Functional implications of pigments bound to a cyanobacterial cytochrome b6f complex

A highly purified cytochrome b(6)f complex from the cyanobacterium Synechocystis sp. PCC 6803 selectively binds one chlorophyll a and one carotenoid in analogy to the recent published structure from two other b(6)f complexes. The unknown function of these pigments was elucidated by spectroscopy and site-directed mutagenesis. Low-temperature redox difference spectroscopy showed red shifts in the chlorophyll and carotenoid spectra upon reduction of cytochrome b(6), which indicates coupling of these pigments with the heme groups and thereby with the electron transport. This is supported by the correlated kinetics of these redox reactions and also by the distinct orientation of the chlorophyll molecule with respect to the heme cofactors as shown by linear dichroism spectroscopy. The specific role of the carotenoid echinenone for the cytochrome b(6)f complex of Synechocystis 6803 was elucidated by a mutant lacking the last step of echinenone biosynthesis. The isolated mutant complex preferentially contained a carotenoid with 0, 1 or 2 hydroxyl groups (most likely 9-cis isomers of beta-carotene, a monohydroxy carotenoid and zeaxanthin, respectively) instead. This indicates a substantial role of the carotenoid - possibly for strucure and assembly - and a specificity of its binding site which is different from those in most other oxygenic photosynthetic organisms. In summary, both pigments are probably involved in the structure, but may also contribute to the dynamics of the cytochrome b(6)f complex.     

b6f-Associated chlorophyll: structural and dynamic contribution to the different cytochrome functions

Cytochromes bc1/b6f complexes catalyze electron transfer from lipid- to water-soluble carriers in both the respiratory and photosynthetic processes. They contain several common redox cofactors, while a chlorophyll a molecule, the function of which is still enigmatic, is only present in b b6f-type complexes. In this work, we describe a mutagenesis approach aimed at characterizing the role of this pigment. Mutants of the binding pocket were constructed to obtain cytochrome (cyt) b6f f complexes altered in chlorophyll position and/or stability. On the basis of a combined biochemical and functional analysis, we conclude that the chlorophyll plays a major structural role in the complex. Moreover, the chlorophyll and its binding pocket may also be implicated in the regulation of photosynthetic state transitions, a function that is specific to cyt b6f complexes.     

Green algal cytochrome b6-f complexes: isolation and characterization from Dunaliella saline, Chlamydomonas reinhardtii and Scenedesmus obliquus

Cytochrome b6-f complexes have been isolated from Chlamydomonas reinhardtii, Dunaliella saline and Scenedesmus obliquus. Each complex is essentially free of chlorophyll and carotenoids and contains cytochrome b6 and cytochrome f hemes in a 2:1 molar ratio. C. reinhardtii and S. obliquus complexes contain the Rieske iron-sulfur protein (present in approx 1:1 molar ratio to cytochrome f) and each catalyzes a DBMIB- and DNP-INT-sensitive electron transfer from duroquinol to spinach plastocyanin. Immunological assays using antibodies to the peptides from the spinach cytochrome complex show varying cross-reactivity patterns except for the complete absence of binding to the Rieske proteins in any of the three complexes, suggesting little structural similarity between the Rieske proteins of algae with those from higher plants. One complex (D. salina) has been uniformly labeled by growth in NaH14CO3 to determine stoichiometries of constituent polypeptide subunits. Results from these studies indicate that all functionally active cytochrome b6-f complexes contain four subunits which occur in equimolar amounts.     

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.     

