Characterization of a prokaryotic haemerythrin from the methanotrophic bacterium Methylococcus capsulatus (Bath) Odd A Karlsen, Linda Ramsevik, Live J Bruseth, Øivind Larsen*, Annette Brenner, Frode S Berven, Harald B Jensen and Johan R Lillehaug Department of Molecular Biology, University of Bergen, Norway Keywords copper regulated; methanotroph; Methylococcus capsulatus; prokaryotic haemerythrin; two-dimensional gel electrophoresis Correspondence O A Karlsen, Department of Molecular Biology, University of Bergen, HIB, Thormøhlensgt 55, 5020 Bergen, Norway Fax: +47 555 89683 Tel: +47 555 84372 E-mail: Odd.Karlsen@mbi.uib.no *Present address Department of Biology, University of Bergen, Norway (Received December 2004, revised 25 February 2005, accepted 15 March 2005) doi:10.1111/j.1742-4658.2005.04663.x For a long time, the haemerythrin family of proteins was considered to be restricted to only a few phyla of marine invertebrates When analysing differential protein expression in the methane-oxidizing bacterium, Methylococcus capsulatus (Bath), grown at a high and low copper-to-biomass ratio, respectively, we identified a putative prokaryotic haemerythrin expressed in high-copper cultures Haemerythrins are recognized by a conserved sequence motif that provides five histidines and two carboxylate ligands which coordinate two iron atoms The diiron site is located in a hydrophobic pocket and is capable of binding O2 We cloned the M capsulatus haemerythrin gene and expressed it in Escherichia coli as a fusion protein with NusA The haemerythrin protein was purified to homogeneity cleaved from its fusion partner Recombinant M capsulatus haemerythrin (McHr) was found to fold into a stable protein Sequence similarity analysis identified all the candidate residues involved in the binding of diiron (His22, His58, Glu62, His77, His81, His117, Asp122) and the amino acids forming the hydrophobic pocket in which O2 may bind (Ile25, Phe59, Trp113, Leu114, Ile118) We were also able to model a three-dimensional structure of McHr maintaining the correct positioning of these residues Furthermore, UV ⁄ vis spectrophotometric analysis demonstrated the presence of conjugated diiron atoms in McHr A comprehensive genomic database search revealed 21 different prokaryotes containing the haemerythrin signature (PROSITE 00550), indicating that these putative haemerythrins may be a conserved prokaryotic subfamily Haemerythrin proteins comprise a family of O2-carrrying proteins mainly found in a few phyla of marine invertebrates Members of this family differ from haemoglobin and haemocyanin in that they contain a nonheme diiron site that reversibly binds one molecule of O2 This oxygen-binding binuclear iron complex is a characteristic feature of haemerythrins The two iron ions are bound to the protein via seven conserved amino acid residues; five histidines, one glutamate and one aspartate [1] All known haemerythrins also share a four-helix bundle fold which surrounds the diiron site Furthermore, the haemerythrins are divided into two subfamilies; the haemerythrins (Hr) and myohaemerythrins (MHr) Hrs are found in coelomic cells and typically exist as homopolymers composed of subunits of 113–117 amino acid residues MHrs are Abbreviations 2DE, two-dimensional gel electrophoresis; Hr, haemerythrin; ICP-MS, inductively coupled plasma atomic emission-mass spectrometry; IPG, immobilized pH gradient; IPTG, isopropyl thio-b-D-galactoside; McHr, Methylococcus capsulatus haemerythrin; MHr, myohaemerythrin; MMO, methane monooxygenase; NMS, nitrate mineral salt; pMMO, particulate methane monooxygenase; sMMO, soluble methane monooxygenase 2428 FEBS Journal 272 (2005) 2428–2440 ª 2005 FEBS O A Karlsen et al monomeric proteins of 118–120 amino acid residues, usually isolated from the muscles of sipunculids [2] MHrs are very similar to the Hr subunit both in structure and function Until recently, the presence of Hrs or MHrs in prokaryotes had not been reported However, Xiong et al [3] described a Hr-like domain in the C-terminal part of the chemotaxis protein Desulfovibrio chemoreceptor H (DcrH), expressed in the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris The DcrH chemoreceptor was proposed to have a membrane-spanning domain, placing the C-terminally located Hr domain in the cytoplasm The biological function of DcrH and its Hr-like domain is not fully elucidated To our knowledge, we are the first to clone a gene encoding a prokaryotic haemerythrin and to characterize the encoded protein The gene was cloned from the methanotrophic Gram-negative bacterium Methylococcus capsulatus (Bath) and encodes a protein of 131 amino acid residues The prokaryotic haemerythrin protein contains the haemerythrin signature typical for members of this family In vivo expression of the putative M capsulatus haemerythrin (McHr) was increased in cells grown at a high copper-to-biomass ratio, indicating an important physiological role under this growth condition The latter observation was also reported by Kao et al [4] McHr was expressed in Escherichia coli and analysed with respect to homology, structure, and metal binding Results Identification and sequence analysis Two-dimensional gel electrophoresis (2DE) analysis of the soluble fraction of M capsulatus grown either at a high or low copper-to-biomass ratio revealed protein spots significantly affected by the growth conditions (Fig 1, spots 1–15) In total, 27 protein spots were identified by MS and N-terminal sequencing combined with genomic information (Table 1) Fifteen of these spots were differentially expressed One of the polypeptides migrated with an apparent molecular mass of 14.4 kDa and a pI value of  5.8 (Fig 1, spot 15) and was only found in cells cultured at high (0.8 lm) copper concentrations MS analysis of the excised spot resulted in seven matching peptides and a 56.5% coverage, which in