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High-resolution crystal structure of the recombinant diheme cytochrome c from Shewanella baltica (OS155) a
b
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Matteo De March , Giulia Di Rocco , Neal Hickey & Silvano Geremia a
Centre of Excellence in Biocrystallography (CEB), Department of Chemical and Pharmaceutical Sciences, University of Trieste, via L.Giorgieri 1, 34127 Trieste, Italy b
Department of Life Sciences, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy Published online: 21 Feb 2014.
Click for updates To cite this article: Matteo De March, Giulia Di Rocco, Neal Hickey & Silvano Geremia (2015) High-resolution crystal structure of the recombinant diheme cytochrome c from Shewanella baltica (OS155), Journal of Biomolecular Structure and Dynamics, 33:2, 395-403, DOI: 10.1080/07391102.2014.880657 To link to this article: http://dx.doi.org/10.1080/07391102.2014.880657
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Journal of Biomolecular Structure and Dynamics, 2015 Vol. 33, No. 2, 395–403, http://dx.doi.org/10.1080/07391102.2014.880657
High-resolution crystal structure of the recombinant diheme cytochrome c from Shewanella baltica (OS155) Matteo De Marcha, Giulia Di Roccob, Neal Hickeya and Silvano Geremiaa* a
Centre of Excellence in Biocrystallography (CEB), Department of Chemical and Pharmaceutical Sciences, University of Trieste, via L.Giorgieri 1, 34127 Trieste, Italy; bDepartment of Life Sciences, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy
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Communicated by Ramaswamy H. Sarma (Received 25 July 2013; accepted 3 January 2014) Multiheme cytochromes c (cyts c) are c-type cyts characterized by non-standard structural and spectroscopic properties. The relative disposition of the heme cofactors in the core of these proteins is conserved and they can be classified from their geometry in two main groups. In one group the porphyrin planes are arranged in a perpendicular fashion, while in the other they are parallel. Orientation of the heme groups is a key factor that regulates the intramolecular electron transfer pathway. A 16.5 kDa diheme cyt c, isolated from the bacterium Shewanella baltica OS155 (Sb-DHC), was cloned and expressed in E. coli and its structure was investigated by X-ray crystallography. Using high-resolution data (1.14 Å) collected at ELETTRA (Trieste), the crystal structure, with an orthorhombic cell (a = 40.81, b = 42.97, c = 82.07 Å), was solved using the homologous diheme from Rhodobacter sphaeroides (Rs-DHC) as the initial model. The electron density map of the refined structure (Rfact of 13.8% and Rfree of 15.4%) shows a two domain structure connected by a central unstructured region (N72-G87). The Sb-DHC, like its homologue (Rs-DHC), folds into a new cyt c class: the N-terminal globular domain, with its three α-helices, belongs to class I of c-type cyts, while the C-terminal domain includes a rare π-helix. The metal centre of the c-type heme groups is axially coordinated by two His residues and it is covalently bound to the protein through two Cys bonds. Keywords: cytochrome c; diheme; electron transfer; biocrystallography; edge to edge
Introduction Recent progress in bacterial genomic analysis has revealed a vast range of genes encoding novel c-type cyts that contain multiple heme cofactors. Shewanella is predicted to encode 42 c-type cyts and it is to date the most sequenced organism, with the exception of Geobacter sulfurreducens (111) (Heidelberg et al., 2002; Methe et al., 2003). Many of these putative cyts are predicted to be multiheme in character (Pitts et al., 2003). The identification of which c-type cyts are in fact present in the whole proteome of Shewanella, and the understanding of how these cyts act under different respiration conditions, will help to elucidate the pathways regarding the metal reduction and pollutant bioremediation. The genomes of Shewanella baltica (strains OS117, OS183, OS195, OS625 and OS678) have been completely sequenced (http://www.jgi.doe.gov/genome-projects) and the gene encoding for the S. baltica diheme cyt c (hereafter SbDHC) was identified by the characteristic double CXXCH motif. This 16.5 kDa diheme cyt c has been recently cloned, expressed and characterized by electrochemical and spectroscopic analyses (Di Rocco et al., 2011). These analyses indicated a thermodynamically *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
stable and kinetically inert axial heme His–His coordination for both hemes. Multiheme cyts c form an extensive class of proteins which act as electron shuttle/storage tools in several metabolic redox processes and respiratory chains (Mowat & Chapman, 2005). In these complex systems, which often operate in a membrane environment, several intramolecular electron transfer (ET) processes occur and these are generally coupled with multiple protonation phenomena and conformational changes (Andersen et al., 2001; Coutinho & Xavier, 1994; Garau, Geremia & Randaccio, 2002; Paquete, Turner, Louro, Xavier, & Caterino, 2007). Their functional properties are in part determined by the relative arrangement of multiple heme cofactors, which in many cases have been found to pack in conserved interaction motifs, namely perpendicular or parallel (Einsle, 2001; Heitmann & Einsle, 2005; Iverson et al., 1998). A good example is found in the crystal structure of the diheme cyt c4 from Pseudomonas stutzeri, a membrane-bound bacterial ET cyt c (Kadziola & Larsen, 1997). This exhibits a tetraheme architecture in which the two functional parallel heme c groups are oriented almost perpendicularly to the other two. An electrochemical study on the immobilized recombinant
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diheme cyt c4 from Pseudolateromans haloplanktis unequivocally showed that the intraheme ET occurs in the protein by a proton exchange at the H-bonded propionate groups of the two facing heme centres. This phenomenon was also reported for the structure of P. stutzeri diheme cyt c4 (Andersen et al., 2001; Andersen, Nørgaard, Jensen, & Ulstrup, 2002; Monari et al., 2009). Furthermore, the two heme c groups in the structure of DHC2 from G. Sulfurreducens (Heitmann & Einsle, 2005) pack into one of the typical heme interaction motifs observed in larger multiheme cyts and, due to the bis–His axial coordination for both hemes, it shows two redox potentials of −135 and −289 mV. Multiheme cyt’s c often have a big heme/protein ratio with few secondary structures elements (Einsle et al., 2000) and despite the low sequence homology, they have strong homologies in the arrangement of their heme groups (Heitmann & Einsle, 2005). In order to elucidate the highly optimized properties of multiheme cyts c, it is necessary to understand the significance of these motifs through spectroscopic and electrochemical investigation. Sb-DHC, like the homologous diheme cyt c from Rhodobacter spaheroides (Rs-DHC), represents an exception, due to the disposition of its two cofactors with non-standard geometry (Gibson et al., 2006). Materials and methods Sample preparation The Sb-DHC gene, isolated from S. baltica (OS155), was cloned into the E. coli cells and its expression and purification were conducted as previously reported (Di Rocco et al., 2011). The protein was 1.25 mM in 50 mM Tris/HCl, pH 7.5. The physical/chemical parameters (C711H1102N210O222S7, Mw = 16368.26 Da, pI = 5.69 and εA280 = 15720 M−1 cm−1) were calculated with ProtParam (www.expasy.org) from the sequence: (red and blue letters refer to negative and positive residues, respectively).
