The kinetics of reactions around the cytochrome bf complex studied in an isolated system

The kinetics of oxidation and reduction of P700, plastocyanin, cytochrome f and cytochrome b-563 were studied in a reconstituted system consisting of Photosystem I particles, cytochrome bf complex and plastocyanin, all derived from pea leaf chloroplasts. Decyl plastoquinol was the reductant of the bf complex. Turnovers of the system were initiated by laser flashes. The reaction between oxidised P700 and plastocyanin was non-homogeneous in that a second-order rate coefficient of c. 5×10^–7 M^–1 s^–1 applied to 80% of the P700⁺ and c. 0.7×10⁷ M^–1 s^–1 to the remainder. In the presence of bf complex, but without quinol, the electron transfer between cytochrome f and oxidised plastocyanin could be described by a second-order rate coefficient of c. 4×10⁷ M^–1 s^–1 (forward), and c. 1.6×10⁷ M^–1 s^–1 (reverse). The equilibrium coefficient was thus 2.5. Unexpectedly, there was little reduction of cytochrome f ⁺ or plastocyanin⁺ by electrons from the Rieske centre. With added quinol, reduction of cytochrome b-563 occurred. Concomitantly, electrons appeared in the oxidised species. It was inferred that either the Rieske centre was not involved in the high-potential chain of electron transfer events, or that, only in the presence of quinol, electrons were quickly passed from the Rieske centre to cytochrome f ⁺. Additionally, the presence of quinol altered the equilibrium coefficient for the cyt f/PC interaction from 2.5 to c. 5. The reaction between quinol and the bf complex was describable by a second-order rate coefficient of about 3×10⁶ M^–1 s^–1. The pattern of the redox reactions around the bf complex could be simulated in detail with a Q-cycle model as previously found for chloroplasts.Abbreviations AQS anthraquinone sulphonate - cyt cytochrome - cyt b-563(H) high-potential cyt b-563 - cyt b-563(L) low potential cyt b-563 - FeS(R) the Rieske protein of the cyt bf complex, containing an Fe₂S₂ centre - PC plastocyanin - PS photosystem - P700 reaction centre in PS I     

Structure and function of the chloroplast cytochrome bf complex

The chloroplast cytochrome bf complex is an intrinsic multisubunit protein from the thylakoid membrane consisting of four polypeptides: cytochrome f, a two heme containing cytochrome b ₆, the Rieske iron-sulfur protein, and a 17 kD polypeptide of undefined function. The complex functions in electron transfer between PSII and PSI, where most mechanisms suggest that the transfer of a single reducing equivalent from plastoquinol to plastocyanin results in the translocation of two protons across the membrane. Primary sequence analyses, dichroism studies, and functional considerations allow the construction of an approximate structural model of a monomeric complex, although some evidence exists for a dimeric structure. Resolution of the properties of the two cytochrome b ₆ hemes has relied upon the availability of purified solubilized complex, while evidence in the thylakoid suggests the difference between the two hemes are not as great in situ. Such variability in the spectroscopic and electrochemical properties of the cytochrome b ₆ is a major concern during the experimental use of the purified complex. There is a general consensus that the complex contains a plastoquinol oxidizing (Q_z) site, although the evidence for a plastoquinone reduction (Q_c) site, called for in most mechanistic hypotheses, is less substantive. Probably the most severe challenge to the so called Q-cycle mechanism comes from experimental observations made with cytochrome b ₆ initially reduced, where proposed interpretations more closely resemble a b-cycle than a Q-cycle. Although functional during cyclic electron transfer, the role of the complex and its possible interaction with other proteins, has not been completely resolved.Abbreviations Cytochrome b _H high potential cytochrome b ₆ - Cytochrome b _L low potential cytochrome b ₆ - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - FNR ferredoxin:NADP oxidoreductase - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - Q_c quinone binding site on the cytochrome bf complex near the outside of the thylakoid membrane, alternatively designated centre i or centre r - Q_z quinone binding site on the cytochrome bf complex near the inside of the thylakoid membrane, alternatively designated centre o     

Effect of Photosystem II inhibitor K-15 on photochemical reactions of the isolated D1/D2 cytochrome b559 complex

