Research Article 1613

Biological identity and diversity in photosynthesis and respiration: structure of the lumen-side domain of the chloroplast Christopher J Carrell, Huamin Zhang, William A Cramer and Janet L Smith*

Background: The b6f complex functions in oxygenic Address: Department of Biological Sciences, photosynthesis as an integral membrane protein complex that mediates coupled Purdue University, West Lafayette, IN 47907-1392, electron transfer and proton translocation. The Rieske [2Fe–2S] protein subunit USA. of the complex functions at the electropositive (p) membrane interface as the *Corresponding author. electron acceptor for plastoquinol and donor for the subunit, and E-mail: [email protected] may have a dynamic role in catalyzing electron and proton transfer at the Key words: membrane interface. There are significant structure/function similarities to the , cytochrome f, energy transduction, plastoquinone, proton cytochrome bc1 complex of the respiratory chain. translocation

Results: The 1.83 Å crystal structure of a 139-residue C-terminal fragment of Received: 19 August 1997 the Rieske [2Fe–2S] protein, derived from the cytochrome b f complex of Revisions requested: 29 September 1997 6 Revisions received: 15 October 1997 spinach chloroplasts, has been solved by multiwavelength anomalous Accepted: 16 October 1997 diffraction. The structure of the fragment comprises two domains: a small ‘cluster-binding’ subdomain and a large subdomain. The [2Fe–2S] cluster- Structure 15 December 1997, 5:1613–1625 binding subdomains of the chloroplast and mitochondrial Rieske proteins are http://biomednet.com/elecref/0969212600501613 virtually identical, whereas the large subdomains are strikingly different despite © Current Biology Ltd ISSN 0969-2126 a common folding topology. A structure-based sequence alignment of the b6f and bc1 groups of Rieske soluble domains is presented.

Conclusions: The segregation of structural conservation and divergence in the cluster-binding and large subdomains of the Rieske protein correlates with the overall relatedness of the cytochrome b6f and bc1 complexes, in which redox domains in the aqueous p phase are dissimilar and those within the membrane are similar. Distinct sequences and surface charge distributions among Rieske large subdomains may provide a signature for interaction with the p-side oxidant protein and for the pH of the intraorganelle compartment.

Introduction photosynthesis. The two complexes are a study of struc- Energy transduction in oxygenic photosynthesis is carried tural identity and diversity. The mitochondrial cytochrome out by four integral membrane protein complexes: the pho- b is a fusion of chloroplast cytochrome b6 and subunit IV tosystem II reaction center, the cytochrome b6 f complex, protomers [2], but the c-type f and c1 are unre- the photosystem I reaction center and the ATP synthase. lated. The relation of the Rieske proteins is discussed in

In addition to mediating electron transfer between the two the present study. In both the b6 f and bc1 cytochrome com- reaction center complexes, the cytochrome b6 f complex plexes, the redox-active domains of the Rieske protein and accomplishes the translocation of protons across the mem- the c-type cytochrome reside in the aqueous phase on the brane. The cytochrome b6 f complex comprises as many as electropositive (p)-side of the energy-transducing mem- seven subunits. Four ‘large’ subunits (molecular weight brane while those of the b-type cytochrome are located [MW] 18 000–32 000 Da) are products of the petA–D genes, within the bilayer. The electron donors to the cytochrome and as many as three small hydrophobic subunits (MW b6 f and bc1 complexes are the related membrane-soluble ~4000 Da) are products of the petG, L and M genes. The small molecules plastoquinol and ubiquinol, respectively. four large subunits are cytochrome f (with one c heme), The electron acceptors are the unrelated soluble proteins cytochrome b6 (with two b hemes), the Rieske protein plastocyanin and cytochrome c. Atomic level structural (containing a high-potential [2Fe–2S] cluster), and subunit information from these integral membrane protein com- IV (Figure 1; summarized in [1]). plexes is limited although high-resolution crystal structures of soluble, redox-active domains of chloroplast cytochrome

The photosynthetic cytochrome b6 f complex is related in f [3,4] and of the mitochondrial Rieske protein [5] have function to the cytochrome bc1 complex that has a central been reported. Preliminary crystal structures of intact role in the mitochondrial respiratory chain and in bacterial cytochrome bc1 have been presented recently [6–9]. In 1614 Structure 1997, Vol 5 No 12

Figure 1 one Fe and two histidines to the other Fe. The two histi- dine ligands are exposed to the solvent.

The three-dimensional structure of the chloroplast Rieske protein, together with the previously determined structure of its electron acceptor cytochrome f, may provide insight into the mechanism of electron transfer in the complex. Electron transfer from membrane-bound plastoquinol through the Rieske [2Fe–2S] cluster to the cytochrome f heme may occur over a long distance because the heme Fe is 45 Å from the site of membrane attachment in the elongate structure of cytochrome f. The distance will depend on the orientation of cytochrome f relative to the membrane surface and the packing arrangement of both Schematic of the membrane topology of a monomer of the cytochrome the Rieske protein and cytochrome f in the complex. b f complex. The 12 transmembrane helices are shown: four 6 Interestingly, there is indirect evidence for a flexible ori- associated with cytochrome b6 (in pink), three with subunit IV (in yellow), and one each with cytochrome f (in green), the Rieske protein entation of both proteins. EPR spectra of cytochrome f in (in red) and the petG, M and L gene products [47–50] (in brown). The oriented membranes indicated a range of orientations of inference of one helix for the Rieske protein is based on the structure the heme relative to the membrane plane, suggestive of of the bovine mitochondrial cytochrome bc complex [6–9]. 1 limited flexibility for the soluble domain [17,18]. The chloroplast Rieske N-terminal membrane anchor is linked to the soluble domain by a very glycine-rich peptide, addition, an 8 Å projection structure of an algal cytochrome which may be unstructured and quite flexible. The rate b6 f complex has been obtained [10]. constant for electron transfer from the Rieske protein to cytochrome f is approximately 104 s–1 [19], which corre- The Rieske [2Fe–2S] protein of oxygenic photosynthesis is sponds to a donor–acceptor distance of 12–17 Å by current the primary oxidant of plastoquinol in the cytochrome b6 f estimates of electron transfer rates in proteins [20,21]. A complex on the p-side (lumen) of the membrane, generat- dynamic role for the Rieske protein is also emerging from ing plastosemiquinone. The protons released in the oxida- structural studies of the cytochrome bc1 complex. Large- tion are ultimately transferred to the p-side bulk aqueous scale motion of the entire Rieske soluble domain about a phase [11], possibly via the Rieske protein and its electron flexible tether to the membrane anchor has been observed acceptor cytochrome f [4]. The mature intact Rieske in cytochrome bc1 [9], apparently in association with the protein in the cytochrome b6 f complex of spinach chloro- occupancy of the ubiquinol site [8]. plasts contains 179 residues [12], and appears to be anchored in the membrane by electrostatic forces [13] and We report here the 1.83 Å crystal structure of the soluble by one relatively hydrophobic N-terminal transmembrane domain of the spinach chloroplast Rieske protein by helix [14]. A soluble 139-residue C-terminal polypeptide multiwavelength anomalous diffraction (MAD) analysis fragment of the Rieske protein has been characterized and utilizing the two intrinsic Fe atoms. The structures of the crystallized [15]. The electron paramagnetic resonance lumen-side domains of the Rieske protein (139 of the 179

