Inhibitor "double occupancy" in the Q(o) pocket of the chloroplast cytochrome b6f complex.

Electron paramagnetic resonance (EPR) spectra of the "Rieske" 2Fe-2S cluster revealed that two molecules of the inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) can bind to each monomer of the spinach cytochrome (cyt) b6f complex, both in isolated form and in intact thylakoid membranes. Binding to the high-affinity site, which accounts for the observed inhibitory effects, caused small shifts in the g(x) transition of the 2Fe-2S cluster EPR spectrum, similar to those induced by stigmatellin or 2-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether (DNP-INT). Occupancy of the low-affinity site was only observed after addition of superstoichiometric amounts of the inhibitor and was accompanied by the appearance of a g = 1.94 EPR signal. The shape of the equilibrium binding titration curve, the effects on the 2Fe-2S EPR spectrum, and the ability of the DBMIB binding to displace DNP-INT were consistent with two molecules of DBMIB binding at the Q(o) pocket, with the strongly binding species binding close to the 2Fe-2S cluster. Possible implications of these findings for so-called "double-occupancy" models for Q(o) site catalysis are discussed.     

Certain metal ions are inhibitors of cytochrome b6f complex 'Rieske' iron-sulfur protein domain movements

Many current models of the Q cycle for the cytochrome (cyt) b6f and the cyt bc1 complexes incorporate 'Rieske' iron-sulfur protein (ISP) domain movements to gate electron transfer and to ensure high yields of proton shuttling. It was previously proposed that copper ions, which bind at a site distant from the quinol oxidase (Q(o)) site, inhibit plastoquinol (PQH2) binding by restraining the hydrophilic head domain of the ISP [Rao B. K., S., Tyryshkin, A. M., Roberts, A. G., Bowman, M. K., and Kramer, D. M. (1999) Biochemistry 38, 3285-3296]. The present work presents evidence that this is indeed the case for both copper ions and Zn2+, which appear to inhibit by similar mechanisms. Electron paramagnetic resonance (EPR) spectra show that Cu2+ and Zn2+ binding to the cyt b6f complex displaces the Q(o) site inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). At high concentrations, both DBMIB and Cu2+ or Zn2+ can bind simultaneously, altering the Rieske 2Fe2S cluster and Cu2+ EPR spectra, suggesting perturbations in their respective binding sites. Both Zn2+ and Cu1+ altered the orientations of the Rieske 2Fe2S cluster with respect to the membrane plane, but had no effect on that of the cyt b6 hemes. Cu2+ was found to change the orientation of the cyt f heme plane, consistent with binding on the cyt f protein. Within conservative constraints, the data suggest that the ISP is shifted into a position intermediate between the ISP(C) position, when the Q(o) site is unoccupied, and the ISP(B) position, when the Q(o) site is occupied by inhibitors such as DBMIB or stigmatellin. These results support the role of ISP domain movements in Q(o) site catalysis.     

Quantitation of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone binding sites in chloroplast membranes: evidence for a functional dimer of the cytochrome b6f complex

The binding of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) to chloroplast thylakoid membranes was investigated by analyzing the inhibition of electron transfer by DBMIB according to a steady-state rate relationship for enzyme-catalyzed reactions in the presence of tightly binding reversible inhibitors. DBMIB interacts with the cytochrome b6f complex in a manner best described by an apparent dissociation constant near 6 nM. The binding site titer is 1 mmol X mol chlorophyll-1. This number of DBMIB binding sites approaches one-half the number of cytochrome b6f complexes present in the membrane. These data suggest that the cytochrome b6f complex may function in electron transfer as a dimer, plastoquinol oxidation being totally inhibited by the binding of a single DBMIB molecule to the dimer.     