Effect of a highly efficient inhibitor of Photosystem II (PS II), K-15 (4-[methoxy-bis-(trifluoromethyl)methyl)-2,6-dinitrophenyl hydrazone methyl ketone), was investigated using the D1/D2/cytochrome b559 reaction centre (RC) complex. A novel approach for photoaccumulating reduced pheophytin (Pheo^–) in the absence of the strong reducing agent, sodium dithionite, was demonstrated which involved illumination in the presence of TMPD (from 5 to 100 M) under anaerobic conditions. The addition of K-15 at concentrations of 0.5 M and 2 M resulted in approx. 50% and near 100%, respectively, inhibition of this photoreaction, while subsequent additions of dithionite eliminated the inhibitory effect of K-15. Methyl viologen induced similar inhibition at much higher concentrations (>1 mM). Moreover, K-15 efficiently quenched the variable part of chlorophyll fluorescence (which is the recombination luminescence of the pair P₆₈₀ ⁺ Pheo^–). A 50% inhibition was induced by 5 M K-15 and the effect was maximal in the range 20 to 200 M. Photooxidation of P₆₈₀ in the presence of 0.1 mM silicomolybdate was also efficiently inhibited by K-15 (50% inhibition at 15 M). The data are consistent with the idea put forward earlier (Klimov et al. 1992) that the inhibitory effect of K-15 is based on facilitating a rapid recombination between Pheo^– and P₆₈₀ ⁺ (or Z⁺) via its redox properties. The inhibitor can be useful for suppressing PS II reactions in isolated RCs of PS II which are resistant to all traditional inhibitors, like diuron, and probably functions by substituting for Q_A missing in the preparation.At a concentration of 0.5–50 M K-15 considerably increased both the rate and extent of cytochrome b559 photoreduction in the presence, as well as in the absence, of 5 mM MnCl₂. Consequently it is suggested that K-15 also serves as a mediator for electron transfer from Pheo^– to cytochrome b559.Abbreviations K-15 4-[methoxy-bis-(trifluoromethyl)methyl]-2,6-dinitrophenyl hydrazone methyl ketone - P₆₈₀ the primary electron donor of PS II - Pheo pheophytin - PS II Photosystem II - Q_A and Q_B the primary and the secondary electron acceptor of PS II - RC reaction centre - SiMo silicomolybdate - TMPD N,N,N,,N,-tetramethyl-p-phenylenediamine - Z secondary electron donor of PS II     

The effect of pre-reduction of cytochrome b-563 on the electron-transfer reactions of the cytochrome bf complex in higher plant chloroplasts

An investigation has been made of the effects of pre-reduction of cytochrome b-563 on electron transfers through the cytochrome bf complex. It has been found that in a system in which anthraquinone-2-sulphonate or anthraquinone-2,6-disulphonate is used as redox buffer, a lipid-soluble mediator must also be present to allow sufficiently rapid equilibration of cytochrome b-563 with the ambient potential. We have found that 1 μM benzyl viologen gives full equilibration of cytochrome b-563 in less than 30 s, while minimizing the side reactions that have been observed with alternative mediators. Pre-reduction of cytochrome b-563 did not prevent turnover of site o (quinol-oxidising site of the cytochrome bc complex), even with fast repetitive flash activation. The site o reaction was accompanied by rapid, 2-nonyl-4-hydroxyquinoline N-oxide-sensitive oxidation of cytochrome b, and by a slow carotenoid bandshift. These results are discussed in conjunction with related results from the cytochrome bc₁ complex; Q-cycle models are considered in which the semiquinone at site o either can reduce an oxidant other than cytochrome b-563, or can migrate to site r (quinone-reducing site of the cytochrome bc complex). Of these possibilities, only the migration of the neutral semiquinone, QH, to site r is compatible with all of the data from the cytochrome bf and bc₁ complexes. Such a scheme would not be compatible with the semiquinone cycle proposed by Wikström and Krab ((1986) J. Bioenerg. Biomembr. 18, 181–193).     

A kinetic model of the cytochrome bf complex. Evaluation of kinetic parameters

A kinetic model of the cytochrome bf complex was developed on the assumption that the Q-cycle operates. The bf complex was considered as a membrane enzyme catalyzing the electron transfer from plastoquinol to plastocyanine, which is coupled with proton translocation from the chloroplast stroma to the thylakoid lumen. The dependence of the electron transfer rates on the value of the transmembrane electric potential was taken into account. The model was applied to describe the experimental data on the flash-induced turnover of cytochromes b, plastocyanine, and the kinetics of proton deposition in the thylakoid lumen. The estimation of model parameters was performed.     