(EPR) spectrum of the reduced fragment (gz = 2.03, residues of the mature protein) and of cytochrome f (252 gy = 1.90, and a broad gx = 1.74) and the room temperature of 285 residues) constitute virtually the entire extrinsic p- midpoint oxidation/reduction potential (Em = 320 mV at side domain of the cytochrome b6 f complex except for pH 7) are similar to the values of those parameters in the connecting loops of the transmembrane α helices of cytochrome b6 f complex. The Em of the fragment is pH- cytochrome b6 and subunit IV. dependent, with Em = 359 mV at pH 6 and pKox = 6.5. Results and discussion A soluble 129-residue C-terminal polypeptide fragment of Structure determination the related Rieske protein from the cytochrome bc1 The crystal structure of the Rieske soluble domain was complex of bovine heart mitochondria was characterized determined by MAD from the iron atoms in the [2Fe–2S] biochemically [16] and a crystal structure determined [5]. cluster and was refined against diffraction data to 1.83 Å.

The EPR spectrum of the reduced fragment (gz = 2.01–2.02, The 2.1 Å electron-density map calculated with MAD gy = 1.90, gx = 1.76–1.80) and the midpoint oxidation- phases was of high quality (Figure 2) and was easily inter- reduction potential (Em = 306 mV at pH 7) are similar to preted in terms of a model for residues 53–179. The first the values of those parameters in the intact cytochrome bc1 twelve residues (41–52) of the soluble Rieske fragment are complex. The [2Fe–2S] center at one end of the Rieske disordered. Residues 41–52 of the proteolytic fragment, soluble domain is coordinated by two cysteine ligands to including six glycines and two prolines, appear to form a Research Article Structure of chloroplast Rieske protein Carrell et al. 1615

Figure 2

Electron density for the soluble domain of the spinach chloroplast Rieske protein. (a) Experimental map with MAD phases at 2.1 Å resolution. (b) 2Fo–Fc map with phases of the refined model at 1.83 Å resolution. The Gly141-Pro142-Ala143-Pro144-Leu145 proline loop, including a cis-peptide at Pro142, is shown in stereo for both maps; atoms are shown in standard colors. Contours are drawn at the root mean square density level. (Maps were drawn in the program O [43].) 1616 Structure 1997, Vol 5 No 12

Table 1 arrangement of anomalous scatterers. This crystallographic special situation presented no problems in MAD phasing. Refined model of the spinach chloroplast Rieske soluble domain. Residue range 53–179 The chloroplast Rieske soluble domain is bilobal with No. nonhydrogen atoms 1110 dimensions of approximately 40 Å × 30Å×20Å. The No. water sites 143 β Data range (Å) 20–1.83 overall fold is dominated by antiparallel secondary struc- R factor (%)* 17.0 ture, with the [2Fe–2S] cluster bound near the top of the No. reflections (all data) 8367 molecule, and the N-terminal connection to the flexible Free R factor (%) 22.0 linker and missing membrane-anchor peptide near the No. reflections 391 α 2 bottom (Figure 3a). The only helix, Ala65–Thr71, is at Average B values (Å ) β ′ β mainchain 10.3 the bottom of the molecule, between 1 and 2 sidechain 11.4 (Figure 3b). The protein has two subdomains. The smaller metal center 7.4 ‘cluster-binding’ subdomain comprises the [2Fe–2S] cluster water 26.9 and residues 105–147, including β4–β8. The large sub- all atoms 12.9 Rms deviations from target values domain includes residues 53–104 and 148–179 (strands bond lengths (Å) 0.008 β1–β4, β9–β10 and β1′). Each subdomain has the topology bond angles (°) 1.92 of a simple antiparallel β barrel such that adjacent β strand 2 B factors between bonded atoms (Å ) 0.76 ‘staves’ of the barrels are also contiguous in the primary Σ Σ β *R factor = h||Fobs |–|Fcalc ||/ h|Fobs | for data with no significance sequence. In the large subdomain, the strands form an cut. irregular six-stranded β barrel in the order β1-β2-β3-β4-β9- β10 with β1 and β10 hydrogen bonded to complete the barrel; strand β1′ and the α helix cap the bottom end of the flexible linker between the soluble domain of the Rieske barrel. The small cluster-binding subdomain is inserted protein and its N-terminal membrane anchor. The final between β4 and β9. Τhe long axis of the molecule is model of the 127-residue Rieske fragment had an R factor spanned by β4 with opposite ends of the strand contribut- of 17.0%, and no residues with disallowed φ/ψ values ing to the β barrels of the two subdomains. The cluster- (Table 1). The Rieske soluble domain is the first structure binding subdomain β barrel, which is incomplete, is formed solved by MAD in space group P1 with a centrosymmetric by β strands in the order β4-β5-β6-β7-β8. An extended

Figure 3

(a) (b)

142 142 110128 110 128 179 179

171 171 163 120 163 120 53 53 78 78 91 91 63 99 63 99 155 155

70 70

Structure

Fold of the soluble domain of the spinach chloroplast Rieske protein. spheres, respectively. (b) Stereo Cα trace of the 127 residues that are (a) Ribbon diagram showing the secondary structure elements and ordered in the crystal structure; the [2Fe–2S] cluster is shown as [2Fe–2S] cluster. The α helix is shown in red, β strands are in blue and black spheres. The view is the same in (a) and (b). (The figures were loops are in yellow; Fe and S atoms are shown as red and yellow drawn in SETOR [51] and MOLSCRIPT [52].) Research Article Structure of chloroplast Rieske protein Carrell et al. 1617

Figure 4

Stereo diagram of the [2Fe–2S] cluster of spinach chloroplast Rieske protein. β Strands are shown and labeled as in Figure 3; Fe and S atoms are in red and yellow, respectively. The ligand loops and proline loop are shown in stick form; hydrogen bonds are depicted as dotted lines. A water molecule (magenta sphere) bridges the β6–β7 ligand loop and the β8–β9 proline loop. The view is approximately perpendicular to Figure 3.

