for the reactivation of the Hill reaction. We have measured the kinetics of the reactivation of electron flow by varying the dark incubation of CO₂-depIeted pea ( Pisum sativum L., cv. Rondo) chloroplasts with bicarbonate. The half-time of this reactivation appears to be 25 s when 2 m M bicarbonate is added.
The dinitrophenol herbicide, i -dinoseb, is shown to be a competitive inhibitor of the bicarbonate dependent Hill reaction with an inhibitor constant (K_i) of 31 n M . In the presence of 100 n M i-dinoseb or 100 n M DCMU the half-time of the reactivation by 2 m M bicarbonate appears to increase to about 58 s. We provide an explanation for these phenomena by analyzing the bicarbonate-thylakoid interaction on the basis of a simple reaction scheme. The binding of bicarbonate to the thylakoids appears to be a second order reaction with pseudo-first order kinetics. According to our analysis, any inhibitor, which is competitive with respect to the bicarbonate stimulation of the Hill reaction, should increase the half-time of the reactivation of the Hill reaction.     

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.     

Identification of a 32–34-kilodalton polypeptide as a herbicide receptor protein in Photosystem II

Photosystem II particles which retained high rates of herbicide-sensitive activity were used to examine the site(s) of action of various herbicides. A polypeptide of 32–34 kdaltons was identified as the triazine-herbicide binding site based upon: (a) parallel loss of atrazine activity and the polypeptide during either trypsin treatment or selective detergent depletion of protein in the Photosystem II complex, and (b) covalent labeling of the polypeptide by a ¹⁴C-labeled photoaffinity triazine.In Photosystem II particles depleted of the 32–34-kdalton polypeptide, electron transport was still active and was slightly sensitive to DCMU and largely sensitive to dinoseb (urea and nitrophenol herbicides, respectively). On the basis of this result it is proposed that the general herbicide binding site common to atrazine, DCMU and dinoseb is formed by a minimum of two polypeptides which determine affinity and/or mediate herbicide-induced inhibition of electron transport on the acceptor side of Photosystem II.     

A chloroplast membrane conformational change activated by electron transport between the region of photosystem II and plastoquinone

Various partial redox reactions involved in photosynthetic electron transport were studied in relation to the electron transport dependent incorporation of the water soluble chemical modifier, diazonium benzene sulfonic acid (DABS)* into chloroplast membranes. This electron transport dependent diazonium incorporation reflects a conformational change (unspecified at this time) in membrane components. The redox reaction(s) responsible for this conformational change was shown to be localized after the site of DCMU inhibition but before plastoquinone by the following evidence:

1. Electron transport from water to lipophilic “Class III” electron acceptors such as dimethyl benzoquinone and high concentrations of dibromothymoquinone potentiate the extra DABS binding to the membranes. These compounds are reduced prior to or at the plastoquinone site.
2. Electron transfer from water to silicomolybdate plus ferricyanide, a DCMU insensitive reaction, does not result in the incremental diazonium binding.
3. Photosystem I cyclic electron flow mediated by menadione (anaerobic), which requires participation of plastoquinone does not give the extra diazonium binding.

The exact redox step responsible for the conformational change is not known for certain, but there is a possibility that cytochrome b-559 may be involved. This is suggested by the observation that diazonium treatment of chloroplasts during illumination but not in darkness, causes the conversion of cytochrome b-559 from the high potential form to the low potential form.     