The effects of quinone analogues on cytochrome b6 reduction and oxidation in a reconstituted system

The reconstituted system containing Photosystem I, plastocyanin and the cytochrome b6-f complex is used to study the effects of various quinone analogues on the redox behavior of cytochrome b6. The effects of DBMIB, DNP-INT and HQNO are compared in an attempt to discern the modes of action of these quinone analogues. Both DBMIB and DNP-INT are potent inhibitors of the plastocyanin reductase activity of the isolated cytochrome complex. However, while DBMIB abolished the oxidant-induced reduction of cytochrome b6, DNP-INT only inhibited about 25% of the net reduction. On the other hand, HQNO does not show any significant inhibition of plastocyanin reductase activity of the isolated cytochrome complex at concentrations up to 20 microM. An enhancement of the net amount of cytochrome b6 reduced is observed in the presence of HQNO. Both DNP-INT and HQNO inhibited the dark oxidation rate of cytochrome b6. The possible identity of the oxidant for cytochrome b6 is discussed. Plastoquinone is concluded to be the most likely candidate. DNP-INT is concluded to have at least two sites of inhibition in the cytochrome complex. The implications of these findings on quinone functions in the cytochrome b6-f complex are discussed.     

New insights on the proton pump associated with cytochrome b6f turnovers from the study of H/D substitution effects on the electrogenicity and electron transfer reactions

We have studied the effect of protium/deuterium substitution on different kinetics associated with the turnovers of cytochrome b(6)f complex in whole cells of Chlamydomonas reinhardtii. Both the oxidation of cytochrome f and the reduction of hemes b were only little affected by the isotopic substitution. Contrasting with this, the initial slope of the electrogenic phase associated with cytochrome b(6)f turnover was slowed by a factor of 4 by H(2)O/D(2)O substitution. Whereas in the presence of H(2)O the electrogenic phase developed concomitantly with cytochrome b reduction, it lagged for a few hundreds of microseconds after cytochrome b reduction in the presence of D(2)O. We propose that a proton pump is triggered by the oxidation of plastoquinol at the Q(o) site. The proton transfer is specifically delayed upon isotopic substitution, accounting for the lack of significant effect on the electron-transfer reaction as well as for the strong decrease of the initial rate of the electrogenic phase.     

Why is it so difficult to construct Q(i) site mutants in Chlamydomonas reinhardtii?

The cytochrome b(6)f complex catalyses electron transfer from plastoquinol to a hydrosoluble acceptor (plastocyanin), while building up an electrochemical proton gradient. Oxidation and reduction of plastoquinol occur respectively at the Q(o) site (exposed on the luminal side of the thylakoid membrane) and at the Q(i) site (facing the stroma). The discovery of an additional c'-type heme in the Q(i) site has cast a new light on the difficulties previously encountered to obtain mutants at this site. In this work, we critically examine our unsuccessful attempts to obtain Q(i) site mutants based on sequence and structure homology between cytochrome b(6)f and bc(1) complexes.     

The inhibitor DBMIB provides insight into the functional architecture of the Qo site in the cytochrome b6f complex

Previously [Roberts, A. G., and Kramer, D. M. (2001) Biochemistry 40, 13407-13412], we showed that 2 equiv of the quinone analogue 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) could occupy the Q(o) site of the cytochrome (cyt) b(6)f complex simultaneously. In this work, a study of electron paramagnetic resonance (EPR) spectra from the oriented cyt b(6)f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Q(o) site. With a single DBMIB at the Q(o) site, the ISP is oriented with the 2Fe-2S cluster toward cyt f, which is similar to the orientation of the ISP in the X-ray crystal structure of the cyt b(6)f complex from thermophilic cyanobacterium Mastigocladus laminosus in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc(1) complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called "proximal niche", near the cyt b(L) heme. These data suggest that the high-affinity DBMIB site is at the proximal niche Q(o) pocket. With >or=2 equiv of DBMIB bound, the Rieske ISP is in a position that resembles the ISP(B) position of the chicken mitochondrial cyt bc(1) complex in the presence of stigmatellin and the Chlamydomonas reinhardtii cyt b(6)f complex in the presence of tridecylstigmatellin (TDS), which suggests that the low-affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe-2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations at which a single binding was expected did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, at which double occupancy was expected, increased the extent of cyt f oxidation to levels similar to that of cyt f oxidation in the presence of a saturating concentration of stigmatellin. Simulations of the EPR g-tensor orientations of the 2Fe-2S cluster versus the physical orientations based on single-crystal studies of the cyt bc(1) complex suggest that the soluble ISP domain of the spinach cyt b(6)f complex can rotate by at least 53 degrees, which is consistent with long-range ISP domain movement. Implications of these results are discussed in the context of the X-ray crystal structures of the chicken mitochondrial cyt bc(1) complex and the M. laminosus and C. reinhardtii cyt b(6)f complexes.     