Crystallization, X-ray diffraction and data collection The protein sample was diluted to 0.3 mM (4.91 mg/mL) using a buffer containing dithionite (0.2 M) as a reducing agent. Small experimental crystals were obtained from automated micro-crystallization trials by the sitting-drop method, mixing 0.5 mL of both protein and reservoir. Final rhombohedra shape crystals, with dimensions 0.13 mm × 0.13 mm × 0.13 mm, grew after 10 days in
0.1 M Tris/HCl (pH 6.5), 0.2 M (NH4)2SO4 and 23% Peg3350w/v. X-ray diffraction experiments were carried out at the ELETTRA synchrotron (Trieste–Italy). Crystals were harvested from the mother liquor, cryoprotected by glycerol 23% and flash-frozen at 100 K. The diffraction data were collected on a Pilatus 2 M detector using monochromatic radiation (λ 1.00 Å) with a Δφ of 1°/image and each reflection has been indexed and integrated using MOSFLM (Leslie & Powell, 2007). The data scaling process was performed using SCALA (Evans, 1993) from the CCP4i suite (Collaborative Computational Project CCP4i, 1994). Sb-DHC crystallized in the P212121 orthorhombic space group (Table 1) with unit cell dimensions a = 40.81, b = 42.97 and c = 82.07 Å. The asymmetric unit was expected to contain one molecule, with a solvent content of 34% and a Matthews’s volume VM (Matthews, 1968) of 1.88 Å3 Da−1. The statistical parameters are listed in Table 1.
Structure determination, refinement and analyses The structure of Sb-DHC was solved by the MR method, starting from the structure of Rs-DHC (pdb: 2FWT). In the apo-protein preliminary model, the 12 residues at N-terminal, the central residues (Gln71-Lys90) and the C-terminal residues Asp147 and Asp148 were missing. The model building combined with several cycles of refinement was carried out using WinCoot (Emsley, Lohkamp, Scott, & Cowtan, 2010) and REFMAC5 (Murshudov, Vagin, & Dodson, 1997) on the initial phases. ARP/wARP 7.1 (Lamzin, Perrakis, & Wilson, 2001) was used to complete the structure by the use of hybrid models. The resulting fragments were docked each other and the side chains were built using the information from the sequence. The starting model was completed introducing the N-(HEC-700) and C-(HEC-500) hemes; the residues Ile9-Gln11, Cys19-Gly20, Gln71, Pro88-Lys90, Asp147 and the solvent molecules.
Alternative side chain conformations were included when disorder was observed. Hydrogen atoms were included at calculated positions. Cycles of anisotropic refinement using ShelxL-97 (Sheldrick & Schneider, 2003) were conducted till Rfact and Rfree values of 0.138 and 0.153, respectively, were obtained (Table 1). The structure was verified with PROCHECK (Laskowski, MacArthur, Moss, & Thornton, 1993). 91.5% of the residues were in
The New Diheme from Shewanella baltica Table 1. Data collection, processing and refinement statistics for the orthorhombic crystals of Sb-DHC (PDBID 3U99). Here, I is the intensity, s is the standard deviation and Rfactor = ∑ | Fo − Fc|/ ∑ Fo and Rmerge = ∑ |I − 〈I〉|/ ∑I, where Fo and Fc are the observed and calculated structure factors, respectively. Sb-DHC (PDBID 3U99) Data collection
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Space group Unit cell (Å) Resolution range (Å) No. of observations No. of reflections Rmerge (I ) Average I > /σ(I ) Completeness % Multiplicity % Refinement Max. Resolution (Å) Rfact % REFMAC5 Rfact % ShelxL-97 Rfree % ShelxL-97 No. of atoms Protein Heme c Water R.M.S. main chain Bond lengths (Å) Bond angles (°) H-bond energy B-factor (Å2) Main chain Side chain Solvent
P 212121 a = 40.81 b = 42.97 c = 82.07 40.53 − 1.14 132,642 45,277 0.079 1.3 91.5 6.3 1.14 16.62 13.83 15.36 1965 140 250 0.012 10.505 0.2 11.44 17.02 39.44
the most favoured regions on the Ramachandran plot and 8.4% in the allowed regions. All glycine residues had energetically favoured Ψ and Φ angles. The root mean square deviation calculated for chi-1 and chi-2 angles are in good agreement with a structural model. The figures were drawn using PyMol (DeLano, 2002). Geometrical analysis of the helices was performed with HeLANAL (Bansal, Kumar, & Velavan, 2000; Kumar & Bansal, 1996). Results and discussion Overall experimental structure The structure consists of two α-helix domains, both of which contain a c-type heme cofactor, connected by a central linker region from Ala69 to Ile91. Ile91 sits in the third helix of the N-terminal domain but marks the first loop of the C-terminal domain, thus establishing a hydrophobic contact between the two hemes via a number of intermediary methyl groups. The distance between atom Cδ1 of residue 91 and atom CMA of the N-heme (HEC-700 in the structure) is 3.9 Å; while atoms Cγ2
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(Ile91) and CMB of the C-heme (HEC-500 in the structure) are separated by 4.4 Å. Within this portion (Ala69- Ile91), the residues between Asn72 and Gly87 are not observed in the electron density map, while in the homologous structure from Rhodobacter sphaeroides (Gibson et al., 2006) this loop is clearly defined, despite the fact that the protein crystals were obtained using the same experimental conditions. In multidomains structures, it is quite frequent to not detect the flexible linker regions in the electron density maps. This can happen when statistical disorder of multiconformational arrangements is present. In such cases, the observed average electron density is very low. Sometimes, the disordered segments fold when they bind to their biological targets; otherwise the role of these flexible linkers is simply in the assembly of macromolecular arrays (Dyson & Wright, 2005). Moreover, in the crystal space, flexible or unstructured regions can be affected by the crystal packing. Our experimental data show that the loop is located in a highly solvent-accessible region, which is free from neighbouring symmetric molecules. Furthermore, we can only speculate that the unsolved loop could act as a linker between the two different domains with a role in the physiological mechanism (Di Rocco et al., 2011), in particular during the interaction with the redox partner. In fact, the gene encoding Sb-DHC is associated with a gene encoding for a putative cyt c with unprecedented metal coordination features, assembled in the same way of the sphaeroides DHC operon (Di Rocco et al., 2011). For this reason, it is believed that Sb-DHC could act as the electron donor for an SHP-like protein, with a recognizing mechanism which can be driven by changes in structure conformations. Despite the unstructured linker, the overall structure is consistent with the structure of the Rhodobacter sphaeroides, confirming that the two domains of Sb-DHC are structurally different from each other, as previously predicted by Di Rocco et al. (2011). Experimentally, we found 67 residues in the N-terminal domain. This domain is globular and presents four helices (Laskowski et al., 1993): H1 (Ala13-Cys19), H2 (Ala28-Leu30), H3 (Ala33-Ala41) and H4 (Pro55-His68), with an additional helix, H5 (Ile91-Glu93) that connects it with that of the C-terminal domain. The C-terminal domain has 54 residues: it is prevalently unfolded with a main helix, H7 (Arg106-Asp111), and two other short helices, H6 (Ala95-His101) and H8 (Cys121-Cys124). The overall geometries of the helices were analysed using the HeLANAL program (Bansal et al., 2000; Kumar & Bansal, 1996). Three helices in the N-terminal domain and two helices in the C- terminal domain were analysed. H2, H5 and H8 were not considered because of their shortness. The analysis confirmed that H1, H3, H4 and H6 are all α-3.613 helices, while the geometrical parameters of H7 indicated an extremely rare π-helix.
