Atomic-resolution structure of reduced cyanobacterial cytochrome c 6 with an unusual sequence insertion Wojciech Bialek 1 , Szymon Krzywda 2 , Mariusz Jaskolski 2,3 and Andrzej Szczepaniak 1 1 Department of Biophysics, Faculty of Biotechnology, University of Wroclaw, Poland 2 Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland 3 Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland Introduction During photosynthesis, electron transfer between two membrane-bound complexes, cytochrome b 6 f and photosystem I, can be accomplished by the copper- containing protein plastocyanin (PC) or the heme protein cytochrome c 6 [1,2]. PC is found as a unique electron carrier in higher plants. Some algae and cyanobacteria express either PC or cytochrome c 6 , whereas others are able to produce both proteins, depending on copper availability [3]. Cytochromes c 6 are water-soluble, low-spin, heme-containing proteins Keywords cyanobacteria; cytochrome; high-resolution structure; photosynthesis; Synechococcus sp. PCC 7002 Correspondence A. Szczepaniak, Department of Biophysics, Faculty of Biotechnology, University of Wroclaw, Przybyszewskiego 63 ⁄ 77, 51-148 Wroclaw, Poland Fax: +48 71 3756234 Tel: +48 71 3756236 E-mail: andrzej.szczepaniak@ibmb.uni.wroc.pl M. Jaskolski, Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland Fax: +48 61 829 1505 Tel: +48 61 829 1274 E-mail: mariuszj@amu.edu.pl Database Atomic coordinates and structure factors are available from the Protein Data Bank under the accession code 3DR0 (Received 5 May 2009, revised 9 June 2009, accepted 11 June 2009) doi:10.1111/j.1742-4658.2009.07150.x The structure of the reduced form of cytochrome c 6 from the mesophilic cyanobacterium Synechococcus sp. PCC 7002 has been determined at 1.2 A ˚ and refined to an R-factor of 0.107. This protein is unique among all known cytochromes c 6 , owing to the presence of an unusual seven-residue insertion, KDGSKSL(44–50), which differs from the insertion found in the recently discovered plant cytochromes c 6A . Furthermore, the present pro- tein is unusual because of its very high content (36%) of the smallest resi- dues (glycine and alanine). The structure reveals that the overall fold of the protein is similar to that of other class I c-type cytochromes, despite the presence of the specific insertion. The insertion is located within the most variable region of the cytochrome c 6 sequence, i.e. between helices II and III. The first six residues [KDGSKS(44–49)] form a loop, whereas the last residue, Leu50, extends the N-terminal beginning of helix III. Several spe- cific noncovalent interactions are found inside the insertion, as well as between the insertion and the rest of the protein. The crystal structure con- tains three copies of the cytochrome c 6 molecule per asymmetric unit, and is characterized by an unusually high packing density, with solvent occupy- ing barely 17.58% of the crystal volume. Abbreviation PC, plastocyanin. 4426 FEBS Journal 276 (2009) 4426–4436 ª 2009 The Authors Journal compilation ª 2009 FEBS involved in the high-potential electron transport chain. They are characterized by low molecular mass (80–90 amino acid residues in the mature protein), and have a covalently bound heme group. Once believed to be absent in plants, cytochrome c 6 -like proteins, now designated cytochrome c 6A , have recently also been discovered in plants [4,5] and in the green alga Chlamydomonas reinhardtii [6]. In general, cytochromes c 6 are characterized by high redox potential (E m,7 of 330–360 mV). However, a number of low redox potential cytochrome c 6 -like pro- teins have been identified. These include plant and algal cytochrome c 6A (E m,7 of $ 70 mV [7]), and cyanobacte- rial cytochrome c 6C (E m,7 of $ 150 mV [8]) and cyto- chrome c 6B , for which a low redox potential has been postulated as well [8]. Members of these recently identi- fied cytochrome c 6 subfamilies are characterized by the presence of Val, Ile or Leu in place of a conserved Gln residue located inside the cytochrome c 6 heme pocket. Because of its low redox potential and the presence of two additional Cys residues, cytochrome c 6A has been postulated to play a role in the formation of disulfide bridges in thylakoid lumen proteins, rather than func- tioning in the photosynthetic electron transport chain [9]. Interestingly, the replacement of the conserved Gln residue by Val resulted in a decrease of the cyto- chrome c 6 redox potential by $ 100 mV [cytochrome c 6 from Phormidium laminosum [7] and Synechococcus sp. PCC 7002 (Bialek, unpublished results)], whereas the opposite Val fi Gln substitution in cytochrome c 6A from Arabidopsis thaliana increased the midpoint potential by approximately 100 mV [7]. The structures of cytochromes c 6 from three cyano- bacteria (Arthrospira maxima [10], Synechococ- cus elongatus [11], and P. laminosum [7]), one from a red alga (Porphyra yezoensis [12]), one from a brown alga (Hizikia fusiformis [13]), and four from green algae (Monoraphidium brauni [14], C. reinhardtii [15], Scenedesmus obliquus [16], and Cladophora