Methylviologen and dibromothymoquinone treatments of pea leaves reveal the role of photosystem I in the Chl a fluorescence rise OJIP

The effects of dibromothymoquinone (DBMIB) and methylviologen (MV) on the Chl a fluorescence induction transient (OJIP) were studied in vivo. Simultaneously measured 820-nm transmission kinetics were used to monitor electron flow through photosystem I (PSI). DBMIB inhibits the reoxidation of plastoquinol by binding to the cytochrome b(6)/f complex. MV accepts electrons from the FeS clusters of PSI and it allows electrons to bypass the block that is transiently imposed by ferredoxin-NADP(+)-reductase (FNR) (inactive in dark-adapted leaves). We show that the IP phase of the OJIP transient disappears in the presence of DBMIB without affecting F(m). MV suppresses the IP phase by lowering the P level compared to untreated leaves. These observations indicate that PSI activity plays an important role in the kinetics of the OJIP transient. Two requirements for the IP phase are electron transfer beyond the cytochrome b(6)/f complex (blocked by DBMIB) and a transient block at the acceptor side of PSI (bypassed by MV). It is also observed that in leaves, just like in thylakoid membranes, DBMIB can bypass its own block at the cytochrome b(6)/f complex and donate electrons directly to PC(+) and P700(+) with a donation time tau of 4.3 s. Further, alternative explanations of the IP phase that have been proposed in the literature are discussed.     

Inhibition of plastoquinol oxidation at the cytochrome b6f complex by dinitrophenyl ether of iodonitrothymol (DNP-INT) depends on irradiance and H+ uptake by thylakoid membranes

Inhibitory analysis is a useful tool for studying reactions in the photosynthetic apparatus. After introducing by Aachim Trebst in 1978, dinitrophenylether of iodonitrothymol (DNP-INT), a competitive inhibitor of plastoquinol oxidation at the cytochrome (cyt.) b₆f complex, has been widely applied to study reactions occurring in the plastoquinone pool and the cyt. b₆f complex. Here we examine the inhibitory efficiency of DNP-INT by implementing three approaches to estimate the extent of blockage of electron flow from the plastoquinone pool to photosystem I in isolated thylakoids from spinach (Spinacia oleracea). We confirm that DNP-INT is a potent inhibitor of electron flow to photosystem I and demonstrate that inhibitory action of DNP-INT depends on irradiance and H⁺ uptake by thylakoid membranes. Based on these findings, we infer that affinity of the quinol-oxidizing site of the cyt. b₆f complex to DNP-INT is increased in the light due to hydrogen bonding between DNP-INT molecules and acidic amino acid residue(s), which is (are) protonated in the light.     

Redox-coupled proton pumping activity in cytochrome b6f,as evidenced by the pH dependence of electron transfer in whole cells of Chlamydomonas reinhardtii