peptide completes the barrel between β4 and β8 without There is a small difference in the redox behavior of the forming hydrogen bonds to either strand and reconnects the soluble Rieske fragment compared with the protein in the cluster-binding subdomain to the large subdomain. intact cytochrome b6 f complex. The midpoint potential of the soluble fragment at pH 6 is approximately 70 mV more [2Fe–2S] cluster positive than that in the complex [15]. This may be due to The [2Fe–2S] cluster is bound by the three, strictly con- the greater solvent exposure of the [2Fe–2S] cluster- served, outermost loops of the cluster-binding subdomain: binding subdomain in the soluble fragment than in the the β4–β5, β6–β7 and β8–β9 loops (Figure 4). The structure intact complex. of the cluster, including protein ligands and hydrogen bonds, is identical within experimental error to that of the Despite the sequence identity of the three cluster-binding bovine mitochondrial Rieske protein [5]. Fe ligands are loops, the peptide bond between Gly141 and Pro142 of the carried on the β4–β5 and β6–β7 loops, which have very proline loop is in the cis conformation in the crystal struc- similar conformations to the cluster-binding peptides of ture of the chloroplast protein and in the trans conformation rubredoxin [22]. The protein Fe ligands are Cys107 and in the mitochondrial protein crystal structure. The observed His109 from the β4–β5 loop, and Cys125 and His128 from peptide conformers in both structures are stabilized by the β6–β7 loop. His109 and His128 ligate the outermost Fe intramolecular interactions. In the chloroplast Rieske atom and are otherwise solvent exposed. Cys107 and soluble domain, the cis-peptide carbonyl oxygen is hydro- Cys125 ligate the innermost Fe atom and are buried in the gen bonded through a well ordered water molecule to the protein. The ligand loops are buttressed by two supporting backbone carbonyl of His128 and to the sidechain of structures. The first of these is a disulfide bridge between Arg140. In the mitochondrial Rieske structure, the trans- Cys112 and Cys127, analogous to the Cys144–Cys160 disul- peptide carbonyl oxygen is directly hydrogen bonded to the fide bridge of the bovine mitochondrial Rieske protein. sidechain of Arg118, which is part of a 23-residue mitochon- The conservation of the disulfide supports an earlier drial insertion with no counterpart in the chloroplast Rieske hypothesis that a disulfide within the Rieske protein con- protein. The observed conformations for this peptide are tributes to the stability of the cytochrome bc1 complex [23]. not obviously affected by crystal packing. In neither struc- On the opposite side of the cluster from the disulfide ture do crystal lattice contacts stabilize the observed con- bridge is the β8–β9 loop, also known as the ‘proline loop’ former, or preclude formation of the other conformer. While because it contains the invariant Gly-Pro-Ala-Pro peptide there is no evidence for a mixture of states in the crystalline (residues 141–144 in the spinach chloroplast protein and chloroplast protein (Figure 2), cis and trans conformers of 174–177 in the bovine mitochondrial protein). The pair of both proteins may co-exist in solution. histidine ligands is flanked on one side by the sidechain of Leu110, which is adjacent to the disulfide bridge, and on Comparison of chloroplast and mitochondrial Rieske the other side by the sidechain of Pro142 in the proline proteins loop. The [2Fe–2S] cluster is enveloped by the tops of the The three-dimensional structures of the Rieske soluble β strands, the backbones of the ligand and proline loops, domains demonstrate that the proteins from the spinach and the sidechains of His107, His128, Leu110 and Pro142. chloroplast (this work) and the bovine mitochondrion [5] 1618 Structure 1997, Vol 5 No 12

Figure 5

(a) Comparison of the spinach chloroplast (green) and bovine mitochondrial (red) Rieske soluble domains. (a) Structural equivalence of the cluster-binding subdomains. The stereo CC Cα trace is based on the superposition of spatially equivalent Cα atoms in the cluster- binding subdomains (residues 105–116 and 122–147 of the spinach chloroplast protein CCwith residues 137–148 and 155–180 of the NNbovine mitochondrial protein). The view is NNapproximately perpendicular to Figure 3. (b) Structural divergence of the large subdomains. The stereo Cα trace is based on the superposition of 25 spatially equivalent Cα atoms in the longer β strands of the large subdomains (residues 70–81, 85–94 and 148–150 of the spinach chloroplast protein with residues 79–90, 93–102 and 181–183 (b) CCof the bovine mitochondrial protein). The superposition yielded a root mean square deviation of 1.64 Å. The view is from the back of Figure 5a. The cluster-binding subdomain, which is behind the large subdomain in this NNNCNC view, has been omitted for clarity.

Structure

are related. Structure conservation, however, is very the C terminus. The chloroplast Rieske soluble fragment unevenly distributed between the cluster-binding and has an eight-residue insertion between β1 and β2 relative large subdomains. The cluster-binding subdomains are to the mitochondrial fragment, and is longer by 15 virtually identical (Figure 5a), whereas the large sub- residues at the C terminus. The mitochondrial fragment domains are substantially different despite the identical has a 23-residue insertion between β3 and β4 relative to folding topology (Figure 5b). Structural alignment and the chloroplast protein. These alignment gaps are modifi- assignment of residue pairs for the chloroplast and mito- cations on the common architecture of the two proteins. chondrial large subdomains is difficult due to a different They are responsible for the bilobal shape of the packing of the shorter β strands (β1-β10-β9) on the longer chloroplast domain and the single-lobed shape of the β strands (β2-β3-β4) in the two proteins. Less than half of mitochondrial domain. These variations are also associated the residues in the large subdomain are spatially equiva- with the largest differences in the three-dimensional lent in the chloroplast and mitochondrial proteins. The structures. For example, the lone α helix occurs in the segregation of conservation and divergence is also evident internal insertion of each protein. The helices are not in the primary sequences (Figure 6). The relationship of spatially equivalent because the internal insertions occur sequence identity to tertiary structural similarity in homol- on opposite sides of the large domain. ogous proteins was quantitated by Chothia and Lesk [24]. The chloroplast and mitochondrial Rieske soluble Conservation and divergence among Rieske proteins domains fit this model well when the subdomains are con- Alignment of the three-dimensional structures of spinach sidered separately, and in one molecule represent the chloroplast and bovine mitochondrial Rieske fragments extremes of similarity (Table 2). (Figure 5; Table 2) revealed the correct alignment of their primary sequences, which differs somewhat from align- The identical lengths of the chloroplast and mitochondrial ments based on sequence data only. Sequences of other soluble fragments (127 amino acids) also belie a lack of Rieske proteins were aligned to the spinach chloroplast simple one-to-one equivalence of residues from the N to and bovine mitochondrion pair, using the structure-based Research Article Structure of chloroplast Rieske protein Carrell et al. 1619