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The π-helix is more loosely constructed than the α-helix: a single turn of a π-helix contains 16 atoms, and it is therefore defined as a 4.416 helix (Weaver, 2000). While π-helices can easily be identified as a “bulge” structure within a longer α-helix and occur in 15% of known proteins (Cooley, Arp, & Karplus, 2010), isolated π-helices on the other hand are extremely rare. Their rarity has been attributed to an inherent instability due to: (i) unfavourable Φ and Ψ dihedral angles; (ii) loss of van der Waals contact of main chain atoms along the helix axis; (iii) the large entropic cost required to align five residues to permit the (i − i + 5) hydrogen bond characteristic of the π-helix. In fact, the π-helix, defined as a 4.416 helix, is more loosely constructed than the α-helix (Weaver, 2000). The critical role of π-helices has been recognized in a limited number of structures, especially in protein active sites. In the structures of fumarase, glycogen phosphorylase and catecholo-methyltransferase, the substrate binding site is stabilized by H-bonds or non-specific interactions with an internal single π-helix (Weaver, 2000). In the lipoxygenase structure, the particular conformation of the π-helix allows coordination of the iron atom. In the serine carboxypeptidase, two residues of the π-helix bind the substrate and position the nucleophile in the active site (Weaver, 2000). Finally, the π-helix in the photoactive yellow protein is responsible for the formation of a thioester bond with the chromophore in the active site; while in the light-chain dimer structures (1MCC/3MCG), it drives the conformational flexibility at the V–V interface after ligand binding (Weaver, 2000). In this particular helix, the number of residues in a pitch and the height of the helix pitch are 4.43 and 1.06 Å, respectively; while the average virtual torsion angle (defined by four Ca atoms) is 28.7°, smaller than the calculated value of about 45° for the α-3.613 helices (Bansal et al., 2000). These observations are in agreement with the main features of a classical π-helix. In the present study, the characteristic H-bond (i − i + 5) of the π-helix is present between the carboxyl group of Arg106 and the amino group of Asp111 (2.9 Å) with the 16 atoms in a loop (Figure 1). Previous analysis, conducted by the superimposition of the modelled structure of Sb-DHC and the X-ray structure of Rs-DHC, revealed that the main structural difference resides in the folding of residues 111–113 of Sb-DHC (Di Rocco et al., 2011). In fact, it was shown that these residues have no correspondence in the Rs-DHC structure and render the C-terminal lobe of Sb-DHC slightly less helical with respect to that of Rs-DHC. Here, we confirm the structural difference located in the same region and we explain it on the basis of the presence of the rare π-helix only in the case of Sb-DHC. The HeLANAL statistical parameters calculated for the two longer helices, H3 and H4, confirmed previous
Figure 1. The π-type helix (Arg106-Asp111) with its typical H-bond in the C-terminal domain of Sb-DHC structure: up (left) and front (right) views.
report that they are α-type helices (Bansal et al., 2000). In fact, the average and maximum bending angles are, respectively, 7.3° and 9.5° for H3, and 2.5° and 3.6° for H4. Furthermore, the curvature radius is 27 Å for H3 and 118 Å for H4. Finally, the respective rms for circle and line fitted to the local helix origin are 0.06 Å and 0.14 Å for H3, and 0.04 Å and 0.26 Å for H4, with r2 correlation values of 0.9 and 1.0, respectively. Many H-bonds and electrostatic interactions connect the two domains. A distance of 2.9 Å is observed between N atoms of Ala25 and Met24 to Oε2 atom of Glu129 and Oε1 atom of Gln120. The distance between Cγ of Pro27 and Cα of Gly131 is 4.1 Å, with the same distance also observed between Sδ of Met24 and Cβ of Ser122. Moreover, Cδ1 of Leu31 interacts with Cγ1 of Ile91 (4.0 Å) and the O of Leu30 with Cε2 of Phe96 (3.6 Å). The heme–heme region Recent studies on the electrochemical and spectroscopic features of the Sb-DHC, together with a theoretical analysis of the data, reported the reduction potential values for the N-domain and C-domain hemes (−257 and −144 mV, respectively) and showed that in both hemes the iron centres have a His–His axial coordination (Di Rocco et al., 2011). In particular, the iron ions are coordinated by four N atoms of the pyrrol rings (A–D) in the equatorial position, and by two His residues in the axial positions though the Nε2 atoms (Figure 2). Both c-type porphyrins are covalently bound to the protein through a cysteinic bond: Cys19 and Cys22 for the heme in the N-domain, Cys 121 and Cys124 for that in the C-domain. No water molecules are present in the C-domain heme pocket, confirming the hydrophobic heme environment, which can explain its higher E° value in comparison with the N-domain heme (Di Rocco et al., 2011). The C-domain heme shows the terminal carboxyl group of the B ring disordered in two close conformations, with the possibility to interact with Nε1 of Trp146 as well as Oε1 of Glu135 (also disordered in two possible side chain conformations), with distances of about 2.5
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Figure 2. (a) Stereo view of the 2Fo−Fc electron density map (blue) at contour level of 0.8σ in the two regions of heme c, visualized as sticks, in the Sb-DHC structure (cartoon). The His–His axial coordination and the Cys covalent bonds (sticks) are shown together with the position of Tyr26 (spheres) acting as a bridge between the N-domain heme c (HEC-700) and the C-domain heme c (HEC-500) with hydrophobic distances of about 3 Å. (b) Significant H-bond networks in the region of N-domain heme c (HEC-700) and C-domain heme c (HEC-500) in the structure Sb-DHC (surface). Cofactors are shown as sticks together with the main interacting residues with distances around 2.5–3 Å. The colour scale for the electrostatic surface of the protein is set from −3 kT/e (red) to + 3 kT/e (blue), where k is the Boltzmann constant, T is the temperature (310 K) and e the electron charge.
and 3 Å, respectively. It should be noted that the proximity to the metal centre of a negatively charged residue (Glu135) would stabilize the ferric state, leading to a more negative E° value (Battistuzzi et al., 2001). For the heme carboxylic groups, three significant H-bonds (Figure 2(b)) must be noted: the first between O1A of the N-domain heme and the N atom of Thr92 (2.9 Å), the second between O1D of the N-domain heme and atom Oγ of Ser119 (3.4 Å) and the last between atom O2A of the C-domain heme and atom N of Glu135 (2.6 Å). These H-bonds are relevant because the other heme– protein interactions are prevalently hydrophobic. Several water molecules are present in the N-domain heme
pocket and four of these are important because they are part of two H-bond networks close to the iron (Figure 2(b)). Wat4 is located at 5.6 Å from the iron and interacts with the O atom of Cys19 (3.0 Å), with Cε1 of His23 (3.4 Å) and with the O atom of Met24 (3.5 Å) that forms a H-bond with Nδ1 of His24 (2.7 Å), with the N atom of Tyr26 (2.9 Å) and with the OH of Tyr15 (2.9 Å) that interacts with the O atom of Tyr 26 (2.6 Å). Wat44 is located at 5.3 Å from Fe and interacts with OH groups of Tyr26 (2.6 Å) and of the disordered Ser119 (2.6 Å), with Cβ of His23 (3.5 Å), with CBD of the N-Heme (3.4 Å), with wat204 (2.5 Å), which in turn interacts with Oε1 of Gln120 (3.4 Å) and with wat90 (2.3 Å).