The pH dependence of cytochrome b(6)f catalytic activity has been measured in whole cells of the green alga Chlamydomonas reinhardtii over the 5-8 range. An acid pH slowed the reactions occurring at the lumenal side of the complex (cytochrome b(6) and f reduction) and affected also the rate and amplitude of the slow electrogenic reaction (phase b), which is supposed to reflect transmembrane electron flow in the complex. On the other hand, a direct measurement of the transmembrane electron flow from the kinetics of cytochrome b(6) oxidation revealed no pH sensitivity. This suggests that a substantial fraction of the electrogenicity associated with cytochrome b(6)f catalysis is not due to electron transfer in the b(6) hemes but to a plastoquinol-oxidation-triggered charge movement, in agreement with previous suggestions that a redox-coupled proton pump operates in cytochrome b(6)f complex. The pH dependence of cytochrome b(6)f activity has also been measured in two mutant strains, where the glutamic 78 of the conserved PEWY sequence of subunit IV has been substituted for a basic (E78K) and a polar (E78Q) residue [Zito, F., Finazzi, G., Joliot, P., and Wollman, F.-A. (1998) Biochemistry 37, 10395-10403]. Their comparison with the wild type revealed that this residue plays an essential role in plastoquinol oxidation at low pH, while it is not required for efficient activity at neutral pH. Its involvement in gating the redox-coupled proton pumping activity is also shown.     

Differential effects of DBMIB and DNP-INT on the kinetic phases of P-700 reduction

In the presence of ferredoxin and NADP, DBMIB abolishes the fast-relaxing portion of P-700 together with the reduction of NADP. The slow-relaxing portion is inhibited at much higher concentrations. Qualitatively similar results have been observed with DNP-INT. However, its action appears to be a light-dependent process. The slow, cyclic turnover of P-700 in the presence of DCMU, ferredoxin and NADPH is inhibited by DBMIB but only slightly by DNP-INT. The data suggest that the inhibitors act at different sites of the electron-transport system.     

质体醌库和细胞色素b6f参与调控蓝细菌 Synechocystis sp. PCC 6803的状态转换

用调制叶绿素荧光研究了对苯醌(1,4-benzoquinone,BQ)和二溴百里香醌(2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone,DBMIB)对蓝细菌Synechocystissp.PCC6803状态转换的作用。BQ和DBMIB是质体醌(PQ)的类似物,两者均可充当PQ的电子受体。其中,DBMIB能够和细胞色素b6f的Qo位点特异结合。在没有作用光的情况下,BQ诱导暗适应的蓝细菌进入状态Ⅰ;相反,DBMIB诱导Syne鄄chocystis6803向状态Ⅱ转换。据此提出,在生理状态下蓝细菌根据PQ库的氧化还原状态调节状态转换;细胞色素b6f参与此调控过程。     

Ascorbate-mediated LHCII protein phosphorylation--LHCII kinase regulation in light and in darkness

A freeze-thaw cycle of isolated thylakoids in darkness in the presence of ascorbate was employed as a novel experimental system to activate the light-harvesting complex (LHC) II kinase. Under these conditions ascorbate reduces Q(A), the primary quinone electron acceptor of photosystem (PS) II, and the subsequent reduction of plastoquinone and the cytochrome (cyt) b(6)f complex results in the activation of the LHCII kinase. Using this activation system, several facets of regulation of LHCII protein phosphorylation were unravelled. (i) Myxothiazol inhibited the activation of LHCII protein phosphorylation, thus being a potent inhibitor of electron flow not only in cyt bc complexes but in darkness also in cyt b(6)f complexes. (ii) Oxygen, the only electron acceptor in darkness, was required for LHCII kinase activation demonstrating that after a full reduction of the cyt b(6)f complex, an additional plastoquinol oxidation cycle in the quinol oxidation (Qo) site is required for LHCII kinase activation. (iii) In the absence of electron flow, when the intersystem electron carriers are reduced, the activated LHCII kinase has a half-life of 40 min, whereas the fully activated LHCII kinase becomes deactivated in a time scale of seconds upon oxidation of the cyt b(6)f complex, indicating that the kinase constantly reads the redox poise of the cyt b(6)f complex. (iv) The LHCII kinase is more tightly bound to the thylakoid membrane than the PS II core protein kinase(s). It is concluded that oxidation of plastoquinol at the Qo site of the reduced cyt b(6)f complex is required for LHCII kinase activation, while rapid reoccupation of the Qo site with plastoquinol is crucial for sustenance of the active state of the LHCII kinase.     