Figure 6

β1 β ′ α1 β2 β3 Rieske (b6f) 1 secondary structure 41 50 60 70 80 90 Spinach F VPPGGGAGT GGTI AKDAL GNDVI AAEWL K THAPGDRTLTQGL KGDPTYLVVES Garden pea LVPPGSGSSTGGTVAKDAVGNDVVATEWL K THAPGDRTLTQGL KGDPTYLVVEK Tobacco FAPPGSGGGSGGTPAKDAL GNDVI ASEWL K THPPGNRTLTQGL KGDPTYLVVEN Synechocystis Sp. LI PPSSGGSGGGVTAKDAL GNDVKVTEFLASHNAGDRVLAQGL KGDPTYI VVQG Synechococcus Sp. FI PPSSGGAGGGVI AKDAL GNDI I VSDYLQTHTAGDRSLAQGL KGDPTYVVVEG Phormidium FI PPSSGGAGGGVTAKDAL GNDVVL KDFLATHTPGERVLAEGLKGDPTYLVVED Nostoc Sp. FI PPASGGAGGGTTAKDEL GNDVSL SKFLENRNAGDRALVQGL KGDPTYI VVEN C. reinhardtii FVPPSSGGGGGGQAAKDAL GNDI KAGEWL K THLAGDRSLSQGL KGDPTYLI VTA Bovine heart VL AMSKI EI KLSDIPEGKNMAFKW RGKPLFVRHRTKKEI DQEAAVEVSQLRDPQHD Rat VL AMSKI EI KLSDIPEGKNMAFKW RGKPLFVRHRTKKEI DQEAAVEVSQLRDPQHD Human VLALAKI EI KLSDIPEGKNMAFKW RGKPLFVRHRTQKEI EQEAAVELSQLRDPQHD N.crassa VLAMAKVEVDLNAIPEGKNVI I KW RGKPVFI RHRTPAEI EEANKVNVATL RDPETD S. cerevisiae VL AMAKVEVNLAAIPLGKNVVVKW QGKPVFI RHRTPHEI QEANSVDMSAL KDPQTD S. pombe VL AMSKAEVDLSKIPEGKNLVVKW QGKPVFI RHRTPEEI QEANSVDI STL RDPQAD Rs. rubrum TLALASTEVDVSAIAEGQAI T VT W RGKPVFVRHRTQKEI VVARAVDPASL RDPQTD B. japonicum I AAGAPI EVDLSPIAEGQDI KVF W RGKPIYISHRTKKQI DEARAVNVASL PDPQSDEA Rp. viridis I AAGAPI DI DISPVT EGQI VRVF W RGKPIFIRHRTAKEI QSEEAADVGAL I DPQPDSA Tobacco bc1 VL ALASLEVDLSSIEPGTTVTVKW RGKPVFI RRRTEDDI NL ANSVDL GSL RDPQQD Potato bc1 VLALASLEVDLSSIEPGSTVTVKW RGKPVFI RRRTDDDI KLANSVDL GTL RDPQQD Z. mays bc1 VL ALASLEVDLSSIEPGTTVTVKW RGKPVFI RRRTEDDI KLANSVDVASL RHPEQD P. denitrificans TMALSSLEVDLSGVEEGTTI TVKW RGKPVFI RHRTDAEI AQSAEVALSELRDPQKD Rb. capsulatus VQALASI QVDVSGVET GT QL T VKW LGKPVFI RRRTEDEI QAGREVDL GQL I DRSAQNSNKPDA Rb. sphaeroides VKAMASI FVDVSAVEVGTQLTVKW RGKPVFI RRRDEKDI ELARSVPLGALRDTSAENANKPGA C. rein. bc1 VQALASI FVDVSSVEPGVQLTVKF LGKPIFIRRRTEADI ELGRSVQL GQL VDTNARNANI DAG T. brucei M V G QMNI EAEVGELEDRECKTVVW RGKPVFVYRRSERQMNDVL GTPLSALKHPETD 68 80 90 100 110 120 Rieske (bc1) secondary structure β1 β2 β3 α1

Ligand loop I Ligand loop II Pro-loop β4 β5 β6 β7 β8 β9 Rieske (b6f) secondary structure 100 110 120 130 140 150 Spinach DKTLATFGI NAVCTHL GCVVPFNAAE NKFI CPCHGSQYNNQGRVVRGPAPLSLALAHCDVDD Garden pea DRTLATFAI NAVCTHL GCVVPFNQAE NKFI CPCHGSQYNDQGRVVRGPAPLSLALAHCDVGV Tobacco DGTLATYGI NAVCTHL GCVVPFNAAE NKFI CPCHGSQYNNQGRVVRGPAPLSLALAHADI DD Synechocystis Sp. DDTIANYGI NAVCTHL GCVVPWNASE NKFMCPCHGSQYNAEGKVVRGPAPLSLALAHATVTD Synechococcus Sp. DNTISSYGI NAI CTHL GCVVPWNTAE NKFMCPCHGSQYDETGKVVRGPAPLSLALVHAEVTE Phormidium AGVDRSYGI NAVCTHL GCVVPWNASE NKFI CPCHGSQYDATGKVVRGPAPLSLALVHTDVTE Nostoc Sp. KQAIKDYGI NAI CTHL GCVVPWNVAE NKFKCPCHGSQYDETGKVVRGPAPLSLALAHANTVD C. reinhardtii DSTIEKYGLNAVCTHL GCVVPWVAAE NKFKCPCHGSQYNAEGKVVRGPAPLSLALAHCDVAE Bovine heart LERVKKPEWV I L I G VCTHLGCVPI ANAGD FGGYYCPCHGSHYDASGRI RKGPAPLNLEVPSYEFTS Rat LERVKKPEWV I L I G VCTHLGCVPI ANAGD FGGYYCPCHGSHYDASGRI RKGPAPLNLEVPTYEFTS Human LDRVKKPEWV I L I G VCTHLGCVPI ANAGD FGGYYCPCHGSHYDASGRI RLGPAPLNLEVPTYEFTS N.crassa ADRVKKPEWL V ML G VCTHLGCVPI GEAGD YGGWF CPCHGSHYDI SGRI RKGPAPLNLEIPLYEFPE S. cerevisiae ADRVKDPQWL I ML G ICTHLGCVPI GEAGD F GGWF CPCHGSHYDI SGRI RKGPAPLNLEIPAYEFDG S. pombe SDRVQKPEWL V MI G VCTHLGCVPI GERGD YGGWF CPCHGSHYDI SGRI RRGPAPLNLAIPAYTFEG Rs. rubrum EARVQQAQWL V MV G VCTHLGCIPLGQKAGDPKGDFDGWF CPCHGSHYDSAGRI RKGPAPLNLPVPPYAFTD B. japonicum RVKSGHEQWL V V I G ICTHLGCIPIAHEGN YDGF F CPCHGSQYDSSGRI RQGPAPANLPVPPYQFVS Rp. viridis RVKPGKAEWL V V Y A SCTHLGCIPLGHQGD WGGWF CPCHGSQYDASGRVRKGPAPTNLPVPPYEFVD Tobacco bc1 AERVKSPEWL V V I G VCTHLGCIPLPNAGD FGGWF CPCHGSHYDI SGRI RKGPAPYNLEVPTYSFLE Potato bc1 AERVKNPEWL V V V G VCTHLGCIPLPNAGD FGGWF CPCHGSHYDI SGRI RKGPAPYNLEVPTYSFLE Z. mays bc1 AERVKNPEWL V V I G VCTHLGCIPLPNAGD FGGWF CPCHGSHYDI SGRI RKGPAPFNLEVPTYSFLE P. denitrificans VDRAI NPKYLVVVGICTHLGCVPI SGAGN YQGWF CPCHGSHYDI SGRI REGPAPYNLEVPEYRFTE Rb. capsulatus PATDEN RTMDEAGEWL V MI G VCTHLGCVPI GDGAGD F GGWF CPCHGSHYDTSGRI RRGPAPQNLHIPVAEFLD Rb. sphaeroides EATDENRTL PAFDGTNTGEWL V ML G VCTHLGCVPMGDKSGD F GGWF CPCHGSHYDSAGRI RKGPAPRNLDIPVAAFVD C. rein. bc1 AEATDQN RTLDEAGEWL V MWG VCTHLGCVPI GGVSGD F GGWF CPCHGSHYDSAGRI RKGPAPENLPIPLAKFID T. brucei EARFPDHREMAVVI AICTHLGCIPIPNEGL FGGF F CPCHGSHYDASGRI RQGPAPLNLEVPPYRWVD 130 140 150 160 170 180 Rieske (bc1) secondary structure β4 β5 β6 β7 β8 β9