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The propionate group (O1 atom) of the A ring in the N-domain heme is directed towards the methyl group of the B ring in the C-domain heme. The iron–iron distance is 17.4 Å. This is slightly longer than that of the Rhodobacter sphaeroides (17.1 Å). However, in both structures the residue of Tyr26 is centrally located to form a bridge between these groups (Figure 2(a)). The hydroxyl group of Tyr26 interacts with CBA of the N-domain heme (3.2 Å) and with CAB of the C-domain heme (3.3 Å). The same critical distances are conserved in the homologous structure. We suggest that Tyr26, located at the interface between two cofactors, can be directly involved in the ET process. A lot of information on the electron tunnelling reaction is present in the literature, and various mechanisms have been proposed. Many different residues could be involved in an intramolecular ET. For example, in the diheme cyt c peroxidises the ET has been proposed to occur by quantum electron tunnelling, involving Trp94 as a bridging structure
(Fülöp, Ridout, Greenwood, & Hajdu, 1995). Other covalent pathways, which mainly involve aromatic residues, can also be ascribed to an ET mechanism (Tan, Balabin, & Onuchic, 2004). Very recent DFT calculations on cyt c551 demonstrated that there are only minimal variations in active site geometry upon oxidation and that the protein environment does not have a direct influence on it (Rajapandian & Subramanian, 2011). Strong supporting evidence on the role of the iron environment in the ET comes from the work of Tan et al. (2004). They showed by molecular dynamic simulations that the coupling between heme a and heme a3 in cyt c peroxidase is mainly driven along a direct pathway between the D propionates of the two heme rings (CMD-HMD), which are located in a strongly hydrophobic environment. Moreover, previous DFT studies on the HOMO–LUMO transitions for heme c derivatives showed that when the propionate groups are modified with electron withdrawing or electron donating groups,
Figure 3. Cluster analysis on the heme–heme geometries in the c-type multiheme proteins (front-up and side-down views). The figure shows the superposition, drawn with PyMol (DeLano, 2002), of the PDB-hemes (lines) on the Sb-DHC hemes (white stick) used as reference. The structural alignments were performed by fixing N-domain heme from Sb-DHC as a template and overlaying the N-domain hemes from the all other structures. (a) Cluster with Fe–Fe distances of about 9.5 Å (green-cyan): 2CZS (green), 2RDZ (light green), 1H21 (green), 1SP3 (dark green), 1FT5 (cyan) and 1JNI (blue). (b) Cluster with Fe–Fe distances of about 12 Å (pink-violet): 3BXU (pink), 1DUW (dark pink), 2FFN (magenta) and 1OS6 (violet). (c) Cluster with Fe-Fe distances of about 18 Å (yellow-brown): 1FW5 (yellow), 2VHD (orange), 1ETP (red) and 2OZ1 (brown).
The New Diheme from Shewanella baltica the electronic density is maintained over their sp3 carbons, avoiding the ET from the substituent to the iron atom (Hernandez et al., 2007).
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The heme–heme geometry It is well known that both heme arrangements (parallel or perpendicular) could play a role to enhance protein stability, to set-up the heme exposure to the solvent and to facilitate the heme packing (Allen, Daltrop, Stevens, & Ferguson, 2003). In addition, either a parallel-stacked arrangement or a perpendicular arrangement may help the rapid ET process and the fine-tuning of redox potentials (Smith, Kahraman, & Thornton, 2010). Structural alignment of heme–heme orientations in c-type multiheme proteins (Figure 3), considering all structures present in PDB determined by X-ray diffraction, shows that there are at least three main clusters, distinguished by their heme–heme geometry. The first cluster (Figure 3(a)) contains structures were the heme– heme planes are parallel with a mean Fe–Fe distance of about 9.5 Å. This cluster contains the majority of multiheme proteins and includes, for example, the diheme cyt c from G. sulfurreducens (2CZS), the E. coli cyt c nitrite reductase (2RDZ), split-Soret cyt c from Desulfovibrio desulfuricans (1H21), the octaheme cyt c from S. oneidensis (1SP3), the cyt c554 from Nitrosomonas europaea (1FT5) and the diheme NapB from Haemophilus influenzae (1JNI). The second cluster (Figure 3(b)) contains structures with heme–heme planes oriented almost perpendicularly, with Fe–Fe distances of about 12 Å. PDB structures from 3BXU (PpcB cyt c7 from G. sulfurreducens), 1DUW (nonaheme cyt c from D. desulfuricans), 2FFN (tetraheme cyt c3 from Desulfovibrio Vulgaris) and 1OS6 (PpcA cyt c7 from G. sulfurreducens) are included in this cluster. This perpendicular arrangement, which can be subdivided into “face-to-edge” and “edge-toedge” heme–heme orientation, has been found in several ET complexes with different functions, such as hydroxylamine oxidoreductase, cyt c oxidase, the photosynthetic reaction centre and fumerate reductase (Smith, Kahraman, & Thornton, 2010). Finally, the third cluster (Figure 3(c)) includes structures that exhibit a wide range of non-parallel heme–heme geometries and long Fe–Fe distances (18 Å). Natural systems often need to transfer electrons with high directional specificity over distances greater than 14 Å (Page, Moser, Chen, & Dutton, 1999). Although shorter distances between the redox centres favour a rapid ET rate (Page et al., 1999), these longer interactions present surprisingly fast redox reactions. This was explained by a multistep ET mechanism, in which the heme proximity within the 14 Å limit is responsible for high tunnelling rates and for a fine tuning of free
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energies (Page et al., 1999). This cluster includes our Sb-DHC structure, its homologue Rs-DHC, the diheme cyt c peroxidase from Pseudomonas aeruginosa (2VHD), the diheme cyt c4 from P. stutzeri (1ETP) and the SoxAX from Rhodovulum sulfidophilum (2OZ1). Excluding the homologue Rs-DHC, the other three structures exhibit different heme–heme dispositions, with a non-optimal Fe–Fe distance for electron tunnelling: from 18.8 Å in the SoxAX from R. sulfidophilum, to 19.2 Å in the cyt c4 from P. stutzeri and 21 Å in the cyt c peroxidase from P. aeruginosa. In the two structures 2VHD and 1ETP, the two propionic groups are oriented towards each other, while in the structure 2OZ1 the same groups display an opposite orientation. In the Sb-DHC, the direction of the two hemes is nearly the same and the angle formed by the porphyrin planes is close to 45°. In conclusion, in the present work we expressed, purified and crystallized the new bacterial diheme cyt c from S. baltica. The solved structure shows singular characteristics regarding the general folding. The unique C-terminal domain is structurally different with respect to the homologous c-type N-terminal domain from Rs-DHC and shows the presence of an extremely rare π-helix. The disposition and the geometry of the two prosthetic groups, together with the distance between the metal centres, were analysed and compared with those found in other multiheme cyts c. Supplementary material The supplementary material for this paper is available online at http://dx.doi.10.1080/07391102.2014.880657. Abbreviations cyt c Sb-DHC Rs-DHC ET VM C-heme N-heme
cytochrome c diheme cytochrome c from Shewanella baltica diheme cytochrome c from Rhodobacter sphaeroides electron transfer Matthews volume C-terminal heme group N-terminal heme group
PDB reference the Sb-DHC X-ray coordinates and structure factors, 3u99.
Acknowledgments We thank the University Federico II–IBB of Naples (Italy), MIUR (PRIN-2009A5Y3N project), CIRCMSB, Friuli-VeneziaGiulia region (DPReg. 120/2007/Pres.) and University of Trieste FRA2012 for financial support and the XRD1 beamline scientists at Elettra Synchrotron for their technical assistance.