Effects of herbicides on binary oscillations of ultraviolet flash-induced absorption changes in chloroplasts

The competition between various herbicides and plastoquinone, at the Q_B site of Photosystem II, has been studied by measuring absorption changes between 300 and 360 nm in spinach and chenopod chloroplasts, in response to a train of saturating short xenon flashes. A complex pattern was observable without addition of chemicals interfering with electron flow. The effect of potassium ferricyanide, of hydroxylamine and of valinomycine-K⁺ permitted to simplify the pattern and, in particular, to observe binary oscillations with flash number, attributable to the functioning of the two-electron gate Q_B. Herbicides belonging to different classes block electron transfer when added at high concentration. At low concentration, however, inverted binary oscillations become observable. When the chloroplasts have been first oxidized with ferricyanide, this behaviour develops progressively in response to illumination. Varying the herbicide concentration, it appears that the concentration inducing maximum binary oscillations correlates with inhibition of linear electron transfer, within each class of herbicides. Phenolic herbicides induce the largest oscillations and ureas the smallest. The binary oscillations have the spectrum of the plastoquinone anion. The results clearly show that the studied herbicides compete efficiently with Q_B, but not with Q⁻_B, at the Q_B binding site. Atrazine-resistant chenopod chloroplasts still display normal binary oscillations in the absence of herbicide, or in the presence of atrazine alone. They are highly sensitive to DCMU and to i-dinoseb, but no inverted binary oscillations could be observed with these herbicides.     

Multiple action sites for photosystem II herbicides as revealed by delayed fluorescence

DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) at concentrations higher than 10 M suppresses the second time range delayed fluorescence (DF) of pea chloroplasts, due to inhibition of the oxidizing side of photosystem II (PS II). The inhibition of the reducing side of PS II resulting in the suppression of millisecond DF takes place at much lower (0.01 M) DCMU concentrations. The variation in the herbicide-affinities of the reducing and oxidizing sides of PS II is not the same for DCMU and phenol-type herbicides. The DCMU-affinity of the oxidizing side considerably increases and approximates that of the reducing side upon mild treatment of chloroplasts with oleic acid. Probably this is a result of some changes in the environment of the binding site at the oxidizing side. At DCMU concentrations higher than 1 mM, the chaotropic action of DCMU leads to the generation of millisecond luminescence which is not related to the functioning of the reaction centres.Abbreviations D-1 The 32 kDa herbicide-binding intrinsic polypeptide of PS II, the apoprotein of Q_B - D-2 The 32–34 kDa intrinsic polypeptide of PS II, probably the apoprotein of Z - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DF Delayed fluorescence - Dinoseb 2,4-dinitro-6-sec-butylphenol - DNOC 4,6-dinitro-o-cresol - F_m Maximal fluorescence yield (when all traps are closed) - F_o Constant fluorescence yield (when all traps are open) - PS Photosystem - Q_A and Q_B The primary and secondary plastoquinone acceptors of PS II, correspondingly - Z A plastoquinol electron donor, presumably associated with the D-2 protein     

Site of bicarbonate effect in Hill reaction. Evidence from the use of artificial electron acceptors and donors.

Using artificial electron donors and acceptors, it is shown here that the major HCO3- effect in the Hill reaction is after the "primary" electron acceptor (Q) of Photosystem II and before the site of action of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (at the plastoquinone pool). Chloroplasts in the presence of both 3-(3',4'-dichlorophenyl)-1,1-dimethylurea, which blocks electron flow from the reduced primary acdeptor Q- to the plastoquinone pool, and silicomolybdate, which accepts electrons from Q-, show no significant bicarbonate stimulation of electron flow. However, a 6-7 fold stimulation is clearly observed when oxidized diaminodurene, as an electron acceptor, and dibromothymoquinone, as an inhibitor of electron flow beyond the plastoquinone pool, are used. In the same chloroplast preparation no measurable effect of bicarbonate is observed in a Photosystem I reaction as monitored by electron flow from reduced diaminodurene to methyl viologen in the presence of 3- (3',4'-dichlorophenyl)-1,1-dimethylurea. The insensitivity of the bicarbonate effect to uncouplers of photophosphorylation and the dependence of this effect on the presence of a weak acid anion and on external pH are also reported.     