Site-directed photochemical coupling of cytochrome b6f-associated chlorophyll

Cytochrome b(6)f complexes contain a molecule of chlorophyll a (Chla), which, in Chlamydomonas reinhardtii, can be exchanged for extraneous chlorophyll during protracted incubation of the purified complex in detergent solution. The specificity of the site and its location in the complex have been studied by photochemical coupling and circular dichroism spectroscopy. Following substitution of the original chlorophyll with [(3)H]Chla, the complex was irradiated in the Soret absorption band of Chla to complete bleaching and the amount of radioactivity covalently bound to each b(6)f subunit determined. Strong labeling was found to be associated with cytochrome f. The labeling originates from [(3)H]Chla molecules bound to a slowly exchanging site and showing the properties of the endogenous Chl, not from molecules dissolved in the detergent belt surrounding the complex. Chlorophyll b (Chlb) can compete with Chla, albeit with a lower affinity. Irradiation of [(3)H]Chlb introduced into the slowly exchanging site yielded the same labeling pattern that was observed with [(3)H]Chla. Proteolytic cleavage showed [(3)H]Chla labeling to be strictly restricted to the C-terminal region of cytochrome f. Circular dichroism spectra of the native complex revealed a bilobed signal characteristic of excitonic interaction between chlorophylls. The structural and evolutionary implications of these findings are discussed.     

Soret-excited Raman spectroscopy of the spinach cytochrome b6f complex. Structures of the b- and c-type hemes, chlorophyll a, and beta-carotene

Soret-excited resonance Raman (RR) spectra of the spinach cytochrome b6f complex (cyt b6f) are reported for the oxidized, native, ascorbate-reduced, and dithionite-reduced forms. Using excitations at 441.6, 413.1, and 406.7 nm, RR contributions of chlorophyll a, beta-carotene, the c-type heme of cytochrome f, and the b-type hemes of cytochrome b6 of the b6f complex were identified and the data compared to those previously obtained for the Rhodospirillum rubrum bc1 complex [Le Moigne, C., Schoepp, B., Othman, S., Verméglio, A., and Desbois, A. (1999) Biochemistry 38, 1066-1076]. RR bands arising from the b(6)f-associated chlorophyll a and beta-carotene pigments were found to be particularly intense in the spectra excited at 441.6 nm. The frequencies of the phorbin skeleton of chlorophyll a at 1606, 1552, and 1525 cm(-1) are typical of a Mg atom with a single axial ligand. Strong RR bands corresponding to stretching or deformation modes of beta-carotene were detected at 1137, 1157, 1191, 1216, and 1531 cm(-1) in the different forms of cyt b6f. This set of frequencies is assigned to an all-trans configuration of the polyene chain. The redox titrations of the b(6)f complex allow the characterization of RR bands of the three hemes. The nu10, nu2, nu3, and nu8 modes of reduced cyt f are detected at 1619, 1591, 1492, and 356 cm(-1), respectively. From this set of frequencies, one can conclude that the particular histidine/amine heme coordination found in the truncated soluble domain of cyt f is a specific feature of the entire cyt f included in the b6f complex. The frequencies of the nu2, nu8, and nu10 marker modes are consistent with different conformations for the two b-type hemes of cyt b6f. One of these hemes is strongly distorted (nu2, nu8, and nu10 at 1581, 351, and 1610 cm(-1), respectively), while the other one is planar (1586, 345, and 1618 cm(-1), respectively). Largely different structures for the b-type hemes appear to be a common property for the bc1/b6f complexes.     

On the structural role of the aromatic residue environment of the chlorophyll a in the cytochrome b6f complex