Architecture of the Qo site of the cytochrome bc1 complex probed by superoxide production

Although several X-ray structures have been determined for the mitochondrial cytochrome (cyt) bc(1) complex, none yet shows the position of the substrate, ubiquinol, in the quinol oxidase (Q(o)) site. In this study, the interaction of molecular oxygen with the reactive intermediate Q(o) semiquinone is used to probe the Q(o) site. It has been known for some time that partial turnover of the cyt bc(1) complex in the presence of antimycin A, a Q(i) site inhibitor, results in accumulation of a semiquinone at the Q(o) site, which can reduce O(2) to superoxide (O(2)(*)(-)). It was more recently shown that myxothiazol, which binds close to the cyt b(L) heme in the proximal Q(o) niche, also induces O(2)(*)(-) production. In this work, it is shown that, in addition to myxothiazol, a number of other proximal Q(o) inhibitors [including (E)-beta-methoxyacrylate-stilbene, mucidin, and famoxadone] also induce O(2)(*)(-) production in the isolated yeast cyt bc(1) complex, at approximately 50% of the V(max) observed in the presence of antimycin A. It is proposed that proximal Q(o) site inhibitors induce O(2)(*)(-) production because they allow formation, but not oxidation, of the semiquinone at the distal niche of the Q(o) site pocket. The apparent K(m) for ubiquinol at the Q(o) site in the presence of proximal Q(o) site inhibitors suggests that the "distal niche" of the Q(o) pocket can act as a fully independent quinol binding and oxidation site. Together with the X-ray structures, these results suggest substrate ubiquinol binds in a fashion similar to that of stigmatellin with H-bonds between H161 of the Rieske iron-sulfur protein and E272 of the cyt b protein. When modeled in this way, mucidin and ubiquinol can bind simultaneously to the Q(o) site with virtually no steric hindrance, whereas progressively bulkier inhibitors exhibit increasing overlap. The fact that partial turnover of the Q(o) site is possible even with bound proximal Q(o) site inhibitors is consistent with the participation of two separate functional Q(o) binding niches, occupied simultaneously or sequentially.     

Cytochrome b6 arginine 214 of Synechococcus sp. PCC 7002, a key residue for quinone-reductase site function and turnover of the cytochrome bf complex

Quinone-reductase (Q(i)) domains of cyanobacterial/chloroplast cytochrome bf and bacterial/mitochondrial bc complexes differ markedly, and the cytochrome bf Q(i) site mechanism remains largely enigmatic. To investigate the bf Q(i) domain, we constructed the mutation R214H, which substitutes histidine for a conserved arginine in the cytochrome b(6) polypeptide of the cyanobacterium Synechococcus sp. SPCC 7002. At high light intensity, the R214H mutant grew approximately 2.5-fold more slowly than the wild type. Slower growth arose from correspondingly slower overall turnover of the bf complex. Specifically, as shown in single flash turnover experiments of cytochrome b(6) reduction and oxidation, the R214H mutation partially blocked electron transfer to the Q(i) site, mimicking the effect of the Q(i) site inhibitor 2-N-4-hydroxyquinoline-N-oxide. The kinetics of cytochrome b(6) oxidation were largely unaffected by hydrogen-deuterium exchange in the mutant but were slowed considerably in the wild type. This suggests that although protonation events influenced the kinetics of cytochrome b(6) oxidation at the Q(i) site in the wild type, electron flow limited this reaction in the R214H mutant. Redox titration of membranes revealed midpoint potentials (E(m,7)) of the two b hemes similar to those in the wild type. Our data define cytochrome b(6) Arg(214) as a key residue for Q(i) site catalysis and turnover of the cytochrome bf complex. In the recent cytochrome bf structures, Arg(214) lies near the Q(i) pocket and the newly discovered c(i) or x heme. We propose a model for Q(i) site function and a role for Arg(214) in plastoquinone binding.     