glomerata [17]) have been determined, as has the structure of cytochrome c 6A from A. thaliana [18]. Together, these data represent a large volume of information about the sequences and structures of cytochrome c 6 . How- ever, the cytochrome c 6 molecule from the mesophilic cyanobacterium Synechococcus sp. PCC 7002 (E m,7 of $ 320 mV [8]) is an exceptional protein in this class, as it contains an unusual heptapeptide insertion beginning after Tyr43. Moreover, the petJ1 gene, which encodes cytochrome c 6 in Synechococcus sp. PCC 7002, could not be inactivated or replaced with petJ (cyto- chrome c 6 ), petE (PC) or cytM (cytochrome c M ) from Synechocystis [19], unlike the petJ gene from Synecho- cystis [20]. Consequently, it appears that only the native cytochrome c 6 of Synechococcus can function properly in electron transport and ensure cell viability. To elucidate the characteristics of this unusual cyto- chrome c 6 molecule, and in particular to shed light on the role of the heptapeptide insertion, the crystal struc- ture of the protein in its reduced form has been deter- mined at a resolution of 1.2 A ˚ . In addition, this study is a contribution towards a better understanding of the molecular determinants of the spectroscopic and electron transfer properties of this class of proteins. Results and Discussion Structure description Overall, the structure of Synechococcus sp. PCC 7002 cytochrome c 6 reveals the characteristic properties of other class I cytochromes c. The molecule is composed of a single polypeptide chain wrapped around the heme group, which is bound to the protein by two thioether links at Cys14 and Cys17, as well as by His18 and Met65, which are the axial ligands in the coordination sphere of the iron atom (Fig. 1). The polypeptide chain consists of four a-helices (I–IV) con- nected by loops. The long N-terminal helix I (Ala3– His18) has a characteristic kink at Cys14. The two Cys residues forming the covalent links to the heme group and the fifth iron-coordinating ligand, His18, are part of this helix. As in other cytochrome c 6 molecules, the three consecutive amino acids Ala19-Gly-Gly21 form a 3 10 helix followed by an X-loop ranging from Asn22 to Lys32. The X-loop separates helix I from the short helix II (Ala33–Tyr39). Residues Leu40–Tyr43 and Gly46–Ser49 form type IV and type VIII b-turns, respectively [21]. The latter turn is formed entirely within the sequence of the heptapeptide insert that is characteristic of this cytochrome c 6 protein only. Leu50, which is also part of the insert, belongs to helix III, formed by Leu50–Asn60. Gln62–Met65 and Gly69–Leu72 in the following region are type II¢ and type IV b-turns, respectively [21]. Helix IV, which is nearly perpendicular to helix I, runs from Asp74 to Glu89. Two residues, Asp86 and Lys92, located near the C-terminus, form a salt bridge stabilizing this part of the protein fold. Conformation of the heptapeptide insertion In general, high overall structural similarity is observed among all the compared cytochrome c 6 molecules (Fig. 2A, Table S1). The most striking structural difference between the present Synechococcus sp. PCC 7002 cytochrome c 6 and all other cyto- W. Bialek et al. Atomic-resolution structure of cytochrome c 6 FEBS Journal 276 (2009) 4426–4436 ª 2009 The Authors Journal compilation ª 2009 FEBS 4427 chrome c 6 molecules is the presence of the specific insertion between helices II and III (Fig. 2C). The insertion consists of seven, mostly polar, amino acids KDGSKSL(44–50). Its major part [GSKS(46–49)] forms a type VIII b-turn. Multiple hydrogen bonds formed within this structure stabilize its conformation. These, as well as hydrogen bonds between the insert residues and residues from other regions of the pro- tein, are listed in Table 1. Additionally, the cationic side chain of Lys48 is also involved in cation–p inter- actions with Tyr56, which, in turn, is adjacent to Gln57, the residue that is implicated in the control of the heme redox potential. Specifically, a structural water molecule inside the heme pocket forms a hydro- gen bond with the Gln57 side chain, and is thus responsible for positioning of the c-amide towards the heme group and within a van der Waals distance of a methine carbon atom (CHA) of the porphyrin ring. In consequence, Gln57 is capable of tuning the redox potential of the heme group [7,8]. An insert of three consecutive Asp residues has been found in the same region in cytochrome c 6 from A. maxima [10], but its length, shape and chemical character are significantly different from those charac- terizing the present insertion of cytochrome c 6 from Synechococcus sp. PCC 7002 (Fig. 2A,C). It is of note that an insertion has also been found in the sequence of the protein from A. thaliana. However, the insertion in that cytochrome c 6A protein is not only longer but also located differently, namely between helices III and IV, and is thus entirely different from the insertion dis- covered in the present protein. Importantly, the cyto- chrome c 6A loop contains two Cys residues that are also found in other cytochrome