Because light is not required for catalytic turnover of the cytochrome b 6 f complex, the role of the single chlorophyll a in the structure and function of the complex is enigmatic. Photodamage from this pigment is minimized by its short singlet excited-state lifetime ( approximately 200 ps), which has been attributed to quenching by nearby aromatic residues ( Dashdorj et al., 2005). The crystal structure of the complex shows that the fifth ligand of the chlorophyll a contains two water molecules. On the basis of this structure, the properties of the bound chlorophyll and the complex were studied in the cyanobacterium, Synechococcus sp. PCC 7002, through site-directed mutagenesis of aromatic amino acids in the binding niche of the chlorophyll. The b 6 f complex was purified from three mutant strains, a double mutant Phe133Leu/Phe135Leu in subunit IV and single mutants Tyr112Phe and Trp125Leu in the cytochrome b 6 subunit. The purified b 6 f complex from Tyr112Phe or Phe133Leu/Phe135Leu mutants was characterized by (i) a loss of bound Chl and b heme, (ii) a shift in the absorbance peak and increase in bandwidth, (iii) multiple lifetime components, including one of 1.35 ns, and (iv) relatively small time-resolved absorbance anisotropy values of the Chl Q y band. A change in these properties was minimal in the Trp125Leu mutant. In vivo, no decrease in electron-transport efficiency was detected in any of the mutants. It was concluded that (a) perturbation of its aromatic residue niche influences the stability of the Chl a and one or both b hemes in the monomer of the b 6 f complex, and (b) Phe residues (Phe133/Phe135) of subunit IV are important in maintaining the short lifetime of the Chl a singlet excited state, thereby decreasing the probability of singlet oxygen formation.     

HCF164 Encodes a Thioredoxin-Like Protein Involved in the Biogenesis of the Cytochrome b6f Complex in Arabidopsis

To understand the biogenesis of the plastid cytochrome b(6)f complex and to identify the underlying auxiliary factors, we have characterized the nuclear mutant hcf164 of Arabidopsis and isolated the affected gene. The mutant shows a high chlorophyll fluorescence phenotype and is severely deficient in the accumulation of the cytochrome b(6)f complex subunits. In vivo protein labeling experiments indicated that the mutation acts post-translationally by interfering with the assembly of the complex. Because of its T-DNA tag, the corresponding gene was cloned and its identity confirmed by complementation of homozygous mutant plants. HCF164 encodes a thioredoxin-like protein that possesses disulfide reductase activity. The protein was found in the chloroplast, where it is anchored to the thylakoid membrane at its lumenal side. HCF164 is closely related to the thioredoxin-like protein TxlA of Synechocystis sp PCC6803, most probably reflecting its evolutionary origin. The protein also shows a limited similarity to the eubacterial CcsX and CcmG proteins, which are required for the maturation of periplasmic c-type cytochromes. The putative roles of HCF164 for the assembly of the cytochrome b(6)f complex are discussed.     

The Q cycle of cytochrome bc complexes: a structure perspective

Aspects of the crystal structures of the hetero-oligomeric cytochrome bc(1) and b(6)f ("bc") complexes relevant to their electron/proton transfer function and the associated redox reactions of the lipophilic quinones are discussed. Differences between the b(6)f and bc(1) complexes are emphasized. The cytochrome bc(1) and b(6)f dimeric complexes diverge in structure from a core of subunits that coordinate redox groups consisting of two bis-histidine coordinated hemes, a heme b(n) and b(p) on the electrochemically negative (n) and positive (p) sides of the complex, the high potential [2Fe-2S] cluster and c-type heme at the p-side aqueous interface and aqueous phase, respectively, and quinone/quinol binding sites on the n- and p-sides of the complex. The bc(1) and b(6)f complexes diverge in subunit composition and structure away from this core. b(6)f Also contains additional prosthetic groups including a c-type heme c(n) on the n-side, and a chlorophyll a and β-carotene. Common structure aspects; functions of the symmetric dimer. (I) Quinone exchange with the bilayer. An inter-monomer protein-free cavity of approximately 30? along the membrane normal×25? (central inter-monomer distance)×15? (depth in the center), is common to both bc(1) and b(6)f complexes, providing a niche in which the lipophilic quinone/quinol (Q/QH(2)) can be exchanged with the membrane bilayer. (II) Electron transfer. The dimeric structure and the proximity of the two hemes b(p) on the electrochemically positive side of the complex in the two monomer units allow the possibility of two alternate routes of electron transfer across the complex from heme b(p) to b(n): intra-monomer and inter-monomer involving electron cross-over between the two hemes b(p). A structure-based summary of inter-heme distances in seven bc complexes, representing mitochondrial, chromatophore, cyanobacterial, and algal sources, indicates that, based on the distance parameter, the intra-monomer pathway would be favored kinetically. (III) Separation of quinone binding sites. A consequence of the dimer structure and the position of the Q/QH(2) binding sites is that the p-side QH(2) oxidation and n-side Q reduction sites are each well separated. Therefore, in the event of an overlap in residence time by QH(2) or Q molecules at the two oxidation or reduction sites, their spatial separation would result in minimal steric interference between extended Q or QH(2) isoprenoid chains. (IV) Trans-membrane QH(2)/Q transfer. (i) n/p-side QH(2)/Q transfer may be hindered by lipid acyl chains; (ii) the shorter less hindered inter-monomer pathway across the complex would not pass through the center of the cavity, as inferred from the n-side antimycin site on one monomer and the p-side stigmatellin site on the other residing on the same surface of the complex. (V) Narrow p-side portal for QH(2)/Q passage. The [2Fe-2S] cluster that serves as oxidant, and whose histidine ligand serves as a H(+) acceptor in the oxidation of QH(2), is connected to the inter-monomer cavity by a narrow extended portal, which is also occupied in the b(6)f complex by the 20 carbon phytyl chain of the bound chlorophyll.     