Cytochrome b6f complex is required for phosphorylation of light-harvesting chlorophyll a/b complex II in chloroplast photosynthetic membranes

The light-harvesting chlorophyll a/b complex (LHC II) and four photosystem II (PS II) core proteins (8.3, 32, 34 and 44 kDa) become phosphorylated in response to reduction of the intersystem electron transport chain of green plant chloroplasts. Previous studies indicated that reduction of the plastoquinone (PQ) pool is the key event in kinase activation. However, we show here that, unlike PS II proteins, LHC II is phosphorylated only when the cytochrome b6f complex is active. Two lines of evidence support this conclusion. (1) 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the 2,4-dinitrophenyl ether of iodonitrothymol (DNP-INT), which are known to block electron flow into the cytochrome complex, selectively inhibit LHC II phosphorylation in spinach thylakoids. (2) The hcf6 mutant of maize, which contains PQ but lacks the cytochrome b6f complex, phosphorylates the four PS II proteins but fails to phosphorylate LHC II in vivo or in vitro.     

Differential inhibition by plastoquinone analogues of photoreduction of cytochrome b-559 in chloroplasts

The high-potential form of cytochrome b-559 (b-559 HP) is closely linked to the oxygenic photosystem (photosystem II) but its relation to other redox components of the photosynthetic apparatus, including plastoquinone, is still obscure. We investigated the photoreduction of cytochrome b-559 HP by isolated chloroplasts in the presence of 3 antagonists of plastoquinone, of which, DBMIB (dibromothymoquinone) and DNP-INT (dinitrophenyl ether of iodonitrothymol) are known to inhibit the oxidation of the plastoquinone pool (PQ) by the FeS-cytochrome ƒ/b₆ complex and one, UHDBT (5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) is known to inhibit the reduction of PQ by Q_B.Q_B is a protein-bound plastoquinone that serves as a two-electron gate for the reduction of PQ. We found that DBMIB and DNP-INT did not inhibit but low concentrations of UHDBT severely inhibited the photoreduction of cytochrome b-559 HP. These results suggest that the electron donor for the reduction of cytochrome b-559 HP was either Q_B or a portion of the PQ pool that was oxidized by a new pathway free of binding sites for DBMIB and DNP-INT.     

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

Photosynthetic electron transfer through the cytochrome b6f complex can bypass cytochrome f

The cytochrome b(6)f complex is an obligatory electron transfer and proton-translocating enzyme in all oxygenic photosynthesis. Its operation has been described by the "Q-cycle." This model proposes that electrons are transferred from plastoquinol to plastocyanin (the reductant of P700 in Photosystem I) through, obligatorily in series, the iron-sulfur and the cytochrome f redox centers in the cytochrome b(6)f complex. However, here we demonstrate that (a) the iron-sulfur center-dependent reductions of plastocyanin and P700 are much faster than cytochrome f reduction, both in Chlamydomonas reinhardtii cytochrome f mutants and in the wild type, and (b) the steady-state photosynthetic electron transport does not correlate with strongly inhibited cytochrome f reduction kinetics in the mutants. Thus, cytochrome f is not an obligatory intermediate for electrons flowing through the cytochrome b(6)f complex. The oxidation equivalents from Photosystem I are delivered to the high potential chain of the cytochrome b(6)f complex both at the cytochrome f level and, independently, at another site connected to the quinol-oxidizing site, possibly the iron-sulfur center.     

Inhibitors in the functional dissection of the photosynthetic electron transport system

The significance of inhibitors and artificial electron acceptor and donor systems as experimental tools for studying the photosynthetic system is described by reviewing early classical articles. The historical development in unravelling the role and sequence of electron carriers and energy conserving sites in the electron transport chain is acknowledged. Emphasis is given to inhibitors of the acceptor side of photosystem II and of the plastoquinol oxidation site in the cytochrome b6/f complex. Their role in regulatory processes under redox control is introduced.