c 6A molecules. In neither case has the function of the insertions been completely elucidated. The heme prosthetic group Figure 3 depicts the hydrophobic heme pocket of the present cytochrome c 6 molecule. By analogy with other cytochrome c prosthetic heme groups, the porphyrin moiety is slightly distorted into a saddle-shaped form in all three molecules in the asymmetric unit. The edge of the pyrrole ring C and the propionic group D are exposed towards the solvent. The heme group is cova- lently linked to the polypeptide chain through thioe- ther bonds from the Cys14 and Cys17 sulfhydryl groups to the two vinyl side chains. The iron of the heme group is in the FeII oxidation state, maintained by the presence of 20 mm dithionate in the mother liquor. The iron ion is coordinated through His18 N e2 and Met65 S d , located at axial positions. The latter residue has a gauche conformation along the C b –C c bond (torsion angle C a –C b –C c –S d of 50.0°, 48.2° and 50.4° in molecules A, B and C, respectively). As in other cytochrome c 6 structures, a hydrogen bond between the N d1 H donor of the axial His18 and the carbonyl oxygen atom of Asn22 serves to maintain the required orientation of the His ring with respect to the heme plane. Interestingly, this is the only His present in the protein. The interactions of the nitrogen atoms of the imidazole ring (N e2 –Fe and N d1 –HÆO) confirm that it is electrically neutral. The structure of reduced as well as oxidized cyto- chrome c 6 from S. obliquus has been described previ- ously [16]. Comparison of both redox states revealed a different conformation of Pro62, which is adjacent to the Met61 axial ligand of the heme group. The posi- tion of C c of this Pro was found to change from exo to endo upon oxidation. The protein used in the pres- ent study was reduced with a 20-fold molar excess of dithionite in the crystallization buffers. Pro66, which is A B Fig. 1. (A) Three-dimensional structure of the reduced cyto- chrome c 6 from Synechococcus sp. PCC 7002. The 3 10 helix (yel- low) is followed by the X-loop (red). The specific insertion is shown in magenta. The N-terminus and C-terminus, as well as the two Cys residues with covalent links to the heme group, are indicated. (B) Electron density omit map contoured at 3.2r, showing the heme group. All structural figures were prepared in PYMOL [39]. Atomic-resolution structure of cytochrome c 6 W. Bialek et al. 4428 FEBS Journal 276 (2009) 4426–4436 ª 2009 The Authors Journal compilation ª 2009 FEBS equivalent to Pro62 of the S. obliquus protein, has the exo pucker, confirming the reduced state of the mole- cule (Fig. S1). Although the conformation of this Pro may serve as an indicator of the protein redox state, the mechanism of this change is currently unknown. Its elucidation will require a comparison of the present structure with a model of the oxidized molecule deter- mined at comparably high resolution. In total, four water molecules are present around the two propionate substituents (at positions 6 and 7) of each heme group (Fig. 3). Two of them (water 20 ⁄ water 33, water 18 ⁄ water 45 and water 24 ⁄ water 30 in molecules A, B, C, respectively) are located between K44 L50 G46 A C B Fig. 2. (A) Structural alignment of 10 cytochrome c 6 and cytochrome c 6A molecules: Synechococcus sp. PCC 7002 (green, this work, 3DR0); P. yezoensis (light pink, 1GDV); H. fusiformis (black, 2ZBO); M. braunii (blue, 1CTJ); S. obliquus (yellow, 1C6O); C. glomerata (magenta, 1LS9); C. reinhardtii (cyan, 1CYI); A. maxima (red, 1F1F); P. laminosum (gray, 2V08); and A. thaliana (orange, 2DGE). The char- acteristic insertions present in the proteins from Synechococcus sp. PCC 7002 and A. maxima (bottom left) and A. thaliana (top middle) are shown. (B) Side chains of the KDGSKSL(44–50) insertion shown in ball-and-stick representation. (C) Alignment of cytochrome c 6 and cytochrome c 6A sequences prepared in ESPRIPT [40]. White letters on a red background represent strictly conserved residues; similar resi- dues are shown as purple letters on a white background in boxes. Insertions of Synechococcus sp. PCC 7002, A. maxima and A. thaliana are shown in green, red and orange, respectively. Above the sequences, the secondary structure elements of the present cytochrome c 6 molecule are shown. Table 1. Hydrogen bonding of amino acids of the insertion (bold), as calculated by WHATIF [38]. Distances are given in A ˚ . Donor Acceptor Molecule A Molecule B Molecule C Lys44 N Leu40 O 2.84 2.89 2.93 Gly46 N Tyr43 O 3.11 3.51 3.21 Ser47 N Asp45 O d1 3.15 3.25 3.14 Ser47 N Gly42 O 2.97 3.04 2.97 Ser47 O c Gly42 O 3.18 3.41 3.27 Ser47 O c Asp45 O d1 2.66 2.70 2.60 Lys48 N Gly42 O 3.08 3.08 3.16 Glu52 N Ser49 O c 3.16 3.07 3.17 Ala53 N Ser49 O 2.93 2.88 2.93 Val54 N Leu50 O 2.86 2.86 2.86 W. Bialek et al. Atomic-resolution structure of cytochrome c 6 FEBS Journal 276 (2009) 4426–4436 ª 2009 The Authors Journal compilation ª 2009 FEBS 4429 the propionate groups and create a network of hydro- gen bonds involving the propionates. Propionate-6 is also hydrogen-bonded with