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.     

Coupled plasmon-waveguide resonance spectroscopy studies of the cytochrome b6f/plastocyanin system in supported lipid bilayer membranes.

The incorporation of cytochrome (cyt) b6f into a solid-supported planar egg phosphatidylcholine (PC) bilayer membrane and complex formation with plastocyanin have been studied by a variant of surface plasmon resonance called coupled plasmon-waveguide resonance (CPWR) spectroscopy, developed in our laboratory. CPWR combines greatly enhanced sensitivity and spectral resolution with direct measurement of anisotropies in refractive index and optical extinction coefficient, and can therefore probe structural properties of lipid-protein and protein-protein interactions. Cyt b6f incorporation into the membrane proceeds in two stages. The first occurs at low protein concentration and is characterized by an increase in total proteolipid mass without significant changes in the molecular order of the system, as demonstrated by shifts of the resonance position to larger incident angles without changing the refractive index anisotropy. The second stage, occurring at higher protein concentrations, results in a decrease in both the mass density and the molecular order of the system, evidenced by shifts of the resonance position to smaller incident angles and a large decrease in the membrane refractive index anisotropy. Plastocyanin can bind to such a proteolipid system in three different ways. First, the addition of plastocyanin before the second stage of b6f incorporation begins results in complex formation between the two proteins with a KD of approximately 10 microM and induces structural changes in the membrane that are similar to those occurring during the second stage of complex incorporation. The addition of larger amounts of plastocyanin under these conditions leads to nonspecific binding to the lipid phase with a KD of approximately 180 microM. Finally, the addition of plastocyanin after the completion of the second phase of b6f incorporation results in tighter binding between the two proteins (KD approximately 1 microM). Quantitation of the binding stoichiometry indicates that two plastocyanin molecules bind tightly to the dimeric form of the cyt b6f complex, assuming random insertion of the cytochrome into the bilayer. The structural basis for these results and formation of the proteolipid membrane are discussed.     

Electron transfer and stability of the cytochrome b6f complex in a small domain deletion mutant of cytochrome f