Mechanism of proton-pumping in the cytochrome b/f complex

Several models have been proposed to interpret the mechanism of proton-pumping associated with the electron transfer reactions in the cytochrome b/f complex. Energetics considerations suggest that the proton pump is coupled to the oxidation of cytochrome b by plastoquinone. Experiments performed in living cells under anaerobic conditions suggest that proton-pumping can occur through two independent mechanisms. When the two b cytochromes are reduced prior to a flash illumination i.e. after a long dark anaerobic incubation (>10 minutes), proton-pumping is very likely associated with the reduction of a semiquinone by cyt b which occurs at a site close to the inner face of the membrane. The electrogenic phase is associated with the tranfer of protons via a transmembrane channel. This process is not inhibited by 2-n-nonyl-4-hydroxyquinoline N-oxide (NQNO). Under repetitive-flash or under aerobic conditions, proton-pumping occurs according to a modified Q-cycle mechanism, which is inhibited by NQNO.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

X-ray structure determination of the cytochrome c2: reaction center electron transfer complex from Rhodobacter sphaeroides

In the photosynthetic bacterium Rhodobacter sphaeroides, a water soluble cytochrome c2 (cyt c2) is the electron donor to the reaction center (RC), the membrane-bound pigment-protein complex that is the site of the primary light-induced electron transfer. To determine the interactions important for docking and electron transfer within the transiently bound complex of the two proteins, RC and cyt c2 were co-crystallized in two monoclinic crystal forms. Cyt c2 reduces the photo-oxidized RC donor (D+), a bacteriochlorophyll dimer, in the co-crystals in approximately 0.9 micros, which is the same time as measured in solution. This provides strong evidence that the structure of the complex in the region of electron transfer is the same in the crystal and in solution. X-ray diffraction data were collected from co-crystals to a maximum resolution of 2.40 A and refined to an R-factor of 22% (R(free)=26%). The structure shows the cyt c2 to be positioned at the center of the periplasmic surface of the RC, with the heme edge located above the bacteriochlorophyll dimer. The distance between the closest atoms of the two cofactors is 8.4 A. The side-chain of Tyr L162 makes van der Waals contacts with both cofactors along the shortest intermolecular electron transfer pathway. The binding interface can be divided into two domains: (i) A short-range interaction domain that includes Tyr L162, and groups exhibiting non-polar interactions, hydrogen bonding, and a cation-pi interaction. This domain contributes to the strength and specificity of cyt c2 binding. (ii) A long-range, electrostatic interaction domain that contains solvated complementary charges on the RC and cyt c2. This domain, in addition to contributing to the binding, may help steer the unbound proteins toward the right conformation.     

Model of a complex between the tetrahemic cytochrome c3 and the ferredoxin I from Desulfovibrio desulfuricans (Norway strain)

A three-dimensional model of an electron-transfer complex between the tetrahemic cytochrome c3 and the ferredoxin I from the sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been generated through computer graphics methods. The model is based on the known X-ray structure of the cytochrome and on a model of the ferredoxin that has been derived through computer graphics modeling and energy minimization methods, from the X-ray structure of the homologous ferredoxin from Peptococcus aerogenes. Four possible models of interaction between the two molecules were examined by bringing in close proximity each of the four hemes and the redox center (4Fe-4S) of the ferredoxin and by optimizing the ion pairs interactions. One of these models shows by far the "best" structure in terms of charges, interactions, and complementarity of the topology of the contact surfaces. In this complex, the distance between the iron atoms of the ferredoxin redox center and the hemic iron atom is 11.8 A, which compares well with those found between redox centers in other complexes. The contact surface area between the two molecules is 170 A2.     

