Biochemical and Biophysical Research Communications 443 (2014) 11311135

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Biochemical and Biophysical Research Communications

Cytochrome c6B of Synechococcus sp. WH 8102 Crystal structure and basic properties of novel c6-like family representative q
Pawel Zatwarnicki a, Jakub Barciszewski c, Szymon Krzywda b, Mariusz Jaskolski b,c, Piotr Kolesinski a, Andrzej Szczepaniak a,
a b c

Department of Biophysics, Faculty of Biotechnology, University of Wroclaw, F. Joliot Curie 14a, 50-383 Wroclaw, Poland Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland

a r t i c l e

i n f o

a b s t r a c t
Cytochromes c are soluble electron carriers of relatively low molecular weight, containing single heme moiety. In cyanobacteria cytochrome c6 participates in electron transfer from cytochrome b6f complex to photosystem I. Recent phylogenetic analysis revealed the existence of a few families of proteins homologous to the previously mentioned. Cytochrome c6A from Arabidopsis thaliana was identied as a protein responsible for disulde bond formation in response to intracellular redox state changes and c550 is well known element of photosystem II. However, function of cytochromes marked as c6B, c6C and cM as well as the physiological process in which they take a part still remain unidentied. Here we present the rst structural and biophysical analysis of cytochrome from the c6B family from mesophilic cyanobacteria Synechococcus sp. WH 8102. Puried protein was crystallized and its structure was rened at 1.4 resolution. Overall architecture of this polypeptide resembles typical I-class cytochromes c. The main features, that distinguish described protein from cytochrome c6, are slightly red-shifted a band of UVVis spectrum as well as relatively low midpoint potential (113.2 2.2 mV). Although, physiological function of cytochrome c6B has yet to be determined its properties probably exclude the participation of this protein in electron trafcking between b6f complex and photosystem I. 2013 Elsevier Inc. All rights reserved.

Article history: Received 15 October 2013 Available online 9 November 2013 Keywords: Photosynthesis Cyanobacteria Synechococcus sp. WH 8102 Cytochrome Cytochrome c6-like Protein crystallography High resolution structure

1. Introduction In cyanobacterial cells, the interaction between different photosynthetic membrane complexes is possible due to the presence of mobile electrons carriers. Plastoquinone molecules transport electrons between PSII and cytochrome b6f complex within phospholipid bilayer. Metalloproteins: cytochrome c6 and/or plastocyanine are responsible for electron transport between cytochrome b6f complex and PSI in thylakoid lumen. The oxidized form of these metalloproteins (Cu2+-carrying plastocyanine as well as Fe3+-containing cytochrome c6) adopts one electron from cytochrome f, being a part of the b6f complex [1]. As a result of this reaction the reduced electron carrier is formed which moves within the lumen

Abbreviations: Cyt, cytochrome; Wat, water. Database: Atomic coordinates and structure factors for Cytochrome c6B of Synechococcus sp. WH 8102 are available from the Protein Data Bank under the accession code 4KMG. Corresponding author. Address: Department of Biophysics, Faculty of Biotechnology, University of Wroclaw, F. Joliot Curie 14a, 50-383 Wroclaw, Poland. E-mail address: Andrzej.Szczepaniak@ibmb.uni.wroc.pl (A. Szczepaniak). 0006-291X/$ - see front matter 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.10.167
q

towards PSI. For specic targeted trafc of mentioned metalloproteins electrostatic and/or hydrophobic interactions are responsible [2]. After docking to PSI, metalloprotein passes an electron onto photooxidized dimer of chlorophyll P700+ molecules. This event closes the electron transfer from b6f to PSI [1]. Photosynthetic electron transport is not the only function of cyanobacterial cytochrome c6 and plastocyanin. In cyanobacteria, photosynthetic and respiratory electron transport chains physically overlap in thylakoid membranes. In opposition, analogous protein complexes localized in the cytoplasmic membranes participate only in respiration process. In cyanobacteria typical cytochromes of type c which would pass electrons to cytochrome c oxidase (in cyanobacteria oxidase type aa3) are absent. Nevertheless, a long time ago hypothesis was formed, that b6f complex, quinone pool or cytochrome c6 are elements common to the processes of photosynthesis and respiration in cyanobacteria. Subsequent data seem to conrm the validity of such supposition over the years [3]. The third process, which involves the cyanobacterial cytochrome c6 is the process of anoxygenic photosynthesis, where hydrogen sulde is a source of electrons. In this process, cytochrome c6 can transport electrons between quinones and iron-sulfur centers during the anaerobic oxidation of sulphides [4].