The lumen segment of cytochrome f consists of a small and a large domain. The role of the small domain in the biogenesis and stability of the cytochrome b(6)f complex and electron transfer through the cytochrome b(6)f complex was studied with a small domain deletion mutant in Chlamydomonas reinhardtii. The mutant is able to grow photoautotrophically but with a slower rate than the wild type strain. The heme group is covalently attached to the polypeptide, and the visible absorption spectrum of the mutant protein is identical to that of the native protein. The kinetics of electron transfer in the mutant were measured by flash kinetic spectroscopy. Our results show that the rate for the oxidation of cytochrome f was unchanged (t(12) = approximately 100 micros), but the half-time for the reduction of cytochrome f is increased (t(12) = 32 ms; for wild type, t(12) = 2.1 ms). Cytochrome b(6) reduction was slower than that of the wild type by a factor of approximately 2 (t(12) = 8.6 ms; for wild type, t(12) = 4.7 ms); the slow phase of the electrochromic band shift also displayed a slower kinetics (t(12) = 5.5 ms; for wild type, t(12) = 2.7 ms). The stability of the cytochrome b(6)f complex in the mutant was examined by following the kinetics of the degradation of the individual subunits after inhibiting protein synthesis in the chloroplast. The results indicate that the cytochrome b(6)f complex in the small domain deletion mutant is less stable than in the wild type. We conclude that the small domain is not essential for the biogenesis of cytochrome f and the cytochrome b(6)f complex. However, it does have a role in electron transfer through the cytochrome b(6)f complex and contributes to the stability of the complex.     

On the spatial organization of hemes and chlorophyll in cytochrome b(6)f. A linear and circular dichroism study

The organization of chromophores in the cytochrome b(6) f from Chlamydomonas reinhardtii has been studied spectroscopically. Linear dichroism (LD) measurements, performed on the complex co-reconstituted into vesicles with photosynthetic reaction centers as an internal standard, allow the determination of the orientations of the chromophore with respect to the membrane plane. The orientations of the b(H)- and b(L)-hemes are comparable to those determined crystallographically on the cytochrome bc(1). The excitonic CD signal, resulting from the interaction between b-hemes, is similar to that reported for the cytochrome bc(1). LD and CD data are consistent with the differences between the b(6) f and bc(1) leaving the orientation of the b-hemes unaffected. By contrast, the LD data yield a different orientation for the heme f as compared either to the heme c(1) in the crystallographic structures or to the heme f as studied by electron paramagnetic resonance. This difference could either result from incorrect assumptions regarding the orientations of the electronic transitions of the f-heme or may point to the possibility of a redox-dependent movement of cytochrome f. The chlorophyll a was observed in a well defined orientation, further corroborating a specific binding site for it in the b(6) f complex.     

A regulatory role of the PetM subunit in a cyanobacterial cytochrome b6f complex

To investigate the function of the PetM subunit of the cytochrome b6f complex, the petM gene encoding this subunit was inactivated by insertional mutagenesis in the cyanobacterium Synechocystis PCC 6803. Complete segregation of the mutant reveals a nonessential function of PetM for the structure and function of the cytochrome b6f complex in this organism. Photosystem I, photosystem II, and the cytochrome b6f complex still function normally in the petM- mutant as judged by cytochrome f re-reduction and oxygen evolution rates. In contrast to the wild type, however, the content of phycobilisomes and photosystem I as determined from 77 K fluorescence spectra is reduced in the petM- strain. Furthermore, whereas under anaerobic conditions the kinetics of cytochrome f re-reduction are identical, under aerobic conditions these kinetics are slower in the petM- strain. Fluorescence induction measurements indicate that this is due to an increased plastoquinol oxidase activity in the mutant, causing the plastoquinone pool to be in a more oxidized state under aerobic dark conditions. The finding that the activity of the cytochrome b6f complex itself is unchanged, whereas the stoichiometry of other protein complexes has altered, suggests an involvement of the PetM subunit in regulatory processes mediated by the cytochrome b6f complex.     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl(2), an inhibitor of cytochrome b(6)f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b(6)f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

HCF153, a novel nuclear-encoded factor necessary during a post-translational step in biogenesis of the cytochrome bf complex