Lys29 and Gln62, whereas propionate-7 has a hydrogen bond contact with Thr30. Additionally, a hydrogen bond between water 20 and the Gln57 side chain is observed. Table 2 presents a complete list of hydrogen bonds within the three heme pockets. Electrostatic surface potential The low isoelectric point of the molecule, calculated as pI 3.8 [8], results from the ratio of acidic (nine) to basic (seven) amino acids (Fig. 4). The only Arg, Arg71, is present in all cytochrome c 6 molecules, except for the protein from S. obliquus. Arg71 creates a minor basic patch on the otherwise highly negative protein surface. The basic centers are predominantly Lys residues. Interestingly, two of the six Lys residues in the protein sequence of Synechococcus sp. PCC 7002 cytochrome c 6 , Lys44 and Lys48, are located within the unique insertion. Their long side chains point away from a bulge with negative surface potential created by the atoms between them (Fig. 4). All of the negative charge centers in this region (Lys44 O, Asp45 O, O d1 and O d2 , Gly46 O, Ser47 O, and O c ) point towards the surface of the protein and contribute to the overall negative surface potential. The major negative patch, however, is created mainly by the side chains of Glu51, Asp86, and Glu89. The surface potential of the molecule shows an additional slightly positive region, near the heme crevice, created by Lys29 and Lys44. Fig. 3. The heme pocket of superposed molecules A (red), B (green), and C (blue). Hydrogen bonds involving the depicted resi- dues are listed in Table 2. Structural water molecules associated with protein molecules A, B and C are shown in the corresponding colors. Table 2. Hydrogen bond distances (A ˚ ) within the heme pockets of molecules A, B, and C. Asterisks indicate water molecules located between the heme propionate groups. Molecule A Molecule B Molecule C His18 N d1 Asn22 O 2.86 2.87 2.84 Heme O2D Water 34 ⁄ 496 ⁄ 137 2.61 2.87 2.64 Heme O2D Water 227 ⁄ 390 ⁄ 69 3.17 2.69 2.51 Heme O1D Water 227 ⁄ 390 ⁄ 69 3.22 3.38 3.16 Heme O1D Gln62 N e2 3.40 3.35 3.37 Heme O1D Lys29 N f 2.78 2.77 2.91 Heme O1D Water 20 ⁄ 18 ⁄ 24 2.69 2.70 2.70 Heme O2A Water 33 ⁄ 45 ⁄ 30 2.85 2.80 2.83 Heme O1A Thr30 O 2.66 2.65 2.63 *Water 20 ⁄ 18 ⁄ 24 *Water 33 ⁄ 45 ⁄ 30 2.58 2.61 2.61 Gln57 O e1 Water 20 ⁄ 18 ⁄ 24 2.70 2.70 2.75 Gln57 N e2 Gln62 O e1 2.94 2.97 3.03 Fig. 4. Electrostatic surface potential distribution for PetJ1. The red zones correspond to negative potential, and blue zones correspond to positive potential. The color range spans )2 to 2 kT. Side chains are shown as sticks. The side chain of Glu89 assumes two confor- mations. The pale blue island on the visible red face of the molecule corresponds to the NH 3 + group of the N-terminus. The figure was prepared in PYMOL using default settings of the APBS plugin [41]. Atomic-resolution structure of cytochrome c 6 W. Bialek et al. 4430 FEBS Journal 276 (2009) 4426–4436 ª 2009 The Authors Journal compilation ª 2009 FEBS Interactions of aromatic residues In total, there are seven aromatic residues in the pro- tein sequence, not including the sole His, which acts as a heme ligand. The side chains of the three conserved aromatic residues, Phe10, Tyr83, and Trp93, form a triangular aromatic packing motif (Fig. 5A) buried in the hydrophobic core of the molecule. In this motif, C–H bonds at the edges of Tyr83 and Trp93 point to the ring center of Phe10 (C–ring centroid distances 4.1–4.2 A ˚ ) (Table S2). Similar C-H–p interactions are also present in other cytochromes c 6 with known crys- tal structures. In some cytochrome c 6A molecules, including that from A. thaliana, whose crystal structure is known, the Tyr of this ‘aromatic triangle’ is replaced by Phe. It is interesting to note that whereas the side chain of Trp93, owing to these interactions, is tightly wedged into a nearly ideally shaped pocket, the C-ter- minus of its main chain shows significant flexibility and has been modeled in two distinct conformations in all three molecules (Fig. 5B). Energetically significant cation–p interactions have been detected between Tyr43, Tyr56 and Phe68 and, respectively, Lys37, Lys48 and Arg71 by the capture software [22]. Tyr56, although pointing with its OH group towards bulk solvent, is firmly held in place by two N-H–p interactions on both sides of the ring, from Lys48 (described above) and from the side chain amide of Asn60. The latter interaction, which is very short (Table S2), may be especially important for stabiliza- tion of Tyr56. Similar interactions stabilizing a key Tyr residue were noted previously in the ultrahigh-resolu- tion structure of bovine pancreatic trypsin inhibitor [23]. In the present case, the amide N–p distance (3.29– 3.37 A ˚ ) is even shorter than in the bovine pancreatic trypsin inhibitor structure (3.44–3.58 A ˚ ). The stability of Tyr56 may be important for the conformation of Gln57, and consequently for tuning the redox potential of the protein. Phe68 forms part of a conserved motif, Pro66-Ala-Phe68. This motif is adjacent to the second heme ligand, Met65, and Pro66 undergoes