Analysis of the nucleus-encoded and chloroplast-targeted rieske protein by classic and site-directed mutagenesis of Chlamydomonas.

Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron-sulfur protein 2Fe-2S subunit of the chloroplast cytochrome b(6)f complex have been characterized. One has a stable deletion that eliminates the protein; two others carry substitutions Y87D and W163R that result in low accumulation of the protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b(6)f complexes of the Y87D and W163R mutant Rieske proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe-2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske protein of photosynthesis.     

The chloroplast ycf7 (petL) open reading frame of Chlamydomonas reinhardtii encodes a small functionally important subunit of the cytochrome b6f complex.

The small chloroplast open reading frame ORF43 (ycf7) of the green unicellular alga Chlamydomonas reinhardtii is cotranscribed with the psaC gene and ORF58. While ORF58 has been found only in the chloroplast genome of C.reinhardtii, ycf7 has been conserved in land plants and its sequence suggests that its product is a hydrophobic protein with a single transmembrane alpha helix. We have disrupted ORF58 and ycf7 with the aadA expression cassette by particle-gun mediated chloroplast transformation. While the ORF58::aadA transformants are indistinguishable from wild type, photoautotrophic growth of the ycf7::aadA transformants is considerably impaired. In these mutant cells, the amount of cytochrome b6f complex is reduced to 25-50% of wild-type level in mid-exponential phase, and the rate of transmembrane electron transfer per b6f complex measured in vivo under saturating light is three to four times slower than in wild type. Under subsaturating light conditions, the rate of the electron transfer reactions within the b6f complex is reduced more strongly in the mutant than in the wild type by the proton electrochemical gradient. The ycf7 product (Ycf7) is absent in mutants deficient in cytochrome b6f complex and present in highly purified b6f complex from the wild-type strain. Ycf7-less complexes appear more fragile than wild-type complexes and selectively lose the Rieske iron-sulfur protein during purification. These observations indicate that Ycf7 is an authentic subunit of the cytochrome b6f complex, which is required for its stability, accumulation and optimal efficiency. We therefore propose to rename the ycf7 gene petL.     

Assembly of the Rieske iron-sulphur protein into the cytochrome bf complex in thylakoid membranes of isolated pea chloroplasts.

The assembly of the Rieske iron-sulphur protein into the cytochrome bf complex was examined following import of 35S-labeled precursor protein by isolated pea chloroplasts. Rieske protein assembled into the cytochrome bf complex was resolved from unassembled Rieske protein and from other membrane complexes by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. Four mutant forms of the Rieske protein were able to assemble into the cytochrome bf complex in isolated chloroplasts. These were a triple substitution mutant, C107S/H109R/C112S, replacing conserved residues involved in the ligation of the [2Fe-2S] centre; the mutant Delta45-52 which removed a glycine-rich region predicted to form a flexible hinge between the hydrophobic membrane-associated region and the hydrophilic lumenal domain; and mutants Delta168-173 and Delta177-179 which removed two C-terminal regions, which are highly conserved in chloroplast and cyanobacterial Rieske proteins. This indicates that the [2Fe-2S] cluster, the glycine-rich region and the C-terminal region are not essential for stable assembly of the Rieske protein into the cytochrome bf complex in isolated chloroplasts.     

Changes to the length of the flexible linker region of the Rieske protein impair the interaction of ubiquinol with the cytochrome bc1 complex.

Crystal structures of the cytochrome bc1 complex indicate that the catalytic domain of the Rieske iron-sulfur protein, which carries the [2Fe-2S] cluster, is connected to a transmembrane anchor by a flexible linker region. This flexible linker allows the catalytic domain to move between two positions, proximal to cytochrome b and cytochrome c1. Addition of an alanine residue to the flexible linker region of the Rieske protein lowers the ubiquinol-cytochrome c reductase activity of the mitochondrial membranes by one half and causes the apparent Km for ubiquinol to decrease from 9.3 to 2.6 microM. Addition of two alanine residues lowers the activity by 90% and the apparent Km decreases to 1.9 microM. Deletion of an alanine residue lowers the activity by approximately 40% and the apparent Km decreases to 5.0 microM. Addition or deletion of an alanine residue also causes a pronounced decrease in efficacy of inhibition of ubiquinol-cytochrome c reductase activity by stigmatellin, which binds analogous to reaction intermediates of ubiquinol oxidation. These results indicate that the length of the flexible linker region is critical for interaction of ubiquinol with the bc1 complex, consistent with electron transfer mechanisms in which ubiquinol must simultaneously interact with the iron-sulfur protein and cytochrome b.     

Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b(6)f complex

In the green alga Chlamydomonas reinhardtii, the ClpP protease is encoded by an essential chloroplast gene. Mutating its AUG translation initiation codon to AUU reduced ClpP accumulation to 25 to 45% of that of the wild type. Both the mature protein and the putative precursor containing its insertion sequence were present in reduced amounts. Attenuation of ClpP did not affect growth rates under normal conditions but restricted the ability of the cells to adapt to elevated CO(2) levels. It also affected the rate of degradation of the cytochrome b(6)f complex of the thylakoid membrane in two experimental situations: (1) during nitrogen starvation, and (2) in mutants deficient in the Rieske iron-sulfur protein. The ClpP level also controls the steady state accumulation of a mutated version of the Rieske protein. In contrast, attenuation of ClpP did not rescue the fully unassembled subunits in other cytochrome b(6)f mutants. We conclude that proteolytic disposal of fully or partially assembled cytochrome b(6)f is controlled by the Clp protease.     

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.     

Topographical studies of the polypeptide subunits of the thylakoid cytochrome b6-f complex

The orientation of specific polypeptides of the cytochrome b6-f complex with respect to the chloroplast stromal phase has been studied using trinitrobenzenesulfonate (TNBS) and pronase E as impermeant modifying reagents. Of the four polypeptides of the complex (33,23,20 and 17 kDa), only cytochrome f was labeled by 14C-TNBS in unfractionated membranes. However, to a varying degree, all of the constituent polypeptides were sensitive to pronase digestion and, in the case of cytochrome f, it was possible, by immunoblotting techniques to identify several degradation products. These results are discussed in relation to the organization of the cytochrome complex in thylakoid membranes and argue for an exposure to the stromal phase of all of the polypeptides, while functional considerations indicate that at least cytochrome f and the Rieske iron-sulfur protein have a possible transmembrane organization.     

Cytochrome f translation in Chlamydomonas chloroplast is autoregulated by its carboxyl-terminal domain

The rate of synthesis of cytochrome f is decreased approximately 10-fold when it does not assemble with the other subunits of the cytochrome b(6)f complex in Chlamydomonas reinhardtii chloroplasts. This assembly-mediated regulation of cytochrome f synthesis corresponds to a regulation of petA mRNA initiation of translation. Here, we demonstrate that cytochrome f translation is autoregulated by its C-terminal domain. Five cytochrome f residues conserved throughout all chloroplast genomes-residue Gln-297 in the transmembrane helix and a cluster of four amino acids, Lys-Gln-Phe-Glu, at positions 305 to 308, in the stromal extension-participate in the formation of a translation repressor motif. By contrast, positively charged residues in the stromal extension have little influence on the autoregulation process. These results do not favor a direct interaction between the repressor motif and the petA 5' untranslated region but suggest the participation of a membrane-bound ternary effector.     

Contrasted effects of inhibitors of cytochrome b6f complex on state transitions in Chlamydomonas reinhardtii: the role of Qo site occupancy in LHCII kinase activation

We have investigated the relationship between the occupancy of the Q(o) site in the cytochrome b(6)f complex and the activation of the LHCII protein kinase that controls state transitions. To this aim, fluorescence emission and LHCII phosphorylation patterns were studied in whole cells of Chlamydomonas reinhardtii treated with different plastoquinone analogues. The analysis of fluorescence induction at room temperature indicates that stigmatellin consistently prevented transition to State 2, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone behaved as an inhibitor of state transitions only after the cells were preilluminated. The same effects were observed on the phosphorylation patterns of the LHCII proteins, while subunit V of the cytochrome b(6)f complex showed a different behavior. These findings are discussed on the basis of a dynamic structural model of cytochrome b(6)f that relates the activation of the LHCII kinase to the occupancy of the Q(o) site and the movement of the Rieske protein.     

The structure of the soluble domain of an archaeal Rieske iron-sulfur protein at 1.1 A resolution

The first crystal structure of an archaeal Rieske iron-sulfur protein, the soluble domain of Rieske iron-sulfur protein II (soxF) from the hyperthermo-acidophile Sulfolobus acidocaldarius, has been solved by multiple wavelength anomalous dispersion (MAD) and has been refined to 1.1 A resolution. SoxF is a subunit of the terminal oxidase supercomplex SoxM in the plasma membrane of S. acidocaldarius that combines features of a cytochrome bc(1) complex and a cytochrome c oxidase. The [2Fe-2S] cluster of soxF is most likely the primary electron acceptor during the oxidation of caldariella quinone by the cytochrome a(587)/Rieske subcomplex. The geometry of the [2Fe-2S] cluster and the structure of the cluster-binding site are almost identical in soxF and the Rieske proteins from eucaryal cytochrome bc(1) and b(6)f complexes, suggesting a strict conservation of the catalytic mechanism. The main domain of soxF and part of the cluster-binding domain, though structurally related, show a significantly divergent structure with respect to topology, non-covalent interactions and surface charges. The divergent structure of soxF reflects a different topology of the soxM complex compared to eucaryal bc complexes and the adaptation of the protein to the extreme ambient conditions on the outer membrane surface of a hyperthermo-acidophilic organism.     