We have isolated the nuclear photosynthetic mutant hcf153 which shows reduced accumulation of the cytochrome b(6)f complex. The levels and processing patterns of the RNAs encoding the cytochrome b(6)f subunits are unaltered in the mutant. In vivo protein labeling experiments and analysis of polysome association revealed normal synthesis of the large chloroplast-encoded cytochrome b(6)f subunits. The mutation resulted from a T-DNA insertion and the affected nuclear gene was cloned. HCF153 encodes a 15 kDa protein containing a chloroplast transit peptide. Sequence similarity searches revealed that the protein is restricted to higher plants. A HCF153-Protein A fusion construct introduced into hcf153 mutant plants was able to substitute the function of the wild-type protein. Fractionation of intact chloroplasts from these transgenic plants suggests that most or all of the fusion protein is tightly associated with the thylakoid membrane. Our data show that the identified factor is a novel protein that could be involved in a post-translational step during biogenesis of the cytochrome b(6)f complex. It is also possible that HCF153 is necessary for translation of one of the very small subunits of the cytochrome b(6)f complex.     

Reconstitution of cytochrome f/b6 and CF0-CF1 ATP synthetase complexes into phospholipid and galactolipid liposomes

Cytochrome f/b6 and ATP synthetase (CF0-CF1) complexes from spinach chloroplasts have been reconstituted into liposomes prepared from soybean phospholipids and purified spinach galactolipids. Freeze- fracture analysis revealed homogeneous populations of particles spanning the lipid bilayers with their elongated axes perpendicular to the membrane plane. The lipid composition of the liposomes had no effect on the size of the reconstituted complexes, the average diameter of cytochrome f/b6 complex measuring 8.5 nm, and of the CF0 base piece of the ATP synthetase, 9.5 nm. When reconstituted cytochrome f/b6 complexes were cross-linked by means of antibodies prepared against the whole complex, the thus aggregated particles formed either hexagonal or square arrays. In both instances the center-to-center spacing of the particles was 8.3 nm, thereby suggesting that this value could be closer to the real diameter of the complexes than the one obtained from measuring individual particles. Assuming an ellipsoidal shape for these particles, and using a measured height of 11 nm, a molecular weight of approximately 280,000 could be calculated for the reconstituted cytochrome f/b6 complex, consistent with a dimeric configuration. In many instances the crystalline sheets of antibody-aggregated cytochrome f/b6 complexes were found to be free in the buffer solution; apparently the antibody-induced strains caused the sheet-like aggregates to pop out of the liposomal membranes. Agglutination studies of inside-out and right-side-out thylakoid vesicles revealed the antigenic determinants of the cytochrome f and cytochrome b6 polypeptides to be exposed on the inner thylakoid surface and to be present in stacked and unstacked membrane regions. The molecular weight calculated from the size of freeze-fractured CF0 base pieces was over twice the value determined by x-ray scattering data. This discrepancy may be caused by significant lipid domains within the base piece, or by an unusual fracturing behavior of the base piece in reconstituted liposomes.     

The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase.

We created a Qo pocket mutant by site-directed mutagenesis of the chloroplast petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY sequence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow in phototrophic conditions although it assembled wild-type levels of cytochrome b6f complexes. We demonstrated a complete block in electron transfer through the cytochrome b6f complex and a loss of plastoquinol binding at Qo. The accumulation of cytochrome b6f complexes lacking affinity for plastoquinol enabled us to investigate the role of plastoquinol binding at Qo in the activation of the light-harvesting complex II (LHCII) kinase during state transitions. We detected no fluorescence quenching at room temperature in state II conditions relative to that in state I. The quantum yield spectrum of photosystem I charge separation in the two state conditions displayed a trough in the absorption region of the major chlorophyll a/b proteins, demonstrating that the cells remained locked in state I. 33Pi labeling of the phosphoproteins in vivo demonstrated that the antenna proteins remained poorly phosphorylated in both state conditions. Thus, the absence of state transitions in the pwye mutant demonstrates directly that plastoquinol binding in the Qo pocket is required for LHCII kinase activation.