Projection structure and oligomeric properties of a bacterial core protein translocase.

The major route for protein export or membrane integration in bacteria occurs via the Sec-dependent transport apparatus. The core complex in the inner membrane, consisting of SecYEG, forms a protein-conducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, purified and crystallized in two dimensions. A 9 A projection structure was calculated using electron cryo-microscopy. The structure exhibits P12(1) symmetry, having two asymmetric units inverted with respect to one another in the unit cell. The map shows elements of secondary structure that appear to be transmembrane helices. The crystallized form of SecYEG is too small to comprise the translocation channel and does not contain a large pore seen in other studies. In detergent solution, the SecYEG complex displays an equilibrium between monomeric and tetrameric forms. Our results therefore indicate that, unlike other known channels, the SecYEG complex can exist as both an assembled channel and an unassembled smaller unit, suggesting that transitions between the two states occur during a functional cycle.

The cytochrome b(6)f complex: structural studies and comparison with the bc(1) complex.

Electron crystallography of the chloroplastic b(6)f complex allowed the calculation of projection maps of crystals negatively stained or embedded in glucose. This gives insights into the overall structure of the extra- and transmembrane domains of the complex. A comparison with the structure of the bc(1) complex, the mitochondrial homologue of the b(6)f complex, suggests that the transmembrane domains of the two complexes are very similar, confirming the structural homology deduced from sequence analysis. On the other hand, the extramembrane organisation of the c-type cytochrome and of the Rieske protein seems quite different. Nevertheless, the same type of movement of the Rieske protein is observed in the b(6)f as in the bc(1) complex upon the binding of the quinol analogue stigmatellin. Crystallographic data also suggest movements in the transmembrane domains of the b(6)f complex, which would be specific of the b(6)f complex.

Kinetic effects of the electrochemical proton gradient on plastoquinone reduction at the Qi site of the cytochrome b6f complex.

We have investigated the effects of the light-induced thylakoid transmembrane potential on the turnover of the b(6)f complex in cells of the unicellular green alga Chlamydomonas reinhardtii. The reduction of the potential by either decreasing the light intensity or by adding increasing concentrations of the ionophore carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) revealed a marked inhibition of the cytochrome b(6) oxidation rate (10-fold) without substantial modifications of cytochrome f oxidation kinetics. Partial recovery of this inhibition could be obtained in the presence of ionophores provided that the membrane potential was re-established by illumination with a train of actinic flashes fired at a frequency higher than its decay. Measurements of isotopic effects on the kinetics of cytochrome b(6) oxidation revealed a synergy between the effects of ionophores and the H(2)O-D(2)O exchange. We propose therefore, that protonation events influence the kinetics of cytochrome b(6) oxidation at the Qi site and that these reactions are strongly influenced by the light-dependent generation of a transmembrane potential.

Conformational changes in the cytochrome b6f complex induced by inhibitor binding.

Binding of stigmatellin, an inhibitor of the Q(o) site of the bc-type complexes, has been shown to induce large conformational changes of the Rieske protein in the respiratory bc(1) complex (Kim, H., Xia, D., Yu, C. A., Xia, J. Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8026-8033; Iwata, S., Lee, J. W., Okada, K., Lee, J. K., Iwata, M., Rasmussen, B., Link, T. A., Ramaswamy, S., and Jap, B. K. (1998) Science 281, 64-71; Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y. I., Kim, K. K., Hung, L. W., Crofts, A. R., Berry, E. A., and Kim, S. H. (1998) Nature 392, 677-684). Such a movement seems necessary to shuttle electrons from the membrane-soluble quinol to the extramembrane heme of cytochrome c(1). To see whether similar changes occur in the related photosynthetic b(6)f complex, we have studied the effect of the binding of stigmatellin to the eukaryotic b(6)f complex by electron crystallography. Comparison of projection maps of thin three-dimensional crystals prepared with or without stigmatellin, and either negatively stained or embedded in glucose, reveals a similar type of movement to that observed in the bc(1) complex and suggests also the occurrence of conformational changes in the transmembrane region.

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.

The 9 A projection structure of cytochrome b6f complex determined by electron crystallography.

Thin three-dimensional crystals of the cytochrome b6 f complex from the unicellular algae Chlamydomonas reinhardtii have been grown by BioBeads-mediated detergent removal from a mixture of protein and lipid solubilized in Hecameg. Frozen-hydrated crystals, exhibiting p22121 plane group symmetry, were studied by electron crystallography and a projection map at 9 A resolution was calculated. The crystals (unit cell dimensions of a=173.5 A, b=70.0 A and gamma=90.0 degrees) showed the presence of dimers, and within each monomer 14 domains of electron density were observed. The combination of the projection map obtained from ice-embedded crystals of cytochrome b6 f with a previous map obtained from negatively stained samples brings new insight in the organization of the complex. For example, it distinguishes some peaks and/or domains that are only extramembrane or transmembrane, and reveals the possible localization of single-stranded transmembrane alpha-helices (Pet subunits). Furthermore, the cross-correlation of our projection map from frozen hydrated samples with the atomic model of the transmembrane part of the cytochrome bc1 complex has allowed us to localize the cytochrome b6 at the dimer interface and to reveal structural differences between the two complexes.

Projection map of cytochrome b6 f complex at 8 A resolution.

The structure of the cytochrome b6 f complex has been investigated by electron microscopy and image analysis of thin three-dimensional crystals. Electron micrographs of negatively stained specimens were recorded and showed optical diffraction peaks to 10 A resolution. A projection map was calculated at 8 A resolution and showed the presence of cytochrome b6 f dimers. The extramembrane part of each monomer featured a C shape, with mean external diameter approximately of 53 A and an internal groove approximately 14 A long and approximately 9 A wide. Within each monomer, strong features were clearly resolved and tentatively attributed to some of the subunits of the cytochrome b6 f complex. The data are consistent with the Rieske iron-sulfur protein lying close to the monomer-monomer interface and the heme-bearing domain of cytochrome f far from it.