β10 Rieske (b6f) secondary structure 160 170 Spinach GKVVF VPWTETDFRTGEAP WWS A Garden pea EDGKVVFVPWVETDFRTGDAP WWS Tobacco GKVVF VPWVETDFRTGEAP WWA Synechocystis Sp. DDKL VL STWTETDFRTDEDP WWA Synechococcus Sp. DDKI SFTDWTETDFRTDEAP WWA Phormidium DGKI AMTPWTETDFRTGDAP WWS Nostoc Sp. DKI I L SPWTETDFRTGDAP WWA C. reinhardtii SGLVTFSTWTETDFRTGL EP WWA Bovine heart DDMVI VG Rat GDVVVVG Human DDMVI VG N.crassa EGKLVI G S. cerevisiae DKVI VG S. pombe SKI I I G Rs. rubrum DTTVLI G B. japonicum DTKI QI G Rp. viridis NTKIRIGAGVA Tobacco bc1 ENKLLI G Potato bc1 ENKLLI G Z. mays bc1 ENKLLVG P. denitrificans GQKVVI G Rb. capsulatus DTTI KL G Rb. sphaeroides ETTI KLG C. rein. bc1 ETTI QLG T. brucei DKTI YL GKL 190 Rieske (bc1) secondary structure β10 Structure

Sequence alignment of the soluble domains of Rieske [2Fe–2S] proteins alignment of the spinach chloroplast and bovine mitochondrial proteins from cytochrome b6f and cytochrome bc1 complexes. Sequences of b6f constrained by the structure alignment. Spatially equivalent residues are Rieske proteins are highlighted in green, with dark shading for invariant enclosed in boxes. Residue numbering is with respect to the mature positions and light shading for sites of conservative substitution. spinach chloroplast protein at the top and the bovine mitochondrial Sequences of bc1 Rieske proteins are highlighted in red, with dark and protein at the bottom. Secondary structure elements for both proteins light shading as for the chloroplast proteins. Yellow highlights are for are indicated with the residue numbering; α helices are shown as residues invariant in both groups of Rieske proteins. The two groups of cylinders and β strands as arrows. The program ALSCRIPT [53] was sequences were aligned simultaneously using ClustalW [46], with used for display of the sequence alignment. 1620 Structure 1997, Vol 5 No 12

Table 2 the archaebacterium Sulfolobus acidocaldarius produces no c- type cytochrome [26,27]. The cytochrome bc complexes of Relationship of sequence identity and structural similarity in chloroplast and mitochondrial Rieske proteins. Bacillus subtilis [28] and Bacillus stearothermophilus [29] do include membrane-bound c-type cytochromes but these

Primary structure Tertiary structure differ from both cytochromes f and c1 and have an N-termi- nal domain resembling subunit IV of the cytochrome b f Identity (%) Length Rmsd (Å) No. Cα 6 complexes. In the familiar pattern of segregated conserva- Cluster-binding tion and divergence, the cluster-binding subdomains of the subdomain 59 43 0.70 38 Chlorobium, Bacillus and Sulfolobus Rieske proteins are

Large subdomain 9 84 1.95 41 highly similar to those of members of the b6 f and bc1 groups Overall 24 127 1.75 79 (33–58% identity). The large subdomains, however, have such low sequence identity with the b6 f and bc1 Rieske Sequence identities are for the full polypeptide length. Structural proteins (3–13% identity) or with each other (6–12% iden- similarities are for spatially equivalent residues only. tity) that the alignments themselves are of dubious accuracy in the absence of three-dimensional structures. alignment as a fixed constraint (Figure 6). As has been Specific interaction of the Rieske large subdomain and established by extensive earlier work, most of the the electron acceptor partner is the simplest inter- sequences segregate into two groups: the b6 f group, which pretation of the correlation between sequence of the includes the chloroplast photosynthetic proteins from Rieske large subdomain and identity of the electron cytochrome b6 f complexes; and the bc1 group, which acceptor. Such an interaction may or may not include the includes the mitochondrial respiratory proteins from the electron transfer pathway between the Rieske protein cytochrome bc1 complexes. The most important conclusions and its electron acceptor. Flexibility of the Rieske from the sequence/structure comparison are that conserva- soluble domain has been demonstrated in preliminary tion between groups is limited to the cluster-binding sub- structures of cytochrome bc1 [8,9] and an extensive, domain and that the large subdomains of the b6 f and bc1 specific protein–protein interaction may be necessary to groups are very dissimilar. Sequences of the cluster-binding ensure productive docking of the Rieske protein with its subdomains of members of the b6 f group are 47–61% iden- electron acceptor. tical to those of the bc1 group. The large subdomains, however, are only 5–18% identical between groups, exactly Distribution of surface charge mirroring the structural comparison (Figure 5; Table 2). The chloroplast and mitochondrial Rieske soluble domains Indeed three-dimensional structures are required for accu- have very different charges at neutral pH. The chloroplast rate sequence alignment when the identity is so low. If the protein has an excess of four acidic amino acid sidechains b6 f and bc1 Rieske proteins have a common ancestor, as (14 Asp + Glu residues and 10 Lys + Arg), whereas the seems likely given their common function and fold, then mitochondrial protein has an excess of two basic sidechains the cluster-binding subdomains have been highly con- (14 Asp + Glu residues and 16 Lys + Arg). The pI of the served while the Rieske large subdomains are considerably chloroplast Rieske soluble domain (4.9, as measured by diverged in cytochromes b6 f and bc1. isoelectric focusing; data not shown) is more acidic than the mitochondrial protein (pI = 6.1, as measured electro- The large subdomain and interaction with the electron chemically; [16]). Anionic regions are prominent on the acceptor surface of the chloroplast protein (Figure 7a), whereas the The structure-based sequence alignment reveals that the mitochondrial protein has several basic surface regions and sequence of the large subdomain is strongly correlated a more globular shape (Figure 7b). The substantial charge with the type of electron acceptor employed by the difference, however, is offset by the substantial difference