Effects of hydrostatic pressure on the kinetics of electron transfer in an isolated system of chloroplast cytochrome bf complex,plastocyanin and P700

The effects of pressure on the kinetics of redox reactions in and around the chloroplast cytochrome bf complex were studied using a reconstituted system consisting of Photosystem I (PS I) particles, cytochrome bf complex and plastocyanin (PC), all derived from pea chloroplasts. There were no significant permanent effects of pressure in the range 0.1–191 MPa on the reaction kinetics, or on the shape of the absorption spectra of components studied. Discernable effects on rate-coefficients of increasing pressure were observed on the reduction of P700⁺ by PC^I, on the reduction of PC^II by ascorbate, and on the oxidation of decyl plastoquinol by the bf complex. The volumes of activation ΔV^# were determined from the dependence of the rate-coefficient on pressure using: $$(\partial lnk/\partial P)_T = - \Delta V^\# /RT.$$ The volume of activation is the difference in partial molar volume between the activated state and the reactants for the redox reaction. Such data was sought to help define in detail those redox reactions and the corresponding activated states. For the reduction of P700⁺ by PC^I and the oxidation of decyl plastoquinol by the bf complex, the rate coefficient decreased with increase in pressure, whilst for the reduction of PC^II by ascorbate it increased. The corresponding volumes of activation were 9.6±0.6×10^-6 m³ mol^-1, 18±2×10^-6 m³ mol^-1 and -14±1×10^-6 m³ mol^-1, respectively. Much of the pressure-dependence of PC^II reduction by ascorbate was ascribed to an increase in ascorbate ionisation with increase in pressure. There was little effect of pressure on the kinetics of oxidation of ferrocytochrome f by PC^II, or on the equilibrium constant of the redox pair ferrocytochrome f/ferricytochrome f: PC^II/PC^I. Possible physical bases for these activation volumes are discussed, and they are compared with literature values.     

Electron transfer through the isolated mitochondrial cytochrome b-c1 complex

(1) A kinetic analysis of electron donation into and through the cytochrome b-c₁ complex isolated from bovine heart mitochondria has been undertaken, using trimethylquinol as the donor. (2) Rate constants of two routes of redox equilibration with quinols have been defined by kinetic measurements and with the use of the inhibitors antimycin A and myxothiazol. (3) A model of electron transfer based upon the original Q-cycle formulation is presented to explain these and related results.     

Repressible chloroplast gene expression in Chlamydomonas: A new tool for the study of the photosynthetic apparatus

A repressible/inducible chloroplast gene expression system has been used to conditionally inhibit chloroplast protein synthesis in the unicellular alga Chlamydomonas reinhardtii. This system allows one to follow the fate of photosystem II and photosystem I and their antennae upon cessation of chloroplast translation. The main results are that the levels of the PSI core proteins decrease at a slower rate than those of PSII. Amongst the light-harvesting complexes, the decrease of CP26 proceeds at the same rate as for the PSII core proteins whereas it is significantly slower for CP29, and for the antenna complexes of PSI this rate is comprised between that of CP26 and CP29. In marked contrast, the components of trimeric LHCII, the major PSII antenna, persist for several days upon inhibition of chloroplast translation. This system offers new possibilities for investigating the biosynthesis and turnover of individual photosynthetic complexes in the thylakoid membranes. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Probing the events of photoinhibition by altering electron-transport activity and light-harvesting capacity in chloroplast thylakoids

Abstract. The effect of photoinhibition on the activity of photosystem II (PSII) in spinach chloroplasts was investigated. Direct light-induced absorbance change measurements at 320 nm (Δ A ₃₂₀) provided a measure of the PSII charge separation reaction and revealed that photoinhibition prevented the stable photoreduction of the primary quinone acceptor Q_A. Sensitivity to photoinhibition was substantially enhanced by treatment of thylakoids with NH₂OH which extracts manganese from the H₂O-splitting enzyme and prevents electron donation to the reaction centre. Incubation with 3-(3,4,-dichlorophenyl)-1,1-dimethylurea (DCMU) during light exposure did not affect the extent of photoinhibitory damage. The chlorophyll (Chl) b -less chlorina (2 mutant of barley displayed a significantly smaller light-harvesting antenna size of PSII (about 20% of that in wild type chloroplasts) and, simultaneously, a lower sensitivity to photoinhibition. These observations suggest that photoinhibition depends on the amount of light absorbed by PSII and that the process of photoinhibition is accelerated when electron donation to the reaction centre is prevented. It is postulated that the probability of photoinhibition is greater when excitation energy is trapped by P680⁺, the oxidized form of the PSII reaction centre. The results are discussed in terms of the D1/D2 heterodimer which contains the functional PSII components P680, pheophytin, Q_A and Q_B.     

A new approach to experimental investigation of dynamic self-organization in reaction centers of purple bacteria

The results of a study of molecular self-organization processes in the reaction centers (RC) ofRb. Sphaeroides purple bacteria by the method of pulsed optical excitation is presented. The existence of a bistability domain for the parameters of RC recovery kinetics is shown. A good agreement between the theory and experimental results is obtained.     