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Bialek and co-workers have identied two new groups of c6-like cytochromes. Due to the earlier discovery of c6A type cytochromes, these groups are named analogously as c6B and c6C [5]. Family of cyanobacterial c6B cytochromes is quite closely related to chloroplastic c6A cytochromes. However, cytochromes of group c6B and c6C do not include in their primary structure 12-residues-long loop, characteristic for c6A family. Another difference is conserved tyrosine residue at position 61 of cytochrome c6B and c6C, which is analogous to phenylalanine or tryptophan occupying corresponding position in c6 and c6A cytochromes. In the c6B group sequences are mainly derived from non-binding nitrogen marine cyanobacteria speciesProcholorococcus and Synechococcus. These species are characterized by about 95% identity to the 16S rRNA sequence, but consists of a number of physiologically and genetically different ecotypes [69]. The presence of genes encoding c6-like cytochromes in genomes of many cyanobacteria provides a basis for extending hypotheses about the role of these proteins in the proper functioning of cells. The diversity of cyanobacteria, in which these genes are present (single-celled green algae, lamentous or nitrogen-xing) indicate that c6-like cytochromes appeared relatively early in the history of the evolution of these microorganisms [5]. Compared to enormity of research projects concerning cytochromes in general, c6-like cytochrome are rarely mentioned in the literature. This fact may be primarily due to their relatively recent discovery. Data presented in this paper extend current knowledge about c6-like cytochromes. Unfortunately, biological function of these proteins still remains unclear.

2.2. Cytochromes absorption spectroscopy and redox titrations All spectroscopic measurements were carried out using a Beckman DU800 spectrophotometer. Measurements were conducted at room temperature using a 1 cm path-length cuvette. Cytochrome was diluted to nal concentration of 5 lM in 10 mM Tris pH 7.5 containing 1 mM potassium ferricyanide (spectra of oxidized cytochromes) or 1 mM sodium dithionate (spectra of reduced cytochromes). Redox titrations were performed as described in [11]. Procedure was repeated three times in both directions in a custom-made anaerobic cuvette, containing platinum electrode and calomel reference electrode, under argon ow, in 50 mM MOPS pH 7.0 and 100 mM KCl in the presence of redox mediators: tetrachlorohydroquinone (TCHQ, Em,7 = 350 mV), 2,3,5,6 tetramethyl-p-phenylenediamine (DAD, 260 mV), 1,2-naphthoquinone-4-sulfonate (NQS, Em,7 = 210 mV), 1,2-naphthoquinone (NQ, 130 mV), phenazine methosulfate (PMS, 80 mV), phenazine ethosulfate (PES, 55 mV), duroquinone (DQ, 5 mV). All redox mediators were at concentration of 45 lM and cytochrome at 5 lM. 50 mM Potassium ferricyanide was used as an oxidant and 50 mM sodium dithionate as a reductant. Spectra (range 400600 nm) were recorded in intervals of 1030 mV. Midpoint potentials were obtained from cytochromes a-band absorbance plotted against corresponding voltage.

2.3. Crystallization Initial screening for cytochrome c6B crystallization conditions was performed at the HTX facility, EMBL-Hamburg using the Classic, Classic 2, PACT and AmSO4 Qiagen suites [12]. The sitting-drop vapour-diffusion technique was used at 292 K, by mixing 0.3 ll protein (7.5 mg/mL in 20 mM sodium citrate, pH 3.2) and 0.3 ll reservoir solution. The optimization of crystallization conditions was carried out using the hanging-drop vapor-diffusion method at 292 K, by mixing 1 ll protein solution and 1 ll reservoir solution. Red crystals suitable for X-ray analysis were obtained from