Co-operation between different targeting pathways during integration of a membrane protein

Membrane protein assembly is a fundamental process in all cells. The membrane-bound Rieske iron-sulfur protein is an essential component of the cytochrome bc₁ and cytochrome b₆f complexes, and it is exported across the energy-coupling membranes of bacteria and plants in a folded conformation by the twin arginine protein transport pathway (Tat) transport pathway. Although the Rieske protein in most organisms is a monotopic membrane protein, in actinobacteria, it is a polytopic protein with three transmembrane domains. In this work, we show that the Rieske protein of Streptomyces coelicolor requires both the Sec and the Tat pathways for its assembly. Genetic and biochemical approaches revealed that the initial two transmembrane domains were integrated into the membrane in a Sec-dependent manner, whereas integration of the third transmembrane domain, and thus the correct orientation of the iron-sulfur domain, required the activity of the Tat translocase. This work reveals an unprecedented co-operation between the mechanistically distinct Sec and Tat systems in the assembly of a single integral membrane protein.     

Overexpression and reconstitution of a Rieske iron-sulfur protein from the higher plant

The iron-sulfur protein subunit, known as the Rieske protein, is one of the central components of the cytochrome b(6)f complex residing in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overexpression in Escherichia coli of full-length and truncated Rieske (PetC) proteins from the Spinacia oleracea fused to MalE. Overexpressed fusion proteins were predominantly found (from 55 to 70%) in cytoplasm in a soluble form. The single affinity chromatography step (amylose resine) was used to purify about 15mg of protein from 1 liter of E. coli culture. The isolated proteins were electrophoretically pure and could be used for further experiments. The NifS-like protein IscS from the cyanobacterium Synechocystis PCC 6803 mediates the incorporation of 2Fe-2S clusters into apoferredoxin and cyanobacterial Rieske apoprotein in vitro. Here, we used the recombinant IscS protein for the enzymatic reconstitution of the iron-sulfur cluster into full-length Rieske fusion and truncated Rieske fused proteins. Characterization by EPR spectroscopy of the reconstituted proteins demonstrated the presence of a 2Fe-2S cluster in both full-length and truncated Rieske fusion proteins.     

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.     

Failure to insert the iron-sulfur cluster into the Rieske iron-sulfur protein impairs both center N and center P of the cytochrome bc1 complex

Mutation of a serine that forms a hydrogen bond to the iron-sulfur cluster of the Rieske iron-sulfur protein to a cysteine results in a respiratory-deficient yeast strain due to formation of iron-sulfur protein lacking the iron-sulfur cluster. The Rieske apoprotein lacking the iron-sulfur cluster is inserted into both monomers of the dimeric cytochrome bc(1) complex and processed to mature size, but the protein lacking iron-sulfur cluster is more susceptible to proteolysis. In addition, the protein environment of center P in one half of the dimer is affected by failure to insert the iron-sulfur cluster as indicated by the fact that only one molecule of myxothiazol can be bound to the cytochrome bc(1) dimer. Although the bc(1) complex lacking the Rieske iron-sulfur cluster cannot oxidize ubiquinol through center P, rates of reduction of cytochrome b by menaquinol through center N are normal. However, less cytochrome b is reduced through center N, and only one molecule of antimycin can be bound at center N in the bc(1) dimer lacking iron-sulfur cluster. These results indicate that failure to insert the [2Fe-2S] cluster impairs assembly of the Rieske protein into the bc(1) complex and that this interferes with proper assembly of both center P and center N in one half of the dimeric enzyme.     

Structure and reaction mechanisms of multifunctional mitochondrial cytochrome bc1 complex.