Rieske protein. Within the b6 f and bc1 groups, the large in acidity of the compartments where the chloroplast and subdomains are highly conserved despite the striking mitochondrial Rieske domains function in vivo. The differences between groups (Figure 6). Cytochrome f is the chloroplast lumen is two to three pH units more acidic electron acceptor for the b6 f Rieske proteins, and than the mitochondrial intermembrane space. Thus the for the bc1 group. Rieske proteins that func- average surface charge of the two Rieske proteins may be tion in energy-transducing membrane protein complexes in similar in situ. Chlorobium, Bacillus and Sulfolobus species are dissimilar to both of the b6 f and bc1 groups of Rieske proteins, and have The distribution of surface charge has relevance to the neither cytochrome f nor cytochrome c1 as electron accep- interactions of the Rieske soluble domain with its redox tors. The cytochrome b complex of the green photo- partners and with other proteins of the membrane protein synthetic bacterium Chlorobium limicola does not include complex. Thus the different distribution of surface charge any membrane-bound c-type cytochrome [25]. Similarly, on the chloroplast and mitochondrial Rieske soluble Research Article Structure of chloroplast Rieske protein Carrell et al. 1621

Figure 7

Divergence of the electrostatic potential surface and overall shape of b6f and bc1 Rieske soluble domains. (a) Spinach chloroplast protein. The left view is the same as in Figure 3, and the right view is of the opposite surface; the [2Fe–2S] cluster is at the top in both views. Electronegative potential is shown in red and electropositive in blue over a range of approximately –8 kT to +8 kT. (b) Bovine mitochondrial protein [5] shown in the same orientations as (a). (The figure was prepared using GRASP [54].)

domains may reflect their interaction with the very differ- is uncertain because of the disorder of the N-terminal 12 ent electron acceptors cytochrome f and cytochrome c1. In residues. A nonpolar surface might be expected for any both the chloroplast and the mitochondrial Rieske soluble part of the Rieske protein that interacts with the interfa- domains, a relatively nonpolar surface surrounds the cial region of the membrane. The lack of such a surface, [2Fe–2S] cluster, and is derived from the strictly conserved excepting the cluster-binding subdomain discussed above, ligand-binding loops and the proline loop. In recently argues against the interaction of the soluble domain with described preliminary crystal structures of cytochrome bc1 hydrophobic regions of the membrane. This is consistent [6–9], the protein surface of the cluster-binding subdomain with the participation of the soluble domain of the Rieske interacts with cytochrome b and forms part of the binding protein of cytochrome bc1 in only protein–protein interac- niche of the ubiquinol/ubiquinone couple that is the elec- tions with cytochrome b and not protein–membrane inter- tron donor to the Rieske protein. On the basis of the actions. Thus the Rieske protein may behave as a soluble similar lack of polarity and the conservation of this region protein, which is prevented from diffusing away from the of the chloroplast Rieske protein, we conclude that the membrane by its long, flexible tether. cluster-binding subdomain of the chloroplast Rieske protein interacts with cytochrome b6 and the plasto- The N-terminal flexible tether quinol/plastoquinone-binding site of cytochrome b6 f in a Residues 41–52 of the spinach chloroplast Rieske soluble similar manner. fragment tether residues 53–179, whose structure we describe here, to the membrane anchor. The 12-residue The Rieske protein contacts the membrane near the N ter- tether peptide is extremely flexible, as evidenced by its minus of the soluble domain. The location of this terminus total disorder in the crystal structure, by the preponderance 1622 Structure 1997, Vol 5 No 12