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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
High-resolution crystal structure of the recombinant diheme cytochrome c from Shewanella baltica (OS155) a
b
a
a
Matteo De March , Giulia Di Rocco , Neal Hickey & Silvano Geremia a
Centre of Excellence in Biocrystallography (CEB), Department of Chemical and Pharmaceutical Sciences, University of Trieste, via L.Giorgieri 1, 34127 Trieste, Italy b
Department of Life Sciences, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy Published online: 21 Feb 2014.
Click for updates To cite this article: Matteo De March, Giulia Di Rocco, Neal Hickey & Silvano Geremia (2015) High-resolution crystal structure of the recombinant diheme cytochrome c from Shewanella baltica (OS155), Journal of Biomolecular Structure and Dynamics, 33:2, 395-403, DOI: 10.1080/07391102.2014.880657 To link to this article: http://dx.doi.org/10.1080/07391102.2014.880657
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Journal of Biomolecular Structure and Dynamics, 2015 Vol. 33, No. 2, 395–403, http://dx.doi.org/10.1080/07391102.2014.880657
High-resolution crystal structure of the recombinant diheme cytochrome c from Shewanella baltica (OS155) Matteo De Marcha, Giulia Di Roccob, Neal Hickeya and Silvano Geremiaa* a
Centre of Excellence in Biocrystallography (CEB), Department of Chemical and Pharmaceutical Sciences, University of Trieste, via L.Giorgieri 1, 34127 Trieste, Italy; bDepartment of Life Sciences, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy
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Communicated by Ramaswamy H. Sarma (Received 25 July 2013; accepted 3 January 2014) Multiheme cytochromes c (cyts c) are c-type cyts characterized by non-standard structural and spectroscopic properties. The relative disposition of the heme cofactors in the core of these proteins is conserved and they can be classified from their geometry in two main groups. In one group the porphyrin planes are arranged in a perpendicular fashion, while in the other they are parallel. Orientation of the heme groups is a key factor that regulates the intramolecular electron transfer pathway. A 16.5 kDa diheme cyt c, isolated from the bacterium Shewanella baltica OS155 (Sb-DHC), was cloned and expressed in E. coli and its structure was investigated by X-ray crystallography. Using high-resolution data (1.14 Å) collected at ELETTRA (Trieste), the crystal structure, with an orthorhombic cell (a = 40.81, b = 42.97, c = 82.07 Å), was solved using the homologous diheme from Rhodobacter sphaeroides (Rs-DHC) as the initial model. The electron density map of the refined structure (Rfact of 13.8% and Rfree of 15.4%) shows a two domain structure connected by a central unstructured region (N72-G87). The Sb-DHC, like its homologue (Rs-DHC), folds into a new cyt c class: the N-terminal globular domain, with its three α-helices, belongs to class I of c-type cyts, while the C-terminal domain includes a rare π-helix. The metal centre of the c-type heme groups is axially coordinated by two His residues and it is covalently bound to the protein through two Cys bonds. Keywords: cytochrome c; diheme; electron transfer; biocrystallography; edge to edge
Introduction Recent progress in bacterial genomic analysis has revealed a vast range of genes encoding novel c-type cyts that contain multiple heme cofactors. Shewanella is predicted to encode 42 c-type cyts and it is to date the most sequenced organism, with the exception of Geobacter sulfurreducens (111) (Heidelberg et al., 2002; Methe et al., 2003). Many of these putative cyts are predicted to be multiheme in character (Pitts et al., 2003). The identification of which c-type cyts are in fact present in the whole proteome of Shewanella, and the understanding of how these cyts act under different respiration conditions, will help to elucidate the pathways regarding the metal reduction and pollutant bioremediation. The genomes of Shewanella baltica (strains OS117, OS183, OS195, OS625 and OS678) have been completely sequenced (http://www.jgi.doe.gov/genome-projects) and the gene encoding for the S. baltica diheme cyt c (hereafter SbDHC) was identified by the characteristic double CXXCH motif. This 16.5 kDa diheme cyt c has been recently cloned, expressed and characterized by electrochemical and spectroscopic analyses (Di Rocco et al., 2011). These analyses indicated a thermodynamically *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
stable and kinetically inert axial heme His–His coordination for both hemes. Multiheme cyts c form an extensive class of proteins which act as electron shuttle/storage tools in several metabolic redox processes and respiratory chains (Mowat & Chapman, 2005). In these complex systems, which often operate in a membrane environment, several intramolecular electron transfer (ET) processes occur and these are generally coupled with multiple protonation phenomena and conformational changes (Andersen et al., 2001; Coutinho & Xavier, 1994; Garau, Geremia & Randaccio, 2002; Paquete, Turner, Louro, Xavier, & Caterino, 2007). Their functional properties are in part determined by the relative arrangement of multiple heme cofactors, which in many cases have been found to pack in conserved interaction motifs, namely perpendicular or parallel (Einsle, 2001; Heitmann & Einsle, 2005; Iverson et al., 1998). A good example is found in the crystal structure of the diheme cyt c4 from Pseudomonas stutzeri, a membrane-bound bacterial ET cyt c (Kadziola & Larsen, 1997). This exhibits a tetraheme architecture in which the two functional parallel heme c groups are oriented almost perpendicularly to the other two. An electrochemical study on the immobilized recombinant
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diheme cyt c4 from Pseudolateromans haloplanktis unequivocally showed that the intraheme ET occurs in the protein by a proton exchange at the H-bonded propionate groups of the two facing heme centres. This phenomenon was also reported for the structure of P. stutzeri diheme cyt c4 (Andersen et al., 2001; Andersen, Nørgaard, Jensen, & Ulstrup, 2002; Monari et al., 2009). Furthermore, the two heme c groups in the structure of DHC2 from G. Sulfurreducens (Heitmann & Einsle, 2005) pack into one of the typical heme interaction motifs observed in larger multiheme cyts and, due to the bis–His axial coordination for both hemes, it shows two redox potentials of −135 and −289 mV. Multiheme cyt’s c often have a big heme/protein ratio with few secondary structures elements (Einsle et al., 2000) and despite the low sequence homology, they have strong homologies in the arrangement of their heme groups (Heitmann & Einsle, 2005). In order to elucidate the highly optimized properties of multiheme cyts c, it is necessary to understand the significance of these motifs through spectroscopic and electrochemical investigation. Sb-DHC, like the homologous diheme cyt c from Rhodobacter spaheroides (Rs-DHC), represents an exception, due to the disposition of its two cofactors with non-standard geometry (Gibson et al., 2006). Materials and methods Sample preparation The Sb-DHC gene, isolated from S. baltica (OS155), was cloned into the E. coli cells and its expression and purification were conducted as previously reported (Di Rocco et al., 2011). The protein was 1.25 mM in 50 mM Tris/HCl, pH 7.5. The physical/chemical parameters (C711H1102N210O222S7, Mw = 16368.26 Da, pI = 5.69 and εA280 = 15720 M−1 cm−1) were calculated with ProtParam (www.expasy.org) from the sequence: (red and blue letters refer to negative and positive residues, respectively).