The cytochrome bc1 complex from bovine heart mitochondria is a multi-functional enzyme complex. In addition to electron and proton transfer activity, the complex also processes an activatable peptidase activity and a superoxide generating activity. The crystal structure of the complex exists as a closely interacting functional dimer. There are 13 transmembrane helices in each monomer, eight of which belong to cytochrome b, and five of which belong to cytochrome c1, Rieske iron-sulfur protein (ISP), subunits 7, 10 and 11, one each. The distances of 21 A between bL heme and bH heme and of 27 A between bL heme and the iron-sulfur cluster (FeS), accommodate well the observed fast electron transfers between the involved redox centers. However, the distance of 31 A between heme c1 and FeS, makes it difficult to explain the high electron transfer rate between them. 3D structural analyses of the bc1 complexes co-crystallized with the Qu site inhibitors suggest that the extramembrane domain of the ISP may undergo substantial movement during the catalytic cycle of the complex. This suggestion is further supported by the decreased in the cytochrome bc1 complex activity and the increased in activation energy for mutants with increased rigidity in the neck region of ISP.     

Orientation of the g-tensor axes of the Rieske subunit in the cytochrome bc1 complex

The orientation of the g-tensors of the Rieske iron-sulfur protein subunit was determined in a single crystal of the bovine mitochondrial cytochrome bc1 complex with stigmatellin in the Qo quinol binding site. The g-tensor principal axes are skewed with respect to the Fe-Fe and S-S atom direction in the 2Fe2S cluster, which is allowed by the lack of rigorous symmetry of the cluster. The asymmetric unit in the crystal is the active dimer, and the g-tensor axes have slightly different orientations relative to the iron-sulfur cluster in the two halves of the dimer. The g approximately 1.79 axis makes an average angle of 30 degrees with respect to the Fe-Fe direction and the g approximately 2.024 axis an average angle of 26 degrees with respect to the S-S direction. This assignment of the g-tensor axis directions indicates that conformations of the Rieske protein are likely the same in the cytochrome bc1 and b6f complexes and that the extent of motion of the Rieske head domain during the catalytic cycle has been highly conserved during evolution of these distantly related complexes.     

Mitochondrial superoxide radical formation is controlled by electron bifurcation to the high and low potential pathways

The generation of oxygen radicals in biological systems and their sites of intracellular release have been subject of numerous studies in the last decades. Based on these studies mitochondria are considered to be the major source of intracellular oxygen radicals. Although this finding is more or less accepted, the mechanism of univalent oxygen reduction in mitochondria is still obscure. One of the most critical electron transfer steps in the respiratory chain is the electron bifurcation at the cytochrome bc 1 complex. Recent studies with genetically mutated mitochondria have made it clear that electron bifurcation from ubiquinol to the cytochrome bc 1 complex requires the free mobility of the head domain of the Rieske iron-sulfur protein. On the other hand, it has been long known that inhibition of electron bifurcation by antimycin A causes leakage of single electrons to dioxygen, which results in the release of superoxide radicals. These findings lead us to study whether hindrance of the interaction of ubiquinol with the cytochrome bc 1 complex is the regulator of single electron diversion to oxygen. Hindrance of electron bifurcation was observed following alterations of the physical state of membrane phospholipids in which the cytochrome bc 1 complex is inserted. Irrespective of whether the fluidity of the membrane lipids was elevated or decreased, electron flow rates to the Rieske iron-sulfur protein were drastically reduced. Concomitantly superoxide radicals were released from these mitochondria, strongly suggesting an effect on the mobility of the head domain of the Rieske iron-sulfur protein. This revealed the involvement of the ubiquinol cytochrome bc 1 redox couple in mitochondrial superoxide formation. The regulator, which controls leakage of electrons to oxygen, appears to be the electron-branching activity of the cytochrome bc 1 complex.     

Iron-sulfur cluster reconstitution of spinach chloroplast Rieske protein requires a partially prefolded apoprotein

The Rieske 2Fe-2S protein is a central component of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for expression in Escherichia coli of full-length and truncated Spinacia oleracea Rieske (PetC) proteins fused to the MalE, maltose binding protein. The expressed Rieske fusion proteins were found predominantly in soluble form in the E. coli cytoplasm. These proteins could be readily purified for further experimentation. In vitro reconstitution of the characteristic, "Rieske-type" 2Fe-2S cluster into these fused proteins was accomplished by a chemical method employing reduced iron and sulfide. Cluster incorporation was monitored by electron paramagnetic resonance and optical circular dichroism (CD) spectroscopy. CD spectral analysis in the ultraviolet region suggests that the spinach Rieske apoprotein must be in a partially folded conformation to incorporate an appropriate iron-sulfur cluster. These data further suggest that upon cluster integration, further folding occurs, allowing the Rieske protein to attain a final, native structure. The data presented here are the first to demonstrate successful chemical reconstitution of the 2Fe-2S cluster into a Rieske apoprotein from higher plant chloroplasts.