of glycine residues in the peptide (six of twelve amino second cluster is approximately 12 Å from the [2Fe–2S] acids), and by the accessibility of residue 41 to the pro- cluster, and even further when through-bond pathways are tease thermolysin when the soluble fragment is prepared considered. In addition, mutagenesis experiments with from the intact cytochrome b6 f complex [15]. A long, flexi- the yeast Rieske protein do not implicate the conserved ble tether for the Rieske protein is consistent with the residues of the first aromatic cluster in electron transfer emerging picture of a large-scale motion of the Rieske function (BL Trumpower, personal communication). protein associated with electron transfer and energy trans- Thus, it is not possible at this time to infer a role for these duction in the cytochrome bc1 complex. The Rieske conserved clusters of aromatic residues in electron transfer protein occupies different positions in three different through the Rieske protein. crystal forms of cytochrome bc1 complexes, dependent on the binding of the drug stigmatellin to the p-side quinol Pathways of proton transfer site of the complex, whereas the distances between the Proton translocation by the cytochrome b6 f complex is cytochrome c1 heme Fe and the cytochrome b heme Fe tightly coupled to electron transfer. The source of protons atoms are constant [6–9]. Efficient electron transfer by the is plastoquinol, which is deprotonated in conjunction with Rieske protein is a problem given the apparently fixed, its oxidation. A simple mechanism for coupling electron greater than 30 Å distance from the quinol-binding site to transfer and proton translocation was proposed to involve the cytochrome c1 redox center in cytochrome bc1. A large- proton transfer to a buried water chain in cytochrome f scale motion by a flexibly tethered Rieske [2Fe–2S] concomitant with reduction of its heme Fe [4]. The most domain is one possible solution. A similar situation may obvious proton source is the cytochrome f reductant, the occur in cytochrome b6 f. The heme Fe of cytochrome f is Rieske protein. The one set of residues in the Rieske distant (45 Å) from its membrane anchor with limited protein that suggest themselves as candidate proton space in the sequence for a flexible tether between the carriers are the histidine ligands His109 and His128. It is soluble domain and the membrane tether [3]. not at all obvious that the pK of either histidine sidechain is appropriate for this function. However, the redox titra- Role of aromatic residues in intraprotein pathways of tion of the Rieske soluble fragment has a pK of 6.5 [15], electron transfer which is most readily associated with a sidechain near the Current discussions of the pathways of electron transfer [2Fe–2S] cluster. The structure of the chloroplast Rieske through proteins focus on whether the pathways are inde- protein soluble domain includes no bound internal water pendent of protein structure [20], or sensitive to local chain analogous to the buried water chain in cytochrome f. structure, occurring with decreasing efficiency through Nor is there an obvious hydrogen-bonded network of covalent bonds, hydrogen bonds and space [21,30]. A role protein groups through the chloroplast Rieske protein. If for the π-electron systems of aromatic amino acids in elec- the water chain in cytochrome f is used in proton trans- tron transfer through proteins has long been discussed location, then it does not connect with a similar structure [31–37], although there currently exists no example of in the Rieske protein. If there is an intraprotein proton electron transfer through a protein facilitated by a chain or pathway involving the Rieske protein that would ulti- cluster of aromatic sidechains. mately donate protons to cytochrome f, then it would seem to involve the interface between cytochrome f and The spinach chloroplast Rieske protein includes a large the Rieske protein during electron transfer. number of conserved aromatic residues (Figure 6). Nine of the conserved aromatic sidechains form two clusters in the Conservation and diversity in cytochrome b6f and protein, suggesting that they may function in conductive cytochrome bc1 pathways of electron transfer. The first cluster includes The structure of the chloroplast Rieske soluble domain Tyr132, Phe123 and Tyr89, which are conserved in all together with other results suggests a striking separation of Rieske protein soluble domains, and Phe116. The second structural conservation and diversity at the membrane cluster, including Trp176, Trp177, Trp164, Phe101 and surface in the cytochrome b6 f and bc1 complexes. The Phe169, is conserved in all b6 f Rieske proteins, but has no energy-transducing b6 f and bc1 complexes are very similar counterpart in the bc1 Rieske proteins. Within each within the membrane bilayer and very different in the cluster, aromatic sidechains are in van der Waals contact. p-side aqueous phase. The cytochrome b6–subunit IV The invariant Tyr132 of the first cluster is connected to tandem pair of cytochrome b6 f has a similar sequence, and the [2Fe–2S] cluster via a hydrogen bond to the Fe ligand by inference a similar structure, throughout its length to γ Cys107 S . Based on results obtained with model aromatic cytochrome b of the cytochrome bc1 complex, although compounds [32,33], it can be hypothesized that either of these proteins differ in some important biochemical details. these aromatic clusters could greatly facilitate electron This is in marked contrast to the soluble domains on the transfer within the Rieske protein, because each cluster p-side of the membrane. Conservation and divergence are traverses a total distance of approximately 15 Å. Other sharply delineated in the Rieske soluble domain, possibly factors, however, argue against this interpretation. The at the membrane border. The invariant cluster-binding Research Article Structure of chloroplast Rieske protein Carrell et al. 1623

subdomain may be conserved to interact with the similar in the chloroplast and mitochondrial complexes. The b-type cytochromes and plastoquinol/ubiquinol-binding Rieske large subdomains, however, appear to have sites within the membrane bilayer. In contrast, the evolved to interact with unrelated redox partners in the Rieske large subdomains are very different and may have p-side aqueous phase, and are dissimilar in the chloroplast evolved to pair with the unrelated membrane-bound and mitochondrial complexes. c-type cytochromes (f and c1) in the p-side aqueous phase of chloroplasts and mitochondria. Materials and methods Crystallization The difference in conservation of the intra- and extramem- The Rieske soluble domain was prepared and crystallized as in Zhang et al. [15]. The Rieske soluble domain was crystallized by the hanging- brane components complicates transfer of experimental drop vapor diffusion method with a reservoir solution of 100 mM results from one complex to the other. Little common sodium acetate (pH 4.6), 100–200 mM ammonium acetate and 30% ground exists for structural comparison of redox com- polyethylene glycol (PEG)-4000. Hanging drops were a 1:1 mixture of plexes between the unrelated c-type cytochromes and protein solution (30 mg/ml Rieske soluble domain, 20 mM morpholino- propanesulfonic acid [MOPS] buffer, pH 7.2) and reservoir solution. their divergent Rieske protein partners. The fundamen- tally different electrostatic potential surfaces of the chloro- Data collection plast and mitochondrial Rieske proteins (Figure 7) reflect Crystals were stabilized in crystallization reservoir solution and cryopro- this fact as well as the large difference in acidity of their in tected in 75 mM sodium acetate (pH 4.6), 150 mM ammonium acetate, vivo compartments. 30% PEG-4000 and 25% ethylene glycol. After a short cryoprotection period (< 5 min), crystals were removed in fiber loops and immediately flash-frozen in liquid ethane. MAD data were collected at three wave- Biological implications lengths on Beamline 19 (BM14) at the European Synchrotron Radia- tion Facility (ESRF), Grenoble, France. X-ray fluorescence from a The integral membrane cytochrome b6 f complex trans- fers electrons between photosystems II and I and con- Rieske protein crystal was recorded from 6.93 keV to 8.36 keV to select wavelengths for data collection. Wavelengths were selected at tributes to the proton gradient of the energy-transducing the peak (maximum f′′, 7.130 keV, 1.7389 Å) and the inflection point photosynthetic membrane. Together with the previously (minimum f′, 7.118 keV, 1.7419 Å) of the Fe K edge and at one remote determined structure of the lumen-side domain of wavelength (8.266 keV, 1.5000 Å). The remote energy was unusually cytochrome f, the 1.83 Å crystal structure of the soluble distant from the Fe K edge in order to obtain high angle, high quality domain of the spinach chloroplast Rieske [2Fe–2S] data for eventual model refinement. Severe air absorption at the low energy Fe K edge limits the quality of data at the edge energies. Data protein, described in the present work, essentially were measured as successive 360° sweeps at the three wavelengths completes an atomic description of the cytochrome b6 f (72 images of 5° each) from one frozen crystal using an 18 cm MARe- complex on the electropositive (p)-side of the mem- search image-plate detector. Diffraction images were processed using brane. Structures of the lumen-side domains of the the HKL program package [38,39] and scaled using SCALA and AGROVATA from the CCP4 program suite [40]. Friedel-pair data at chloroplast Rieske protein and cytochrome f suggest each wavelength were scaled to a reference data set constructed from possibilities for intra- and interprotein pathways of elec- fully merged data at λ = 1.5000 Å. Data quality is summarized in tron and proton transfer in cytochrome b6 f that are dis- Table 1. tinct from those of the related respiratory cytochrome bc complex. The interaction between the Rieske and The crystals belong to the space group P1 with cell parameters 1 a = 29.05 Å, b = 31.87 Å, c = 35.79 Å, α = 95.6°, β = 106.1° and cytochrome f redox partner domains in the chloroplast γ = 117.3°. The asymmetric unit contains one molecule of the Rieske lumen remains to be determined. soluble domain, with a calculated solvent content of 31%.