Molecular dynamics simulations of the cytochrome c3-rubredoxin complex from Desulfovibrio vulgaris.

Molecular dynamics simulations have been carried out on the complex formed between the tetraheme cytochrome c3 and the iron protein rubredoxin from the sulfate-reducing bacterium Desulfovibrio vulgaris. These simulations were performed both with explicit solvent water molecules included, and without solvent molecules using a distance-dependent dielectric constant to approximate the screening effects of solvent. The results of both simulations are strikingly different, indicating that the representation of environmental effects is important in such simulations. For example, a striking adaptation of the two proteins seen in the nonsolvated simulation is not seen when explicit solvent water is included; in fact, the complex appears to become weaker in the solvated simulation. Nonetheless, the iron-iron distance decreases more significantly in the solvated simulation than in the nonsolvated simulation. It was found that in both cases molecular dynamics optimized the structures further than energy minimization alone.     

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.     

On the mode of integration of plastid-encoded components of the cytochrome bf complex into thylakoid membranes

Four distinct integration/translocation routes into/across thylakoid membranes have recently been deduced for nuclear-encoded polypeptides of the photosynthetic membrane. Corresponding information for the plastid-encoded protein complement is lacking. We have investigated this aspect with in-organello assays employing chimeric constructs generated with codon-correct cassettes for genes of plastid-encoded thylakoid proteins, and appropriate transit peptides from six nuclear genes, representing three targeting classes, as a strategy. The three major plastid-encoded components of the cytochrome b ₆  f complex, namely pre-apocytochrome f, (including apocytochrome f, and pre-apocytochrome f lacking the C-terminal transmembrane segment), cytochrome b ₆ , and subunit IV, which differ in the number of their transmembrane segments, were studied. Import into chloroplasts could be observed in all instances but with relatively low efficiency. Thylakoid integration can occurr post-translationally, but only components with secretory/secretory pathway (SEC)-route-specific epitopes were correctly assembled with the cytochrome complex, or competed with this process. Inhibitor studies were consistent with these findings. Imported cytochrome b ₆ and subunit IV operated with uncleaved targeting signals for thylakoid integration. The corresponding determinant for cytochrome f is its signal peptide; its C-terminal hydrophobic segment did not, or did not appreciably, contribute to this process. The N-termini of cytochrome b ₆ and subunit IV appear to reside on the same (lumenal) side of the membrane, consistent with the currently favored four-helix model for the cytochrome, but in disagreement with the topography proposed for both components. The impact of the findings for protein routing, including for applied approaches such as compartment-alien transformation, is discussed. Received: 18 September 1996 / Accepted: 15 October 1996     

A needle in a haystack: the active site of the membrane-bound complex cytochrome c nitrite reductase

Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.     

Ferredoxin:NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b6f complex

Purified detergent-soluble cytochrome b6f complex from chloroplast thylakoid membranes (spinach) and cyanobacteria (Mastigocladus laminosus) was highly active, transferring 300-350 electrons per cyt f/s. Visible absorbance spectra showed a red shift of the cytochrome f alpha-band and the Qy chlorophyll a band in the cyanobacterial complex and an absorbance band in the flavin 450-480-nm region of the chloroplast complex. An additional high molecular weight (M(r) approximately 35,000) polypeptide in the chloroplast complex was seen in SDS-polyacrylamide gel electrophoresis at a stoichiometry of approximately 0.9 (cytochrome f)(-1). The extra polypeptide did not stain for heme and was much more accessible to protease than cytochrome f. Electrospray ionization mass spectrometry of CNBr fragments of the 35-kDa polypeptide was diagnostic for ferredoxin:NADP+ oxidoreductase (FNR), as were antibody reactivity to FNR and diaphorase activity. The absence of FNR in the cyanobacterial complex did not impair decyl-plastoquinol-ferricyanide activity. The activity of the FNR in the chloroplast b6f complex was also shown by NADPH reduction, in the presence of added ferredoxin, of 0.8 heme equivalents of the cytochrome b6 subunit. It was inferred that the b6f complex with bound FNR, one equivalent per monomer, provides the membrane protein connection to the main electron transfer chain for ferredoxin-dependent cyclic electron transport.