2. Materials and methods 2.1. Protein expression and purication Escherichia coli strain DH5a was co-transformed with pUC_c6B and pEC86 plasmids. The former harbors a gene encoding mature cyt c6B from Synechococcus sp. WH8102, whereas the latter the heme maturation gene [10]. 5 mL of overnight culture were used to inoculate 1.75 L of TB medium in a 2 L ask. Cultures were grown for 8 h at 37 C with vigorous agitation and then IPTG was added to nal concentration of 0.75 mM. Subsequently, cultures were grown for 7296 h at 30 C with agitation at 80 rpm. Cells were harvested at 5000g, 4 C, washed in 30 mM Tris, pH 8.0, 0.1 M NaCl, 20% sucrose, 1 mM EDTA. After centrifugation and resuspension in the same buffer the periplasmic proteins were released by lysozyme treatment (0.2 mg/mL) at RT for 20 min with shaking and then centrifuged at 25,000g for 25 min at 4 C. The supernatant, which contains the periplasmic protein fraction, was incubated with ammonium sulfate (45% saturation, 30 min, 4 C) and centrifuged at 20,000g for 20 min at 4 C, and the pellet was discarded. Ammonium sulfate was added to the supernatant to 95% saturation and treated as described above. The red pellets were resuspended in 20 mM phosphate buffer pH 6.2, 1 mM PMSF and dialyzed against the same buffer, overnight at 4 C. Solution after dialysis was loaded onto a HiTrap SP HP column (GE Healthcare) connected to AKTA Purier system and equilibrated with the same buffer. Proteins were eluted with linear 0200 mM NaCl gradient. Fractions collected from the rst column were dialyzed against 50 mM ethanolamine pH 9.0. After overnight dialysis the sample was applied to a HiTrap Q HP column (GE Healthcare) equilibrated with the same buffer. Proteins were eluted with linear 100500 mM NaCl gradients. Puried cyt c6B was characterized by SDSPAGE and heme staining as described in [5]. The A557/A280 ratio of puried cyt c6C was 0.623. The proteins were aliquoted and stored at 20 C.

Fig. 1. Crystal structure of cytochrome c6B from Synechococcus sp. WH 8102. (A) Crystals of cytochrome c6B from Synechococcus WH 8102 obtained in 2.2 M sodium malonate, pH 7.0, 19 C, over a period of 10 days. Average dimension of 200 lm. (B) Overall structure of cytochrome c6B from Synechococcus sp. WH 8102 (PDB:4KMG). Ia-helix I; IIa-helix II; IIIa-helix III; IVa-helix IV. The structure resembles characteristic features of cytochromes of class c6. Polypeptide chain wraps around the heme moiety (grey). Secondary structure is described by four a-helices (cyan), several loops (magenta) and one b-hairpin (red). The N- and C-terminus are indicated.

P. Zatwarnicki et al. / Biochemical and Biophysical Research Communications 443 (2014) 11311135 Table 1 Data-collection and renement statistics. Data collection Space group Unit cell parameters (, ) X-ray source Wavelength () Temperature (K) Mosaicity () Resolution range ()a Rinta,b (%) <I/r(I)>a Reections Measured Unique Redundancya Completeness (%)a Wilson B-factor (2) Renement Resolution range () No. of reections Working set Test set Rwork/Rfreec (%) No. of atoms Protein Solvent <B>(2) Protein Solvent R.m.s. deviations from ideal Bond lengths () Bond angles () Ramachandran statistics (%) Favored Additional
a b

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P21 a = 106.11, b = 28.98, c = 24.68 b = 93.30 BL 14.2 BESSY, Berlin 0.91841 100 0.28 30.001.40 (1.491.40) 5.9 (55.9) 13.8 (2.0) 52939 14664 3.6 (3.1) 97.9 (90.1) 20.1 27.971.40 13712 950 12.0/18.1 687 95 15.1 22.3 0.016 1.769 96.5 3.5

Values in parentheses are for the highest resolution shell. Rint = RhklRi|Ii(hkl) - <I(hkl)>| / RhklRi Ii(hkl), where Ii(hkl) is the ith measurement of the intensity of reection hkl and <I(hkl)> is the mean intensity of reection hkl. c R = R||Fo|-|Fc|| / R|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. Rfree is calculated analogously for the test reections, which were randomly selected and excluded from the renement. Fig. 2. Biophysical properties of cytochrome c6B from Synechococcus sp. WH 8102. (A) Redox titration of cytochrome c6B from Synechococcus sp. WH 8102. Absorbance of fully reduced cytochrome was taken as 100%. Absorbance recorded during stepby-step oxidation was taken as corresponding percent. Midpoint potential was obtained from cytochrome a-band absorbance plotted against corresponding voltage and tted to one-component Nernst equation with aid of SigmaPlot. Cytochrome c6B midpoint potential is Em,7 = 113.2 2.2 mV. (B) UVVis spectra of cytochrome c6B from Synechococcus sp. WH 8102. UVVis spectra of reduced cyt c6B is characteristic for c6-like cytochromes. a-band is red-shifted in comparison to c6 cyts (maximum absorbance at 557.0 nm). Maximum absorbances of b-, c-, d-bands are at 523.5, 418.3 and 316.9 nm, respectively. Cyt c6B oxspectra of oxidized cyt c6B; Cyt c6B redspectra of reduced cyt c6B.