In general, elucidation of the structure of an integral MAD structure determination The data at the three wavelengths were put on an approximately membrane protein with extended peripheral domains is absolute scale using SCALEIT from the CCP4 suite. The positions of greatly facilitated by the determination of accurate struc- the two Fe atoms were determined by inspection of an anomalous tures for the isolated soluble domains. Comparison of difference Patterson map from data at the wavelength of maximal f′′ structures and sequences of the soluble domains of (1.7389 Å). Refinement of the Fe partial structure and MAD phasing chloroplast and mitochondrial Rieske proteins, together were performed by the pseudo-isomorphous approach using MLPHARE [41]. The data at the edge-remote energy (λ = 1.5000 Å) with preliminary structural data for the mitochondrial were designated as the ‘native’ data, and data at the other two wave- complex, has already highlighted important differences in lengths as ‘derivatives’. Phasing statistics are summarized in Table 3. Phases were refined by density modification in the form of solvent the protein subunits of the cytochrome b6 f and bc1 com- plexes. The complexes are highly similar for domains flattening and histogram matching using the program DM [42] with a 30% solvent mask. The real-space free residual decreased from within the membrane bilayer and dissimilar in domains 0.273 to 0.161, and the interpretability of the electron-density map that function on the p-side of the membrane. Diversity is improved somewhat. most dramatic in the unrelated p-side cytochrome f and c1 subunits. Segregation of conservation and divergence is Model building and refinement especially evident in the Rieske protein. The Rieske The atomic model was constructed using the program O [43] and refined against the higher quality, more extensive data at λ = 1.5000 Å [2Fe–2S] cluster-binding subdomains, which interact with using XPLOR [44,45]. The initial model of 127 amino acids had an R electron donors within the bilayer, are virtually identical work of 0.349 and R free of 0.376 for data from 20.0 Å to 2.1 Å. After 1624 Structure 1997, Vol 5 No 12

Table 3 4. Martinez, S.E., Huang, D., Ponomarev, M., Cramer, W.A. & Smith, J.L. (1996). The heme redox center of chloroplast cytochrome f is linked to MAD data collection and phasing. a buried five-water chain. Protein Sci. 5, 1081–1092. 5. Iwata, S., Saynovits, M., Link, T.A. & Michel, H. (1996). Structure of a water-soluble fragment of the ‘Rieske’ iron–sulfur protein of the bovine λ = 1.7389 Å λ = 1.7419 Å λ = 1.5000 Å heart mitochondrial cytochrome bc1 complex determined by MAD phasing at 1.5 Å resolution. Structure 4, 567–579. No. observations 22 187 18 692 28 882 6. Xia, D., et al., & Deisenhofer, J. (1997). Crystal structure of the cytochrome bc complex from bovine heart mitochondria. Science No. unique reflections 5633 5603 8758 1 277, 60–66. d (Å) 2.1 2.1 1.83 7. Deisenhofer, J., et al., & Yu, L. (1997). The 3-D structure of the min cytochrome bc complex from bovine heart. FASEB J. 11, A1279. Redundancy 3.9 3.3 3.3 1 8. Kim, H., et al., & Yu, L. (1997) X-ray crystallographic studies on in Completeness (%) 91.6 91.1 89.7 inhibitor-induced conformational change of the iron–sulfur protein in (68.5) (65.1) (57.3) mitochondrial bc1 complex. FASEB J. 11, A1084. R (%)* 5.2 4.3 4.8 9. Berry, E.A., Huang, L.-S., Zhang, Z., Chi, Y.-I., Hung, L.-W. & Kim, sym S.-H. (1997). Structure of the redox-active subunits of the (8.5) (7.2) (10.8) mitochondrial cytochrome bc1 complex. Variable conformation of the Fe anomalous Rieske ironsulfur protein. FASEB J. 11, A1279. scattering factors 10. Mosser, G., Breyton, C., Olofsson, A., Popot, J.L. & Rigaud, J.-L. (1996). Projection map of cytochrome b f complex at 8 Å resolution. ′ – 6 f (e ) –7.790 –9.499 –1.118 J. Biol. Chem. 272, 20263–20268. ′′ – f (e ) 4.060 1.949 3.020 11. Trumpower, B.L. (1990). The protonmotive . Energy † transduction by coupling of proton translocation to electron transfer by RCullis the cytochrome bc1 complex. J. Biol. Chem. 265, 11409–11412. isomorphous 0.69 0.57 – 12. Steppuhn, J., et al., & Herrmann, R.G. (1987). The complete amino- anomalous 0.71 0.82 0.72 acid sequence of the Rieske FeS-precursor protein from spinach chloroplasts deduced from cDNA analysis. Mol. Gen. Genet. Phasing power‡ 1.76 2.30 210, 171–177. *R = Σ | I – 〈I 〉 |/ Σ I . †R = root mean square (rms) lack of 13. Szczepaniak, A., Huang, D., Keenan, T.W. & Cramer, W.A. (1991). sym h,i h,i h h,i h,i Cullis Electrostatic destabilization of the cytochrome b f complex in the closure/rms isomorphous difference. ‡Phasing power = 〈|F |〉/rms lack 6 H thylakoid membrane. EMBO J. 10, 2757–2764. of closure. The overall figure of merit = 0.70. Values in parentheses 14. Karnauchov, I., Herrmann, R.G. & Klösgen, R.B. (1997). Transmembrane pertain to the outermost shell of data, 2.21–2.10 Å for the first two topology of the Rieske Fe/S protein of the cytochrome b6f complex from wavelengths and 1.90–1.83 Å for the third wavelength. spinach chloroplasts. FEBS Lett. 408, 206–210. 15. Zhang, H., et al., & Cramer, W.A. (1996). Characterization and crystallization of the lumen-side domain of the chloroplast Rieske one round of simulated annealing, followed by four iterations of conven- iron–sulfur protein. J. Biol. Chem. 271, 31360–31366. tional refinement and model building, data were extended to the limit of 16. Link, T.A., Hagen, W.R., Pierik, A.J., Assmann, C. & von Jagow, G. 1.83 Å. After six more iterations of refinement and model building, the (1992). Determination of the redox properties of the Rieske [2Fe–2S] final R work and R free were 0.170 and 0.221, respectively. The model cluster of bovine heart bc1 complex by direct electrochemistry of a water-soluble fragment. Eur. J. Biochem. 208, 685–691. includes 127 amino acids (residues 53–179), the [2Fe–2S] cluster 17. Bergström, J. & Vanngärd, T. (1982). EPR signals and orientation of and 143 water molecules. Model quality is summarized in Table 1. cytochromes in the spinach chloroplast thylakoid membrane. 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