conforma- tional changes upon oxidation ⁄ reduction [16]. Whereas in cyanobacterial, plant and red and brown algal cyto- chrome c 6 this Phe is conserved, it is replaced by Trp in green algae (Fig. 2C). It is of note that Phe68 interacts with the single Arg found in cytochromes c 6 , Arg71 (Fig. 5C). This Arg is required for cytochrome f oxida- tion [24] and photosystem I reduction [25]. In addition, Phe68 forms an evident C–H–p interaction with the heme group itself (Table S2). All of the remaining aro- matic residues (Phe10, Tyr39, Tyr83, and Trp93) are involved in C–H–p interactions (Table S2). Comparison of existing models of cytochrome c 6 The three independent molecules of Synechococcus cytochrome c 6 in the asymmetric unit are similar, with pairwise rmsd values of 0.30 (A ⁄ C), 0.32 (B ⁄ C) and 0.36 A ˚ (A ⁄ B) for the Ca atoms (Fig. 6). In general, the structures superpose very closely, but there are two regions with larger discrepancies. The first one is the single Gly63, and the second is a stretch of six residues from Gly70 to Ala75, with the largest difference of 1.68 A ˚ between Gly70 of chains B and C. This large A B C Fig. 5. Interactions of aromatic residues. (A) The three aromatic residues involved in a ‘triangular’ interaction in the protein core. (B) A pocket around Trp93. Note the tightly wedged aromatic side chain of Trp93 and the dual conformation of its C-terminal group. (C) N–H p interaction between the only Arg and Phe68. (A) and (C) show 2F o À F c electron density contoured at 1.2r. W. Bialek et al. Atomic-resolution structure of cytochrome c 6 FEBS Journal 276 (2009) 4426–4436 ª 2009 The Authors Journal compilation ª 2009 FEBS 4431 difference can be attributed to a peculiarity of the packing arrangement involving molecules B and C, in which the side chain of Asp74 of molecule C accepts two strong hydrogen bonds from the main chain N–H groups of Gly70 and Arg71 of molecule B. In this interaction, the carbonyl oxygen of Gly69 of mole- cule B flips by 180°, bringing the main chain nitrogen atom of residue 70 much closer to the O d atom of the adjacent Asp74. The changed geometry of this frag- ment should be classified as reverse turn type III. The corresponding fragment in the other two molecules, as in all known X-ray structures of c 6 -type cytochromes, is classified as b-turn type II. The higher lability of this region in the otherwise very rigid and well-ordered structure is not surprising, because, in general, the region on the C-side of the heme moiety (i.e. including residues from the loop between helices II and III to the C-terminus) is more flexible, and the acidic region Asp73-Glu74-Asp75-Glu76 is a hot spot in the struc- ture of cytochrome c 6 from M. braunii [14]. There are 13 side chains that do not align in molecu- lar superpositions, adopting different rotamers in one or more of the three molecules. With two exceptions, all of these side chains are on the surface, and some of them are clearly perturbed by crystal contacts. The first exception is Pro27, which in molecule C has the exo pucker, whereas in the other two cases it is endo. The second exception is Ile84, which is buried inside the molecular core but in molecule B has C d in two conformations. Only 10 of the 261 side chains (3.8%) in the asymmetric unit have the side chains in double conformation. Such a small number of alternative con- formations is related to the unusual amino acid com- position, where Ala accounts for 25.8% and Gly for 9.7% of all the residues in the amino acid sequence of this cytochrome c 6 . Table S1 shows both the percentage of sequence identity and the overall Ca rmsd values for pairs of different cytochrome c 6 structures. The lowest Ca backbone deviations are observed in the regions of helices I, III, and IV, as well as the 3 10 helix and the X-loop. consurf analysis [26] has revealed several resi- dues that are most conserved (data not shown). These residues are in the heme pocket area and are involved in electron transfer. Whereas they are located within regions of the lowest Ca discrepancies, the shortest helix II is sequentially the least conserved element among the cytochrome c 6 molecules. The largest devia- tions are observed around the specific insertion of this cytochrome c 6 molecule, i.e. in the loop connecting helices II and III. Intermolecular contacts As a consequence of the very low solvent content (17.58%) of the Synechococcus cytochrome c 6 crystals, their structure is very densely packed, and a number of surface residues are involved in direct contacts with neighboring protein molecules. The contacts between molecules A and B involve three hydrogen bonds from the main chain amide of Ala75 and the side chains of Thr28 and Lys29 to the main chain carbonyl groups of, respectively, Asp45 (B), Gly70 (A), and Arg71 (A). There are only two direct hydrogen bonds between molecules A and C involving main chain atoms of molecule A, namely oxygen of Asn60 and nitrogen of Gly63, and the side chains of Lys29 and Tyr39 of mol- ecule C. Three of the four direct hydrogen bonds between molecules B and C