Site of bicarbonate effect in Hill reaction. Evidence from the use of artificial electron acceptors and donors

Using artificial electron donors and acceptors, it is shown here that the major HCO₃^? effect in the Hill reaction is after the “primary” electron acceptor (Q) of Photosystem II and before the site of action of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (at the plastoquinone pool). Chloroplasts in the presence of both 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, which blocks electron flow from the reduced primary acceptor Q^? to the plastoquinone pool, and silicomolybdate, which accepts electrons from Q^?, show no significant bicarbonate stimulation of electron flow. However, a 6–7-fold stimulation is clearly observed when oxidized diaminodurene, as an electron acceptor, and dibromothymoquinone, as an inhibitor of electron flow beyond the plastoquinone pool, are used. In the same chloroplast preparation no measurable effect of bicarbonate is observed in a Photosystem I reaction as monitored by electron flow from reduced diaminodurene to methyl viologen in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. The insensitivity of the bicarbonate effect to uncouplers of photophosphorylation and the dependence of this effect on the presence of a weak acid anion and on external pH are also reported.     

Charge accumulation at the reducing side of system 2 of photosynthesis

A study was made of the reactions between the primary and secondary electron acceptors of Photosystem 2 by measurements of the increase of chlorophyll fluorescence induced in darkness by dithionite or by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The experiments were done either with chloroplasts to which hydroxylamine or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) was added, or with chloroplasts treated with tris(hydroxymethyl)aminomethane (Tris) to which phenylenediamine and ascorbate were added as donor system. Under these conditions the fluorescence increase induced by dithionite or DCMU added after illumination with short light flashes was dependent on the flash number with a periodicity of two; it was large after an uneven number of flashes, and small after a long darktime or after an even number of flashes. The results are interpreted in terms of a model which involves a hypothetical electron carrier situated between Q and plastoquinone; this electron carrier is thought to equilibrate with plastoquinone in a two-electron transfer reaction; the results obtained with DCMU are explained by assuming that its midpoint potential is lowered by this inhibitor.     

The site of manganese function in photosynthetic electron transport system

The effects of Mn²⁺ on aerobic photobleaching of carotenoids, on photoreduction of 2,6-dichlorophenolindophenol (DCIP) and on fluorescence above 600 mμ of spinach chloroplasts washed with 0.8 M Tris-HC1 buffer were investigated. Carotenoids (mostly carotenes, lutein and violaxanthin) in the Tris-washed chloroplasts were irreversibly bleached by illumination with red light, while carotenoids in normal chloroplasts prepared with a low concentration of Tris-HC1 underwent no bleaching upon illumination. The photobleaching of carotenoids observed with Tris-washed chloroplasts was inhibited by Mn²⁺ (MnCl₂ or MnSO₄) as well as by some inhibitors of the Hill reaction such as dichlorophenyl-1,1-dimethylurea (DCMU), methylthio-4,6-bis-isopropylamino-s-triazine and o-phenanthroline or by reducing agents such as ascorbate plus tetramethyl-p-phenylene diamine (TMPD). DCIP photoreduction, which was deactivated by Tris, was reactivated to 50–80% of the rate for normal chloroplasts upon addition of Mn²⁺. The restored photoreduction of DCIP was inhibited by DCMU and carbonylcyanide m-chlorophenylhydrazone (CCCP). The steady-state fluorescence yield of normal chloroplasts measured at room temperature was lowered by Tris treatment, and the decreased yield was restored by adding Mn²⁺ as well as ascorbate plus TMPD. CCCP also lowered the yield; the yield was recovered by adding ascorbate plus TMPD. Determination of manganese in normal and Tris-washed chloroplasts showed that 30% of the manganese in chloroplast was removed with Tris. It was postulated that Mn²⁺ functions in the electron transport on the oxidizing side of Photosystem II at a site between water and an electron carrier (Y). CCCP as well as Tris inhibits the reduction of Y⁺ by Mn²⁺, and carotenoids are oxidized by Y⁺ which is reduced by ascorbate plus TMPD.     