2.2 M sodium malonate, pH 7.0 over a period of 10 days (see Fig. 1A). 2.4. Data collection and processing The mother liquor was amenable to cryo-cooling. A single crystal was scooped in a nylon loop and ash-frozen in a nitrogen gas stream at 100 K. Diffraction data were measured on a Rayonics MX-225 CCD detector on beamline BL 14.2 at the BESSY II synchrotron facility, Berlin. Integration, scaling and merging of the intensity data was accomplished using the XDS package [13]. An overall B-factor of 20.1 2 was estimated from the Wilson plot using the CORRECT program from the XDS package [13]. The data were checked for diffraction anisotropy [14]. A very low spread in values of the three principal components (4.42 2) indicated almost no anisotropy. Space group, unit-cell and data-collection parameters are given in Table 1. 2.5. X-ray structure solution and renement The structure was solved by molecular replacement using the MOLREP program from the CCP4 suite [15,16] and the structure of c6-like cytochrome from Synechococcus sp. PCC 7002 (PDB code 4EIE) as the search model. The amino-acid sequence of the model shows 47.7% identity and 75% similarity (LALIGN; [17]) with cyt c6B. Maximum-likelihood structure-factor renement was carried out in REFMAC [18] using all intensity data, with the exception

of 950 reections (6.5%) agged for cross-validation purposes. No r cutoff was applied. To account for diffuse solvent effects, a correction according to the Babinet principle was applied. Manual rebuilding of the model was performed in COOT [19]. The main steps of the renement included (1) isotropic renement with manually added water molecules, (2) anisotropic renement, (3) addition of H atoms according to geometrical criteria as implemented in REFMAC. Full renement statistics are given in Table 1. 3. Results and discussion 3.1. Structure description Overall, the structure of Synechococcus sp. WH 8102 cyt c6B reveals the characteristic properties of other class I cytochromes c. In the electron density map, the rst amino acid residue (Gly)

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is missing. Cytochrome c6B consists of a single polypeptide chain of 88 amino acid residues (Fig. 1B). The heme ring is covalently bound to the polypeptide chain as in other cyts of c6 familyby two thioether bonds formed by sidechains of Cys14 and Cys17. The central iron cation of the heme moiety is axially coordinated by the imidazole ring of His18 and the side chain of Met58. Secondary structures found in the cyt c6B molecule (four a-helices, one 310-helix, several b-turns and b-hairpin motif) are separated by more or less organized loops. a-Helix I is formed by amino acid residues Glu5His18. This long N-terminal structural element, like in the other cyts c6, possesses a characteristic kink at position Cys14. A part of this helix is the heme-binding motif CXXCH-. Subsequent 310-helix (Val19-Gly21) is localized right next to it. The second a-helix is formed by residues Leu33-Arg38. The succeeding a-helix III consists of residues Thr44-Lys53. The last, fourth a-helix is formed by residues Gly69-Gln82. The relatively long regions between helices are well described by the different b- and c- type turns. The N-terminus of this cytochrome is arranged in a b-turn of type II (residues Thr2-Glu5). Further b-turns are present between helix I and IIwithin a well-dened in the cyt c6 family, conservative X-loop. In this region b-turns of type IV (Asn23-Arg26), type VIII (Ile24-Arg27), type I (Arg26-Lys29) as well as type IV (Arg27-Asn30) are localized. Inside the loop separating the second and third a-helices, reversed c-turn is located, formed by residues Leu41-Ser43. In the context of comparison of cyts c6 structures, the most interesting motifs are located in the long loop separating helices III and IV (region between residues Gly54 and Gly68). In this region, structure of a b-hairpin of class 2:2 IIP is present. The rst arm of this b-hairpin is formed by residues Gly54-Ile55, subsequent bend is formed by Gly56-Gln57 and the second arm by Met58-Ser59. The side chain of residue Met58, part of this b-hairpin, is also one of the heme iron axial ligands. Furthermore, within the discussed loop, residues Gly62-Leu65 are arranged in the pattern of an a-helix. The last b-turn in this region is a b-turn type II formed by residues Gly66-Gly69. The C-terminal fragment of the molecule is arranged in a b-turn formed by residues Asn83-Thr86. 3.2. Heme binding pocket environment In contrast to typical cyts c, the environment of the heme binding pocket in cyt c6B is hydrophobic, as in other cytochromes referred to c6-like, e.g. c6A from Arabidopsis thaliana [20] or c6C from