Crystallization, X-ray diffraction and data collection The protein sample was diluted to 0.3 mM (4.91 mg/mL) using a buffer containing dithionite (0.2 M) as a reducing agent. Small experimental crystals were obtained from automated micro-crystallization trials by the sitting-drop method, mixing 0.5 mL of both protein and reservoir. Final rhombohedra shape crystals, with dimensions 0.13 mm × 0.13 mm × 0.13 mm, grew after 10 days in
0.1 M Tris/HCl (pH 6.5), 0.2 M (NH4)2SO4 and 23% Peg3350w/v. X-ray diffraction experiments were carried out at the ELETTRA synchrotron (Trieste–Italy). Crystals were harvested from the mother liquor, cryoprotected by glycerol 23% and flash-frozen at 100 K. The diffraction data were collected on a Pilatus 2 M detector using monochromatic radiation (λ 1.00 Å) with a Δφ of 1°/image and each reflection has been indexed and integrated using MOSFLM (Leslie & Powell, 2007). The data scaling process was performed using SCALA (Evans, 1993) from the CCP4i suite (Collaborative Computational Project CCP4i, 1994). Sb-DHC crystallized in the P212121 orthorhombic space group (Table 1) with unit cell dimensions a = 40.81, b = 42.97 and c = 82.07 Å. The asymmetric unit was expected to contain one molecule, with a solvent content of 34% and a Matthews’s volume VM (Matthews, 1968) of 1.88 Å3 Da−1. The statistical parameters are listed in Table 1.
Structure determination, refinement and analyses The structure of Sb-DHC was solved by the MR method, starting from the structure of Rs-DHC (pdb: 2FWT). In the apo-protein preliminary model, the 12 residues at N-terminal, the central residues (Gln71-Lys90) and the C-terminal residues Asp147 and Asp148 were missing. The model building combined with several cycles of refinement was carried out using WinCoot (Emsley, Lohkamp, Scott, & Cowtan, 2010) and REFMAC5 (Murshudov, Vagin, & Dodson, 1997) on the initial phases. ARP/wARP 7.1 (Lamzin, Perrakis, & Wilson, 2001) was used to complete the structure by the use of hybrid models. The resulting fragments were docked each other and the side chains were built using the information from the sequence. The starting model was completed introducing the N-(HEC-700) and C-(HEC-500) hemes; the residues Ile9-Gln11, Cys19-Gly20, Gln71, Pro88-Lys90, Asp147 and the solvent molecules.
Alternative side chain conformations were included when disorder was observed. Hydrogen atoms were included at calculated positions. Cycles of anisotropic refinement using ShelxL-97 (Sheldrick & Schneider, 2003) were conducted till Rfact and Rfree values of 0.138 and 0.153, respectively, were obtained (Table 1). The structure was verified with PROCHECK (Laskowski, MacArthur, Moss, & Thornton, 1993). 91.5% of the residues were in
The New Diheme from Shewanella baltica Table 1. Data collection, processing and refinement statistics for the orthorhombic crystals of Sb-DHC (PDBID 3U99). Here, I is the intensity, s is the standard deviation and Rfactor = ∑ | Fo − Fc|/ ∑ Fo and Rmerge = ∑ |I − 〈I〉|/ ∑I, where Fo and Fc are the observed and calculated structure factors, respectively. Sb-DHC (PDBID 3U99) Data collection
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Space group Unit cell (Å) Resolution range (Å) No. of observations No. of reflections Rmerge (I ) Average I > /σ(I ) Completeness % Multiplicity % Refinement Max. Resolution (Å) Rfact % REFMAC5 Rfact % ShelxL-97 Rfree % ShelxL-97 No. of atoms Protein Heme c Water R.M.S. main chain Bond lengths (Å) Bond angles (°) H-bond energy B-factor (Å2) Main chain Side chain Solvent
P 212121 a = 40.81 b = 42.97 c = 82.07 40.53 − 1.14 132,642 45,277 0.079 1.3 91.5 6.3 1.14 16.62 13.83 15.36 1965 140 250 0.012 10.505 0.2 11.44 17.02 39.44
the most favoured regions on the Ramachandran plot and 8.4% in the allowed regions. All glycine residues had energetically favoured Ψ and Φ angles. The root mean square deviation calculated for chi-1 and chi-2 angles are in good agreement with a structural model. The figures were drawn using PyMol (DeLano, 2002). Geometrical analysis of the helices was performed with HeLANAL (Bansal, Kumar, & Velavan, 2000; Kumar & Bansal, 1996). Results and discussion Overall experimental structure The structure consists of two α-helix domains, both of which contain a c-type heme cofactor, connected by a central linker region from Ala69 to Ile91. Ile91 sits in the third helix of the N-terminal domain but marks the first loop of the C-terminal domain, thus establishing a hydrophobic contact between the two hemes via a number of intermediary methyl groups. The distance between atom Cδ1 of residue 91 and atom CMA of the N-heme (HEC-700 in the structure) is 3.9 Å; while atoms Cγ2
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(Ile91) and CMB of the C-heme (HEC-500 in the structure) are separated by 4.4 Å. Within this portion (Ala69- Ile91), the residues between Asn72 and Gly87 are not observed in the electron density map, while in the homologous structure from Rhodobacter sphaeroides (Gibson et al., 2006) this loop is clearly defined, despite the fact that the protein crystals were obtained using the same experimental conditions. In multidomains structures, it is quite frequent to not detect the flexible linker regions in the electron density maps. This can happen when statistical disorder of multiconformational arrangements is present. In such cases, the observed average electron density is very low. Sometimes, the disordered segments fold when they bind to their biological targets; otherwise the role of these flexible linkers is simply in the assembly of macromolecular arrays (Dyson & Wright, 2005). Moreover, in the crystal space, flexible or unstructured regions can be affected by the crystal packing. Our experimental data show that the loop is located in a highly solvent-accessible region, which is free from neighbouring symmetric molecules. Furthermore, we can only speculate that the unsolved loop could act as a linker between the two different domains with a role in the physiological mechanism (Di Rocco et al., 2011), in particular during the interaction with the redox partner. In fact, the gene encoding Sb-DHC is associated with a gene encoding for a putative cyt c with unprecedented metal coordination features, assembled in the same way of the sphaeroides DHC operon (Di Rocco et al., 2011). For this reason, it is believed that Sb-DHC could act as the electron donor for an SHP-like protein, with a recognizing mechanism which can be driven by changes in structure conformations. Despite the unstructured linker, the overall structure is consistent with the structure of the Rhodobacter sphaeroides, confirming that the two domains of Sb-DHC are structurally different from each other, as previously predicted by Di Rocco et al. (2011). Experimentally, we found 67 residues in the N-terminal domain. This domain is globular and presents four helices (Laskowski et al., 1993): H1 (Ala13-Cys19), H2 (Ala28-Leu30), H3 (Ala33-Ala41) and H4 (Pro55-His68), with an additional helix, H5 (Ile91-Glu93) that connects it with that of the C-terminal domain. The C-terminal domain has 54 residues: it is prevalently unfolded with a main helix, H7 (Arg106-Asp111), and two other short helices, H6 (Ala95-His101) and H8 (Cys121-Cys124). The overall geometries of the helices were analysed using the HeLANAL program (Bansal et al., 2000; Kumar & Bansal, 1996). Three helices in the N-terminal domain and two helices in the C- terminal domain were analysed. H2, H5 and H8 were not considered because of their shortness. The analysis confirmed that H1, H3, H4 and H6 are all α-3.613 helices, while the geometrical parameters of H7 indicated an extremely rare π-helix.