are formed by the carbox- ylic group of Asp74 of molecule C, which interacts with the N of Gly70, and both the N g atoms of Arg71. The fourth bond is created between the Asp74 nitrogen atom (molecule B) and Gln52 O e2 . There are five direct hydrogen bonds between the three molecules in the asymmetric units and their sym- metry counterparts. Three of them are created by Asn91, which uses its N d (two bonds) and O for inter- actions with, respectively, the main chain atoms of 10 30 10 Fig. 6. Stereoview of the superposition of the main chain traces of the three indepen- dent molecules in the asymmetric unit, with residue numbering added for orientation. The side chains of the two iron-coordinating residues (His18 and Met65) are shown as sticks. Color code as in Fig. 3. Atomic-resolution structure of cytochrome c 6 W. Bialek et al. 4432 FEBS Journal 276 (2009) 4426–4436 ª 2009 The Authors Journal compilation ª 2009 FEBS Ala7 and Gly20, and the side chain of Tyr83. The latter side chain is also a hydrogen bond donor to the Gln89 O. Gln87 and Asn90 are linked through an N e ÆO d hydrogen bond. Because of the low solvent content, the surface area buried on intermolecular contacts (including asym- metric unit and symmetry-related protein molecules) is quite high, 41.7% (molecule A), 45.8% (molecule B), and 47.1% (molecule C). Crystal packing and water structure With three independent molecules of cytochrome c 6 in the crystallographic asymmetric unit, the volume-to- mass ratio V M is 1.49 A ˚ 3Æ Da )1 , corresponding to a solvent content of merely 17.58%. There are only four protein structures deposited in the Protein Data Bank that have lower reported solvent content (V S ) (1XEK, 2J70, 2DUY, and 3C00). However, the number of wrongly attributed Matthews coefficients and solvent volumes in structures deposited in the Protein Data Bank is astonishingly high. Searching the Protein Data Bank for protein structures with V S between 5.00% and 17.57% gave 29 structures. Calculations of the V M and V S values (Matthews program, CCP4 [27]) for those structures returned only four cases with V S within the search interval. Therefore, we would not be surprised if the actual number of structures with lower V S was higher. The solvent molecules modeled in the electron den- sity maps correspond to 252 H 2 O sites with full occu- pancy and 272 with partial occupancy, which are equivalent to 404 effective water molecules. Although the number of partial water molecules may seem high, the average water occupancy is 0.76, which is very close to the value of 0.78 determined for a protein structure at ultimate resolution (0.65 A ˚ ) where solvent occupancies were refined [28]. Considering the low sol- vent content of this crystal, one can conclude that essentially all water molecules in the solvent region have been accounted for in this atomic model of the crystal structure. Additionally, there are two partially occupied sulfate ions. One of them forms two hydro- gen bonds with the main chain nitrogen atoms of Asp74 (2.80 A ˚ ) and Ala67 (2.86 A ˚ ) and with a water molecule (2.43 A ˚ ). The other one forms two hydrogen bonds with N g2 (3.12 A ˚ ) and N e (3.03 A ˚ ) of Arg71 and with two water molecules (2.79 and 2.77 A ˚ ). Conclusions In summary, cytochrome c 6 from Synechococcus sp. PCC 7002 has an overall structure that is similar to that of other previously characterized cyto- chrome c 6 molecules. However, unlike other cyto- chrome c 6 molecules, the Synechococcus sp. PCC 7002 protein has a unique insertion of seven amino acid residues [KDGSKSL(44–50)], which is entirely differ- ent from the insertion found in type c 6A cyto- chromes. The presence of the insertion in the most variable region of cytochrome c 6 might suggest that it does not play any role in the cell. On the other hand, the inability to inactivate or replace the gene encoding this cytochrome c 6 by PC or cytochrome c 6 from Synechocystis [19] is a strong indication that the presence of the insertion is essential for this organism. The question of whether the Synechococcus sp. PCC 7002 insertion does indeed have a biological function is currently being studied by site-directed mutagenesis. Experimental procedures Protein expression and purification Escherichia coli strain DH5a was cotransformed with pUCJ1 and pEC86 plasmids. The former harbors a gene encoding mature cytochrome c 6 from Synechococcus sp. PCC 7002, whereas the latter harbors the heme matura- tion genes [29]. Protein was expressed basically as described elsewhere [8]. Briefly, 5 mL of overnight culture was used to inoculate 1.7 L of LB medium supplemented with 1 mm FeCl 3 in a 2 L flask. Cultures were grown for 72–96 h at 30 °C with agitation at 150 r.p.m. and harvested, and the periplasmic proteins were released by lysozyme treatment (1 mgÆmL )1 ). Subsequently, cytochrome c 6 was purified as outlined in [8]. Protein crystallization The hanging-drop vapor diffusion method was used for the crystallization experiments. Cytochrome c 6 (1 mm in 10 mm Tris buffer, pH 7.5) was reduced by the addition of sodium dithionate