Synechococcus sp. PCC 7002 [Krzywda & Zatwarnicki et al. in preparation]. This feature results from the presence of a hydrophobic amino acid side chain (i.e. leucine, isoleucine, valine) in position occupied by a conservative glutamine in typical cyts c6 [5]. Furthermore, presence of such polar residue inside heme binding pocket seems to have crucial inuence on midpoint redox potential as well as for spectral properties, which is in agreement with previous literature reports [21]. The present cyt c6B possesses hydrophobic side chain of isoleucine-50 in the heme binding pocket, which probably is the major factor affecting its midpoint potential (Em,7 = 113.2 2.2 mV; Fig. 2A) which is very low in comparison to other cyts c6. Additionally, maximum absorption of a-band occurs at 557.0 nm, which is approx. 4 nm red-shifted in comparison to classical c6 cytochromes (Fig. 2B). As previously mentioned the heme ring of cyt c6B is covalently bound via two thioether bonds with side chains of Cys14 and Cys17. Heme iron is coordinated by a nitrogen atom Ne2 of His18 and sulfur atom Sd of Met58. The bond angle is 175.4. The distances between the atoms are as follows: Ne2 His18Fe3+ = 2.0 , Sd Met58Fe3+ = 2.3 . His18 imidazole ring is stabilized by a hydrogen bond with the carbonyl oxygen atom of Gly22. The network of hydrogen bonds in cyt c6B heme binding pocket (Fig. 3A) is completely different when compared to other structures of cyanobacterial cyts c6 (Fig. 3B) [22]. In close proximity to the heme ring, only one water molecule (wat-218) is present. It occupies two positions (A and B) with a 0.5:0.5 ratio. Additionally, the side chain of glutamine Gln39 may also adopt two different conformations-interaction in between this side chain and heme ring is possible only with the adoption of a conformation designated as A. As a result of such conformational change the hydrogen bonds are formed between propionate hp-7 and Ne atoms and Oe of Gln39 side chain. Moreover, wat-218 in position A218 forms hydrogen bond with Oe atom of Gln39. Furthermore, like in other cyanobacterial cyts c6 and c6-like [Krzywda & Zatwarnicki et al. in preparation] heme propionate hp-7 is stabilized by a hydrogen bond with the carbonyl oxygen of Asn30. The heme propionate hp-6 forms hydrogen bond network insulated from the two bonds between the hp-7 propionate oxygen atoms and atom Ng1 of Arg26 side chain and in addition, hydrogen bond between oxygen atom of hp-6 and carbonyl nitrogen of Gln57. The water molecule wat-218, when manning the B218 position, changes slightly hydrogen bonds network in the heme binding pocket. Compared to network discussed above, bond

Fig. 3. Comparison of heme-binding pockets of cytochrome c6B from Synechococcus sp. WH 8102 and cytochrome c6 from Synechococcus sp. PCC 7002. (A) Heme binding pocket of cytochrome c6B. In the closest proximity of the heme moiety, only one water molecule is present: wat-218. This molecule partly occupies positions A218 and B218 (ratio 0.5:0.5). Hydrogen bonds formed by side chains of Arg26, Gln37 (in position A), Gln57 and carbonyl oxygen of Asn30 stabilize heme moiety. hp-6,7heme propionate 6 and 7, respectively. (B) Heme binding pocket of cytochrome c6 of Synechococcus sp. PCC 7002; PDB:3DR0 [22]. In the closest proximity to heme ring, 3 water molecules are present (marked as 103, 105 and 106). These water molecules create hydrogen bond network, together with side chains of glutamine-57, glutamine-62, lysine-29 and carbonyl oxygen of threonine-30.

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between wat-218 and Gln39 are no more present. On the other hand, a bond between wat-218 and heme propionate hp-6 appears, in result creating interaction chain hp-6wat-218hp-7.

Acknowledgments This work was funded in part by grant N N303 817640 from the Ministry of Science and Higher Education awarded to AS. The research leading to these results has received funding from the European Communitys Seventh Framework Programme (FP7/ 2007-2013) under BioStruct-X (grant agreement N 283570). We thank HZB for the allocation of synchrotron radiation beamtime.

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