Mode of action of Photosystem II herbicides studied by thermoluminescence

The mode of action of chemically different herbicides (ureas, pyridazinones, phenylcarbamates, triazines, hydroxyquinolines, hydroxybenzonitriles and dinitrophenols) on photosynthetic electron transport was investigated by measurements of oxygen evolution and thermoluminescence. Depending on the particular herbicide used the thermoluminescence band related to Q (the primary acceptor of Photosystem II) appears at +5, 0 or −14°C. It was shown that these three different peak positions can be ascribed to various redox states of Q, the shifts being due to the binding of herbicides to the chloroplast membrane. Both displacement experiments and additive inhibition of herbicide pairs measured by thermoluminescence and oxygen evolution suggested that the sites of action of these herbicides are on the same protein. However, herbicide treatment of trypsinized chloroplasts showed that there were three different binding sites on the same protein, in agreement with the classification of herbicides into three groups based on thermoluminescence measurements. Our results suggest that the primary and secondary acceptors of Photosystem II (Q and B, respectively) are in close proximity and form a common complex with the herbicide-binding protein within the chloroplast membrane.     

Comparative study of the action of ultraviolet-C and ultraviolet- B radiation on photosynthetic electron transport

The effect of ultraviolet-C (UV-C, mainly 254 nm radiation) and ultraviolet-B (UV-B, 290-320 nm) radiation on the photosynthetic electron transport reactions has been investigated. The rates of Hill activity mediated by ferricyanide and dichlorodimethoxy-p-benzoquinone (DCDMQ) were differently sensitive to UV-C but equally inhibited by UV-B. Replacement of water with diphenylcarbazide was ineffective in restoring the activity of dichlorophenol indophenol (DCPIP) Hill reaction in UV-B treated chloroplasts, but had significant effect in UV-C treated chloroplasts.
Photobleaching of carotenoids in the presence of carbonyl cyanide-m-chlorophenyl-hydrazone, an indicator of the photochemical reaction associated with the reaction centre of photosystem II, was suppressed and is paralleled by the changes in Hill activity only in UV-B-treated chloroplasts. Carotenoid photobleaching occurred even in UV-C treated chloroplasts showing no measurable Hill activity. UV-C and UV-B irradiation diminished variable fluorescence. With UV-B treated, but not with UV-C treated chloroplasts, an increase in the fluorescence yield was observed upon the addition of 3-(3,4-dichIorophenyl)-l,l-dimethylurea (DCMU) and/or Na dithionite.
Photosystem I activity was found to be unaffected by both UV-C and UV-B radiation at the fluences tested. Kinetics of P700 photooxidation and dark reversal in UV treated chloroplasts indicate that only the electron flow from photosystem II to photosystem I is impaired. It is concluded that while UV-B radiation inactivates specifically the photosystem II reaction centre, UV-C radiation acts at plastoquinone, the quencher Q, and the water oxidizing enzyme system.     

Inhibition of the reoxidation of the secondary electron acceptor of Photosystem II by bicarbonate depletion

In bicarbonate-depleted chloroplasts, the chlorophyll a fluorescence decayed with a halftime of about 150 ms after the third flash, and appreciably faster after the first and second flash of a series of flashes given after a dark period. After the fourth to twentieth flashes, the decay was also slow. After addition of bicarbonate, the decay was fast after all the flashes of the sequence. This indicates that the bicarbonate depletion inhibits the reoxidation of the secondary acceptor R²⁻ by the plastoquinone pool; R is the secondary electron acceptor of pigment system II, as it accepts electrons from the reduced form of the primary electron acceptor (Q⁻). This conclusion is consistent with the measurements of the DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea)-induced chlorophyll a fluorescence after a series of flashes in the presence and the absence of bicarbonate, if it is assumed that DCMU not only causes reduction of Q if added in the state QR⁻, but also if added in the state QR²⁻.     