to a final concentration of 20 mm. Drops con- taining equal volumes (1 lL) of protein and reservoir solu- tions were equilibrated against 1 mL of reservoir solution in 24-well Linbro plates at 19 °C. Red crystals suitable for X-ray analysis were obtained from 10 mm sodium Hepes (pH 6.2) and 2.2 m ammonium sulfate over a period of 1 week. X-ray diffraction data collection A single crystal was scooped through a solution of 30% glycerol and 70% reservoir solution (v ⁄ v) and placed in a stream of cold N 2 gas at 100 K. Diffraction data were col- lected on the X11 synchrotron beamline (k = 0.8170 A ˚ )at W. Bialek et al. Atomic-resolution structure of cytochrome c 6 FEBS Journal 276 (2009) 4426–4436 ª 2009 The Authors Journal compilation ª 2009 FEBS 4433 the EMBL Outstation, c ⁄ o DESY, Hamburg, using a MAR 165 mm CCD detector. Two passes were collected, using different crystal-to-detector distances, oscillations (1° and 0.5°) and exposure times to ensure reliable measure- ment of high-resolution data as well as of the strong low-angle reflections. The Synechococcus sp. PCC 7002 cytochrome c 6 crystals belong to space group P3 2 with unit cell dimensions a = 82.88 A ˚ and c = 28.28 A ˚ and diffract to at least 1.226 A ˚ . The mean overall redundancy is 5.0, and it is 2.8 in the highest-resolution shell (1.260–1.226 A ˚ ). The data are 98.7% complete with an overall R int of 0.076. Post-refinement of the crystal parameters during scaling indicated low mosaicity throughout the data collection (0.29°). The diffraction data were indexed, integrated and scaled using the hkl2000 [30] package, and the final statis- tics are included in Table 3. Structure solution and refinement The structure was solved by the molecular replacement method as programmed in molrep [31], using diffraction data in the 20.0–3.5 A ˚ resolution range and the cyto- chrome c 6 model from the green alga M. brauni [14] (Protein Data Bank accession number: 1CTJ) as the molecular probe. Analysis of the Matthews volume [32] indicated the pres- ence of two or three protein molecules in the asymmetric unit (2.24 or 1.49 A ˚ 3Æ Da )1 , solvent content 45.05% or 17.58%). Molecular replacement calculations confirmed that there were three independent protein chains. The cor- rect space group enantiomorph, P3 2 , was deduced from a comparison of the translation function solutions in the two alternative space groups (P3 2 ⁄ P3 1 with CC = 0.430 ⁄ 0.235, R = 0.454 ⁄ 0.526). The structure was refined anisotropically using the maxi- mum-likelihood algorithm as implemented in refmac [33], including the TLS parameters [34]. For free R-factor calcu- lations [35], 1274 reflections were randomly selected from the full resolution range of 50.0–1.226 A ˚ . After the first round of refmac refinement (isotropic, no TLS parame- ters), the model was rebuilt in quanta [36]. The final refined model is characterized by an R-factor of 0.107 and an R free of 0.138 for all 62 908 reflections between 50 and 1.226 A ˚ , and has an rmsd from ideal bond lengths of 0.020 A ˚ . The very high quality of the electron density maps allowed the identification of 524 (including 252 fully occu- pied) water sites and of two partially occupied sulfate ions. Stereochemical analysis of the final model using procheck [37] indicated that there are no residues with generously allowed or unfavorable backbone dihedral angles, and that 87.1% of all residues are in the core region of the Ramachandran plot. The statistics of the refinement are shown in Table 3. Acknowledgements Some of the calculations were carried out in the Poz- nan Metropolitan Supercomputing and Networking Center. This work was funded in part by grants N N204 245635 and N N303 3856 33 from the Minis- try of Science and Higher Education. References 1 Cramer WA, Furbacher PN, Szczepaniak A & Tae GS (1991) Electron Transport between Photosystem II and Photosystem I. Academic Press, San Diego. 2 Kallas T (1994) The cytochrome b6f complex. In The Molecular Biology of Cyanobacteria: Advances in Photosynthesis and Respiration (Bryant DA, ed.), pp. 559–579. Springer, Dordrecht. 3 Herva ´ s M, Navarro JA & De la Rosa MA (2003) Elec- tron transfer between membrane complexes and soluble Table 3. Data collection and refinement statistics. Data collection Radiation source X11, EMBL Hamburg Wavelength (A ˚ ) 0.8170 Temperature of measurements (K) 100 Space group P3 2 Cell dimensions (A ˚ ) a = 82.88, c = 28.28 Mosaicity (°) 0.29 Resolution range (A ˚ ) 50.00–1.226 (1.260–1.226) a Crystal-to-detector distance (mm) 160 ⁄ 100 Oscillation step (°) 1.0 ⁄ 0.5 No. of images 120 ⁄ 240 Reflections collected 313 876 Unique reflections 62 960 (5567) Completeness (%) 98.7 (87.7) Redundancy 5.0 (2.8) <I>=<rI> 23.1 (2.4) R int b 0.076 (0.389) Refinement Independent protein molecules 3 Matthews coefficient (A ˚ 3Æ Da )1 ) 1.49 No. of reflections in work ⁄ test set 61 634 ⁄ 1274 Rejection criterion None R c ⁄ R free (%) 0.107 ⁄ 0.138 Number of atoms (protein ⁄ solvent) rmsd from ideal 2708 (2174 ⁄ 534) Bond lengths (A ˚ ) 0.020 Bond angles (°) 1.76 Chiral volumes (A ˚ 3 ) 0.104 Average B-factor (protein ⁄ solvent) (A ˚ 2 ) 11.40 (9.59 ⁄ 18.83) Residues in Ramachandran plot (%) Most favored 86.7 Additional 13.3 a Values in parentheses correspond to the last resolution shell. b R int ¼ R h R j I hj À <I h >     =R h R j I hj , where I hj is the intensity of obser- vation j of reflection h. c R ¼ R h F o jjÀF c jjjj=R h F o jjfor all reflec- tions, where F o and F c are observed and calculated structure factors, respectively. 