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The π-helix is more loosely constructed than the α-helix: a single turn of a π-helix contains 16 atoms, and it is therefore defined as a 4.416 helix (Weaver, 2000). While π-helices can easily be identified as a “bulge” structure within a longer α-helix and occur in 15% of known proteins (Cooley, Arp, & Karplus, 2010), isolated π-helices on the other hand are extremely rare. Their rarity has been attributed to an inherent instability due to: (i) unfavourable Φ and Ψ dihedral angles; (ii) loss of van der Waals contact of main chain atoms along the helix axis; (iii) the large entropic cost required to align five residues to permit the (i − i + 5) hydrogen bond characteristic of the π-helix. In fact, the π-helix, defined as a 4.416 helix, is more loosely constructed than the α-helix (Weaver, 2000). The critical role of π-helices has been recognized in a limited number of structures, especially in protein active sites. In the structures of fumarase, glycogen phosphorylase and catecholo-methyltransferase, the substrate binding site is stabilized by H-bonds or non-specific interactions with an internal single π-helix (Weaver, 2000). In the lipoxygenase structure, the particular conformation of the π-helix allows coordination of the iron atom. In the serine carboxypeptidase, two residues of the π-helix bind the substrate and position the nucleophile in the active site (Weaver, 2000). Finally, the π-helix in the photoactive yellow protein is responsible for the formation of a thioester bond with the chromophore in the active site; while in the light-chain dimer structures (1MCC/3MCG), it drives the conformational flexibility at the V–V interface after ligand binding (Weaver, 2000). In this particular helix, the number of residues in a pitch and the height of the helix pitch are 4.43 and 1.06 Å, respectively; while the average virtual torsion angle (defined by four Ca atoms) is 28.7°, smaller than the calculated value of about 45° for the α-3.613 helices (Bansal et al., 2000). These observations are in agreement with the main features of a classical π-helix. In the present study, the characteristic H-bond (i − i + 5) of the π-helix is present between the carboxyl group of Arg106 and the amino group of Asp111 (2.9 Å) with the 16 atoms in a loop (Figure 1). Previous analysis, conducted by the superimposition of the modelled structure of Sb-DHC and the X-ray structure of Rs-DHC, revealed that the main structural difference resides in the folding of residues 111–113 of Sb-DHC (Di Rocco et al., 2011). In fact, it was shown that these residues have no correspondence in the Rs-DHC structure and render the C-terminal lobe of Sb-DHC slightly less helical with respect to that of Rs-DHC. Here, we confirm the structural difference located in the same region and we explain it on the basis of the presence of the rare π-helix only in the case of Sb-DHC. The HeLANAL statistical parameters calculated for the two longer helices, H3 and H4, confirmed previous
Figure 1. The π-type helix (Arg106-Asp111) with its typical H-bond in the C-terminal domain of Sb-DHC structure: up (left) and front (right) views.
report that they are α-type helices (Bansal et al., 2000). In fact, the average and maximum bending angles are, respectively, 7.3° and 9.5° for H3, and 2.5° and 3.6° for H4. Furthermore, the curvature radius is 27 Å for H3 and 118 Å for H4. Finally, the respective rms for circle and line fitted to the local helix origin are 0.06 Å and 0.14 Å for H3, and 0.04 Å and 0.26 Å for H4, with r2 correlation values of 0.9 and 1.0, respectively. Many H-bonds and electrostatic interactions connect the two domains. A distance of 2.9 Å is observed between N atoms of Ala25 and Met24 to Oε2 atom of Glu129 and Oε1 atom of Gln120. The distance between Cγ of Pro27 and Cα of Gly131 is 4.1 Å, with the same distance also observed between Sδ of Met24 and Cβ of Ser122. Moreover, Cδ1 of Leu31 interacts with Cγ1 of Ile91 (4.0 Å) and the O of Leu30 with Cε2 of Phe96 (3.6 Å). The heme–heme region Recent studies on the electrochemical and spectroscopic features of the Sb-DHC, together with a theoretical analysis of the data, reported the reduction potential values for the N-domain and C-domain hemes (−257 and −144 mV, respectively) and showed that in both hemes the iron centres have a His–His axial coordination (Di Rocco et al., 2011). In particular, the iron ions are coordinated by four N atoms of the pyrrol rings (A–D) in the equatorial position, and by two His residues in the axial positions though the Nε2 atoms (Figure 2). Both c-type porphyrins are covalently bound to the protein through a cysteinic bond: Cys19 and Cys22 for the heme in the N-domain, Cys 121 and Cys124 for that in the C-domain. No water molecules are present in the C-domain heme pocket, confirming the hydrophobic heme environment, which can explain its higher E° value in comparison with the N-domain heme (Di Rocco et al., 2011). The C-domain heme shows the terminal carboxyl group of the B ring disordered in two close conformations, with the possibility to interact with Nε1 of Trp146 as well as Oε1 of Glu135 (also disordered in two possible side chain conformations), with distances of about 2.5
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Figure 2. (a) Stereo view of the 2Fo−Fc electron density map (blue) at contour level of 0.8σ in the two regions of heme c, visualized as sticks, in the Sb-DHC structure (cartoon). The His–His axial coordination and the Cys covalent bonds (sticks) are shown together with the position of Tyr26 (spheres) acting as a bridge between the N-domain heme c (HEC-700) and the C-domain heme c (HEC-500) with hydrophobic distances of about 3 Å. (b) Significant H-bond networks in the region of N-domain heme c (HEC-700) and C-domain heme c (HEC-500) in the structure Sb-DHC (surface). Cofactors are shown as sticks together with the main interacting residues with distances around 2.5–3 Å. The colour scale for the electrostatic surface of the protein is set from −3 kT/e (red) to + 3 kT/e (blue), where k is the Boltzmann constant, T is the temperature (310 K) and e the electron charge.
and 3 Å, respectively. It should be noted that the proximity to the metal centre of a negatively charged residue (Glu135) would stabilize the ferric state, leading to a more negative E° value (Battistuzzi et al., 2001). For the heme carboxylic groups, three significant H-bonds (Figure 2(b)) must be noted: the first between O1A of the N-domain heme and the N atom of Thr92 (2.9 Å), the second between O1D of the N-domain heme and atom Oγ of Ser119 (3.4 Å) and the last between atom O2A of the C-domain heme and atom N of Glu135 (2.6 Å). These H-bonds are relevant because the other heme– protein interactions are prevalently hydrophobic. Several water molecules are present in the N-domain heme
pocket and four of these are important because they are part of two H-bond networks close to the iron (Figure 2(b)). Wat4 is located at 5.6 Å from the iron and interacts with the O atom of Cys19 (3.0 Å), with Cε1 of His23 (3.4 Å) and with the O atom of Met24 (3.5 Å) that forms a H-bond with Nδ1 of His24 (2.7 Å), with the N atom of Tyr26 (2.9 Å) and with the OH of Tyr15 (2.9 Å) that interacts with the O atom of Tyr 26 (2.6 Å). Wat44 is located at 5.3 Å from Fe and interacts with OH groups of Tyr26 (2.6 Å) and of the disordered Ser119 (2.6 Å), with Cβ of His23 (3.5 Å), with CBD of the N-Heme (3.4 Å), with wat204 (2.5 Å), which in turn interacts with Oε1 of Gln120 (3.4 Å) and with wat90 (2.3 Å).