Further characterization of a photosystem ii particle isolated from spinach chloroplasts by triton treatment delayed light emission

Delayed light emission from the Triton-fractionated Photosystem II subchloroplast fragments (TSF-IIa) was measured between 0.5 and 10 ms after the termination of illumination. The delayed light emission was diminished by Photosystem II inhibitors, DCMU and o-phenanthroline, which act between the reduced primary acceptor and the plastoquinone pool.Secondary electron donors to Photosystem II, diphenylcarbazide, phenylenediamine, Mn²⁺, and ascorbate inhibited delayed light emission. Secondary electron acceptors such as ferricyanide, dichlorophenol indophenol, and dimethyl benzoquinone enhanced delayed light emission. The addition of secondary electron acceptors to TSF-IIa particles containing Mn²⁺ restored delayed light emission to almost the control level. The plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl p-benzoquinone, increased delayed light emission at low concentrations but decreased it at higher concentrations. Silicomolybdate enhanced the delayed light emission of TSF-IIa particles markedly, and reversed the inhibition by DCMU. Silicomolybdate showed a similar stimulatory effect on the delayed-light intensity in broken spinach chloroplasts at shorter times after the termination of illumination. Carbonyl cyanide m-chloro (or p-trifluoromethoxy) phenylhydrazones inhibited the delayed light emission, but NH₄Cl had no effect.     

Stimulation of electron transport from Photosystem II to Photosystem I in spinach chloroplasts

Electron transport from Photosystem II to Photosystem I of spinach chloroplasts can be stimulated by bicarbonate and various carbonyl or carboxyl compounds. Monovalent or divalent cations, which have hitherto been implicated in the energy distribution between the two photosystems, i.e., spillover phenomena at low light intensities, show a similar effect under high light conditions employed in this study. A mechanism for this stimulation of forward electron transport from Photosystem II to Photosystem I could involve inhibition of two types of Photosystem II partial reactions, which may involve cycling of electrons around Photosystem II. One of these is the DCMU-insensitive silicomolybdate reduction, and the other is ferricyanide reduction by Photosystem II at pH 8 in the presence of dibromothymoquinone. Greater stimulation of forward electron transport reactions is observed when both types of Photosystem II cyclic reactions are inhibited by bicarbonate, carbonyl and carboxyl-type compounds, or by certain mono- or divalent cations.Abbreviations used: DCMU, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea; DCIP, 2,6-dichloroindophenol; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; FeCN, potassium ferricyanide; MV, methylviologen; PS I, photosystem I; PS II, photosystem II; SM, silicomolybdic acid.     

Interactions of herbicides and azidoquinones at a Photosystem II binding site in the thylakoid membrane

6-Azido-5-decyl-2,3-dimethoxy-p-benzoquinone (6-azido-Q₀C₁₀) was found to replace the native plastoquinone at B (the second stable electron acceptor to Photosystem II (PS II)). The 6-azido-Q₁₀C₁₀ would accept electrons from the primary electron-accepting quinone, Q, thus allowing electron transport through PS II to the plastoquinone pool in thylakoids. The synthetic azidoquinone also competes with the PS II herbicides ioxynil and atrazine for binding. This observation strongly favors the hypothesis that PS II herbicides block electron transport by replacing the native quinone which acts as the second electron carrier on the reducing side of PS II (termed B). Covalent linkage of 6-azido-Q₀C₁₀ to its binding environment by ultraviolet irradiation greatly reduces herbicide-binding affinity but does not lead to a loss in herbicide-binding sites. We take this as evidence that covalent attachment of 6-azido-Q₀C₁₀ allows some freedom of quinone head-group movement such that the herbicides can enter the binding site. This indicates that the protein determinants which regulate quinone and herbicide binding are very closely related, but not identical. A compound somewhat related to 6-azido-Q₀C₁₀ is 2-azido-3-methoxy-5-geranyl-6-methyl-p-benzoquinone (2-azido-Q₂). This compound was found to be an ineffective competitor with respect to herbicide binding. Thus, interactions with protein-binding determinants are highly dependent on the molecular structure of quinones. The 2-azido-Q₂ was an inhibitor of electron flow in the intersystem portion of the chain.