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Biochem Biophys Res Commun 251, 744–747. W. Bialek et al. Atomic-resolution structure of cytochrome c 6 FEBS Journal 276 (2009) 4426–4436 ª 2009 The Authors Journal compilation ª 2009 FEBS 4435 [...]... plastocyanin Structure 3, 1159–1169 Kerfeld CA, Anwar HP, Interrante R, Merchant S & Yeates TO (1995) The structure of chloroplast cyto˚ chrome c6 at 1.9 A resolution: evidence for functional oligomerization J Mol Biol 250, 627–647 Schnackenberg J, Than ME, Mann K, Wiegand G, Huber R & Reuter W (1999) Amino acid sequence, crystallization and structure determination of reduced Atomic-resolution structure of. .. Szczepaniak A (2008) Deeply branching c6- like cytochromes of cyanobacteria Biochemistry 47, 5515–5522 Schlarb-Ridley BG, Nimmo RH, Purton S, Howe CJ & Bendall DS (2006) Cytochrome c6A is a funnel for thiol oxidation in the thylakoid lumen FEBS Lett 580, 2166–2169 Sawaya MR, Krogmann DW, Serag A, Ho KK, Yeates TO & Kerfeld CA (2001) Structures of cytochrome c-549 and cytochrome c6 from the cyanobacterium... in quanta [36] The final refined model is characterized by an R-factor of 0.107 and an Rfree of 0.138 for all 62 908 reflections between 50 and ˚ 1.226 A, and has an rmsd from ideal bond lengths of ˚ 0.020 A The very high quality of the electron density maps allowed the identification of 524 (including 252 fully occupied) water sites and of two partially occupied sulfate ions Stereochemical analysis of the... expression and purification of cytochrome c6 from the brown alga Hizikia fusiformis and complete X-ray diffraction analysis of the structure Acta Crystallogr Sect F Struct Biol Cryst Commun 64, 674–680 Frazao C, Soares CM, Carrondo MA, Pohl E, Dauter Z, Wilson KS, Hervas M, Navarro JA, De la Rosa MA & Sheldrick GM (1995) Ab initio determination of the crystal structure of cytochrome c6 and comparison with. .. are no residues with generously allowed or unfavorable backbone dihedral angles, and that 87.1% of all residues are in the core region of the Ramachandran plot The statistics of the refinement are shown in Table 3 Acknowledgements Some of the calculations were carried out in the Poznan Metropolitan Supercomputing and Networking Center This work was funded in part by grants N N204 245635 and N N303 3856... crystal-to-detector distances, oscillations (1° and 0.5°) and exposure times to ensure reliable measurement of high-resolution data as well as of the strong low-angle reflections The Synechococcus sp PCC 7002 cytochrome c6 crystals belong to space group P32 with unit ˚ ˚ cell dimensions a = 82.88 A and c = 28.28 A and diffract ˚ to at least 1.226 A The mean overall redundancy is 5.0, ˚ and it is 2.8 in the... Bendall DS, Luisi BF & Howe CJ (2006) Structure of cytochrome c6A, a novel dithio -cytochrome of Arabidopsis thaliana, and its reactivity with plastocyanin: implications for function J Mol Biol 360, 968–977 Nomura C & Bryant DA (1999) Cytochrome c6 from Synechococcus sp PCC 7002 In The Phototrophic Prokaryotes (Peschek G, ed.), pp 269–274 Kluwer Academic ⁄ Plenum, New York Zhang L, Pakrasi HB & Whitmarsh J... Functional relationship of cytochrome c6 and plastocyanin in Arabidopsis Nature 417, 567–571 Wastl J, Purton S, Bendall DS & Howe CJ (2004) Two forms of cytochrome c6 in a single eukaryote Trends Plant Sci 9, 474–476 Worrall JA, Schlarb-Ridley BG, Reda T, Marcaida MJ, Moorlen RJ, Wastl J, Hirst J, Bendall DS, Luisi BF & Howe CJ (2007) Modulation of heme redox potential in the cytochrome c6 family J Am Chem... 245635 and N N303 3856 33 from the Ministry of Science and Higher Education References 1 Cramer WA, Furbacher PN, Szczepaniak A & Tae GS (1991) Electron Transport between Photosystem II and Photosystem I Academic Press, San Diego 2 Kallas T (1994) The cytochrome b6f complex In The Molecular Biology of Cyanobacteria: Advances in Photosynthesis and Respiration (Bryant DA, ed.), pp 559–579 Springer, Dordrecht... of cytochrome c6 17 18 19 20 21 22 23 24 25 26 27 28 29 and oxidized cytochrome c6 from the green alga Scenedesmus obliquus J Mol Biol 290, 1019–1030 Dikiy A, Carpentier W, Vandenberghe I, Borsari M, Safarov N, Dikaya E, Van Beeumen J & Ciurli S (2002) Structural basis for the molecular properties of cytochrome c6 Biochemistry 41, 14689–14699 Marcaida MJ, Schlarb-Ridley BG, Worrall JA, Wastl J, Evans . Atomic-resolution structure of reduced cyanobacterial cytochrome c 6 with an unusual sequence insertion Wojciech Bialek 1 ,. determination of the crystal structure of cytochrome c6 and comparison with plastocyanin. Structure 3, 1159–1169. 15 Kerfeld CA, Anwar HP, Interrante R, Merchant