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The propionate group (O1 atom) of the A ring in the N-domain heme is directed towards the methyl group of the B ring in the C-domain heme. The iron–iron distance is 17.4 Å. This is slightly longer than that of the Rhodobacter sphaeroides (17.1 Å). However, in both structures the residue of Tyr26 is centrally located to form a bridge between these groups (Figure 2(a)). The hydroxyl group of Tyr26 interacts with CBA of the N-domain heme (3.2 Å) and with CAB of the C-domain heme (3.3 Å). The same critical distances are conserved in the homologous structure. We suggest that Tyr26, located at the interface between two cofactors, can be directly involved in the ET process. A lot of information on the electron tunnelling reaction is present in the literature, and various mechanisms have been proposed. Many different residues could be involved in an intramolecular ET. For example, in the diheme cyt c peroxidises the ET has been proposed to occur by quantum electron tunnelling, involving Trp94 as a bridging structure
(Fülöp, Ridout, Greenwood, & Hajdu, 1995). Other covalent pathways, which mainly involve aromatic residues, can also be ascribed to an ET mechanism (Tan, Balabin, & Onuchic, 2004). Very recent DFT calculations on cyt c551 demonstrated that there are only minimal variations in active site geometry upon oxidation and that the protein environment does not have a direct influence on it (Rajapandian & Subramanian, 2011). Strong supporting evidence on the role of the iron environment in the ET comes from the work of Tan et al. (2004). They showed by molecular dynamic simulations that the coupling between heme a and heme a3 in cyt c peroxidase is mainly driven along a direct pathway between the D propionates of the two heme rings (CMD-HMD), which are located in a strongly hydrophobic environment. Moreover, previous DFT studies on the HOMO–LUMO transitions for heme c derivatives showed that when the propionate groups are modified with electron withdrawing or electron donating groups,
Figure 3. Cluster analysis on the heme–heme geometries in the c-type multiheme proteins (front-up and side-down views). The figure shows the superposition, drawn with PyMol (DeLano, 2002), of the PDB-hemes (lines) on the Sb-DHC hemes (white stick) used as reference. The structural alignments were performed by fixing N-domain heme from Sb-DHC as a template and overlaying the N-domain hemes from the all other structures. (a) Cluster with Fe–Fe distances of about 9.5 Å (green-cyan): 2CZS (green), 2RDZ (light green), 1H21 (green), 1SP3 (dark green), 1FT5 (cyan) and 1JNI (blue). (b) Cluster with Fe–Fe distances of about 12 Å (pink-violet): 3BXU (pink), 1DUW (dark pink), 2FFN (magenta) and 1OS6 (violet). (c) Cluster with Fe-Fe distances of about 18 Å (yellow-brown): 1FW5 (yellow), 2VHD (orange), 1ETP (red) and 2OZ1 (brown).
The New Diheme from Shewanella baltica the electronic density is maintained over their sp3 carbons, avoiding the ET from the substituent to the iron atom (Hernandez et al., 2007).
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The heme–heme geometry It is well known that both heme arrangements (parallel or perpendicular) could play a role to enhance protein stability, to set-up the heme exposure to the solvent and to facilitate the heme packing (Allen, Daltrop, Stevens, & Ferguson, 2003). In addition, either a parallel-stacked arrangement or a perpendicular arrangement may help the rapid ET process and the fine-tuning of redox potentials (Smith, Kahraman, & Thornton, 2010). Structural alignment of heme–heme orientations in c-type multiheme proteins (Figure 3), considering all structures present in PDB determined by X-ray diffraction, shows that there are at least three main clusters, distinguished by their heme–heme geometry. The first cluster (Figure 3(a)) contains structures were the heme– heme planes are parallel with a mean Fe–Fe distance of about 9.5 Å. This cluster contains the majority of multiheme proteins and includes, for example, the diheme cyt c from G. sulfurreducens (2CZS), the E. coli cyt c nitrite reductase (2RDZ), split-Soret cyt c from Desulfovibrio desulfuricans (1H21), the octaheme cyt c from S. oneidensis (1SP3), the cyt c554 from Nitrosomonas europaea (1FT5) and the diheme NapB from Haemophilus influenzae (1JNI). The second cluster (Figure 3(b)) contains structures with heme–heme planes oriented almost perpendicularly, with Fe–Fe distances of about 12 Å. PDB structures from 3BXU (PpcB cyt c7 from G. sulfurreducens), 1DUW (nonaheme cyt c from D. desulfuricans), 2FFN (tetraheme cyt c3 from Desulfovibrio Vulgaris) and 1OS6 (PpcA cyt c7 from G. sulfurreducens) are included in this cluster. This perpendicular arrangement, which can be subdivided into “face-to-edge” and “edge-toedge” heme–heme orientation, has been found in several ET complexes with different functions, such as hydroxylamine oxidoreductase, cyt c oxidase, the photosynthetic reaction centre and fumerate reductase (Smith, Kahraman, & Thornton, 2010). Finally, the third cluster (Figure 3(c)) includes structures that exhibit a wide range of non-parallel heme–heme geometries and long Fe–Fe distances (18 Å). Natural systems often need to transfer electrons with high directional specificity over distances greater than 14 Å (Page, Moser, Chen, & Dutton, 1999). Although shorter distances between the redox centres favour a rapid ET rate (Page et al., 1999), these longer interactions present surprisingly fast redox reactions. This was explained by a multistep ET mechanism, in which the heme proximity within the 14 Å limit is responsible for high tunnelling rates and for a fine tuning of free
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energies (Page et al., 1999). This cluster includes our Sb-DHC structure, its homologue Rs-DHC, the diheme cyt c peroxidase from Pseudomonas aeruginosa (2VHD), the diheme cyt c4 from P. stutzeri (1ETP) and the SoxAX from Rhodovulum sulfidophilum (2OZ1). Excluding the homologue Rs-DHC, the other three structures exhibit different heme–heme dispositions, with a non-optimal Fe–Fe distance for electron tunnelling: from 18.8 Å in the SoxAX from R. sulfidophilum, to 19.2 Å in the cyt c4 from P. stutzeri and 21 Å in the cyt c peroxidase from P. aeruginosa. In the two structures 2VHD and 1ETP, the two propionic groups are oriented towards each other, while in the structure 2OZ1 the same groups display an opposite orientation. In the Sb-DHC, the direction of the two hemes is nearly the same and the angle formed by the porphyrin planes is close to 45°. In conclusion, in the present work we expressed, purified and crystallized the new bacterial diheme cyt c from S. baltica. The solved structure shows singular characteristics regarding the general folding. The unique C-terminal domain is structurally different with respect to the homologous c-type N-terminal domain from Rs-DHC and shows the presence of an extremely rare π-helix. The disposition and the geometry of the two prosthetic groups, together with the distance between the metal centres, were analysed and compared with those found in other multiheme cyts c. Supplementary material The supplementary material for this paper is available online at http://dx.doi.10.1080/07391102.2014.880657. Abbreviations cyt c Sb-DHC Rs-DHC ET VM C-heme N-heme
cytochrome c diheme cytochrome c from Shewanella baltica diheme cytochrome c from Rhodobacter sphaeroides electron transfer Matthews volume C-terminal heme group N-terminal heme group
PDB reference the Sb-DHC X-ray coordinates and structure factors, 3u99.
Acknowledgments We thank the University Federico II–IBB of Naples (Italy), MIUR (PRIN-2009A5Y3N project), CIRCMSB, Friuli-VeneziaGiulia region (DPReg. 120/2007/Pres.) and University of Trieste FRA2012 for financial support and the XRD1 beamline scientists at Elettra Synchrotron for their technical assistance.
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