Dimer to monomer conversion of the cytochrome b6 f complex. Causes and consequences.

The molecular weight of the cytochrome b6 f complex purified from Chlamydomonas reinhardtii thylakoid membranes has been determined by combining velocity sedimentation measurements, molecular sieving analyses, and determination of its lipid and detergent content. The complex in its enzymatically active form is a dimer. Upon incubation in detergent solution, it converts irreversibly into an inactive, monomeric form that has lost the Rieske iron-sulfur protein, the b6 f-associated chlorophyll, and, under certain conditions, the small 32-residue subunit PetL. The results are consistent with the view that the dimer is the predominant form of the b6f in situ while the monomer observed in detergent solution is a breakdown product. Indirect observations suggest that subunit PetL plays a role in stabilizing the dimeric state. Delipidation is shown to be a critical factor in detergent-induced monomerization.

On the presence and role of a molecule of chlorophyll a in the cytochrome b6 f complex.

Highly purified preparations of cytochrome b6 f complex from the unicellar freshwater alga Chlamydomonas reinhardtii contain about 1 molecule of chlorophyll a/cytochrome f. Several lines of evidence indicate that the chlorophyll is an authentic component of the complex rather than a contaminant. In particular, (i) the stoichiometry is constant; (ii) the chlorophyll is associated with the complex at a specific binding site, as evidenced by resonance Raman spectroscopy; (iii) it does not originate from free chlorophyll released from thylakoid membranes upon solubilization; and (iv) its rate of exchange with free, radioactive chlorophyll a is extremely slow (weeks). Some of the putative functional roles for a chlorophyll in the b6f complex are experimentally ruled out, and its possible evolutionary origin is briefly discussed.

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.

The 4-kDa nuclear-encoded PetM polypeptide of the chloroplast cytochrome b6f complex. Nucleic acid and protein sequences, targeting signals, transmembrane topology.

The 4-kDa subunit of cytochrome b6f complex encoded by the nuclear PetM gene in Chlamydomonas reinhardtii has been characterized. 38 of the 39 residues of the mature protein have been established by Edman degradation, a cDNA clone encoding the complete precursor has been isolated and sequenced, and a 0.6-kb transcript detected. The deduced amino acid sequence of the precursor includes an N-terminal transit peptide of 60 amino acids with stromal targeting features. Examination of the sequence suggests that PetM spans the membrane as a single transmembrane alpha-helix, which is supported by its non-extractability following dissociating treatments. When PetM and PetG, another small subunit of the b6f complex, are folded into alpha-helices, an array of identical residues becomes apparent. Proteolysis data, charge distribution, and homology with PetG are consistent with a lumenal localization of the N terminus of PetM.

Purification and characterization of the cytochrome b6 f complex from Chlamydomonas reinhardtii.

A protocol has been developed for the purification of the cytochrome b6 f complex from the unicellular alga Chlamydomonas reinhardtii. It is based on the use of the neutral detergent Hecameg (6-O-(N-heptylcarbamoyl)-methyl-alpha-D-glycopyranoside) and comprises only three steps: selective solubilization from thylakoid membranes, sucrose gradient sedimentation, and hydroxylapatite chromatography. The purified complex contains two b hemes (alpha bands, 564 nm; Em,8 = -84 and -158 mV) and one chlorophyll alpha (lambda max = 667-668 nm) per cytochrome f (alpha band, 554 nm; Em,8 = +330 mV). It is highly active in transferring electrons from decylplastoquinol to oxidized plastocyanin (turnover number 250-300 s-1). The purified complex contains seven subunits, whose identity has been established by N-terminal sequencing and/or peptide-specific immunolabeling, namely four high molecular weight subunits (cytochrome f, Rieske iron-sulfur protein, cytochrome b6, and subunit IV) and three approximately 4-kDa miniproteins (PetG, PetL, and PetX). Stoichiometry measurements are consistent with every subunit being present as two copies per b6 f dimer.

Membrane association of cytochrome b6f subunits. The Rieske iron-sulfur protein from Chlamydomonas reinhardtii is an extrinsic protein.

The mode of membrane attachment of five subunits from Chlamydomonas reinhardtii cytochrome b6f complex has been studied using biochemical approaches. Antisera specific for cytochrome f, cytochrome b6, the Rieske iron-sulfur protein, subunit IV, and a 4-kDa subunit (product of the petG gene) were used to quantify the degree of extraction of each of these polypeptides following various treatments. In contrast to the other four subunits, the Rieske protein was extracted to extents varying between 50 and 100% following two cycles of freezing and thawing in the presence of chaotropic agents (KSCN, urea, or NaI). The Rieske protein was not extracted by 2 M NaCl and was rather resistant to alkaline treatments, being extracted by 20 mM 3-(cyclohexylamino)propanesulfonic acid buffer only at pH > 11.5. The hydrodynamic behavior of the isolated Rieske protein was examined in the absence and presence of detergent by ultracentrifugation and by molecular sieving. The extracted protein bound neither to laurylmaltoside nor to C12E8 micelles. Its sedimentation coefficient (D20,w = 9.6 x 10(-11) m2 x s-1), diffusion coefficient (s20,w = 2S), an deduced molecular mass (20.0 +/- 1.7 kDa) are those expected for the monomeric protein. We conclude that the Rieske protein is extrinsic and therefore does not cross the membrane, although its association with the rest of the complex involves primarily hydrophobic interactions, and that the other four subunits analyzed are intrinsic.