combination with M capsulatus genome sequence information identified the protein as being encoded by the gene MCA0715 (Accession no AE01782) This gene has previously FEBS Journal 272 (2005) 2428–2440 ª 2005 FEBS Characterization of prokaryotic haemerythrin been annotated by us as a haemerythrin family protein [5] MCA0715 was located to a single transcriptional unit, between the conserved hypothetical protein MCA0714 and the hypothetical protein MCA0716 (Fig 2) After the MS-based gene identification, specific PCR primers were designed and the differential expression in cells cultured at high or low copper concentrations was verified by northern blot analysis (Fig 3) Sequence analyses of the MCA0715-encoding protein using scanprosite [6] and conserved domain search [7,8], identified the conserved H-F-x(2)-[EQ][ENQ]-x(2)-[LMF]-x(4)-[FY]-x(5,6)-H-x(3)-[HR] motif (PROSITE 00550) This signature pattern is characteristic for members of the haemerythrin family and it is located in the central region of these proteins We therefore named the MCA0715-encoded protein Methylococcus capsulatus haemerythrin (McHr) The haemerythrin family motif contains four conserved iron ligands: the three histidines and the first glutamate ⁄ glutamine of the motif When using multiple clustalx [9] alignments of the MCA0715-encoded protein with members of the Hr (Fig 4A) and MHr (Fig 4B) families, several conserved amino acids were identified in the McHr protein sequence, as indicated in Fig The regions of the McHr that contained the highest level of identical residues and conservative replacements corresponded to the known helices in resolved eukaryotic haemerythrin structures In contrast, the loops between helices showed low degree of identity, thus representing the regions with more amino acid variation In the helical segments, the amino acids H, E, Q, F, D were particularly well conserved In addition, all the amino acid residues that are candidates for conjugating a diiron complex, and the amino acids forming the O2-binding pocket were found to be conserved (Fig 4) The putative M capsulatus haemerythrin sequence (131 amino acids) exceeds the sequence length of both haemerythrin subfamilies making McHr the largest known haemerythrin Thus, assuming that the corresponding a helices are of equal length in the different haemerythrins, the loops (random coils) between the helices in McHr are longer than those of the eukaryotic haemerythrins Furthermore, a clustalx alignmentbased phylogenetic tree branched McHr distantly to the members of both the eukaryotic Hr and MHr subfamilies (Fig 5) The highest similarity between members of the haemerythrin family and McHr was observed with the Hr alpha chain of Lingula reevii, in which the sequence identity and sequence similarity were calculated to 29 and 42%, respectively 2429 Characterization of prokaryotic haemerythrin O A Karlsen et al A B Fig 2DE patterns of the soluble fraction from low- (A) and high-copper (B) cultured M capsulatus The soluble fractions were analysed using overlapping 18 cm narrow-range IPGs spanning the pH range of 4.0–5.0, 4.5–5.5, 5.0–6.0, 5.5–6.7 and broad range pH 3–10 IPGs (Amersham) Both (A) and (B) are composite of such individual 2D gels Numbered arrows refer to N-terminally or MS-identified spots Approximate molecular masses and pI values are indicated to the left and at the top of the gels, respectively Phylogenetic distribution The known phylogenetic distribution of haemerythrins has long been limited to a relatively small group of marine invertebrates However, in light of the many genomic sequencing projects of prokaryotes, we also searched the databases SWISSPROT and TrEMBL for putative prokaryotic proteins containing a haemerythrin-like domain (PROSITE PS00550) (Table 2) [6] Interestingly, unlike the very restricted distribution in eukaryotes, the haemerythrin motif seems to be more widespread in the prokaryotic kingdom, and to date has been found in 21 different bacteria from five major genera A majority of these putative haemerythrins are identified from Proteobacteria Until now, none of these putative prokaryotic haemerythrins has been characterized In fact, apart from the McHr [4], there has been only one report of micro2430 bial proteins containing a haemerythrin signature [3] This haemerythrin-like motif was found in the C-terminal end of DcrH, isolated from D vulgaris DcrH is a member of the dcr gene family which sense and respond to specific states of its environment By expressing only this DcrH C-terminal part, Xiong et al [3] demonstrated the presence of an oxo-bridged diiron(III) site whose structure was very similar to that found in haemerythrins Most interestingly, a pairwise alignment of this C-terminal end and McHr revealed a very strong resemblance, containing 45 identical residues and 23 conservative replacements (Fig 6) Cloning and purification of McHr Comparative bioinformatics data on the MCA0715 protein suggested that McHr is the first characterized microbial homologue of the eukaryotic haemerythrins FEBS Journal 272 (2005) 2428–2440 ª 2005 FEBS O A Karlsen et al Characterization of prokaryotic haemerythrin Table Summary of identified proteins of M capsulatus soluble fractions Spot no 1–15 represent differentially expressed polypeptides between cells cultured at high- and low copper-to-biomass ratio Spot no 16–27 were found equally expressed in these culture conditions +, indicates expression in given growth condition (+), indicate less abundant expression Spot number Gene Annotation 1, 2, 3, 12, 13 10 14 6, 11 15 16, 17 18 26 24 21 mopE (MCA2589) mmoX (MCA1194) mmoY (MCA1195) mmoZ (MCA1198) mmoB (MCA1196) mmoC (MCA1200) groEL-2 (MCA1202) (mmoG) groEL-3 (MCA1704) groES-2 (MCA1705) MCA0715 mxaF (MCA0779) mxaI (MCA0782) MCA0789 Ndk (MCA2886) dapD (MCA1490) 22 19, 20 27 25 23 rpe (MCA2582) MCA1023 Tkt2 (MCA3046) Tuf1 ⁄ Tuf2 (MCA1059 ⁄ MCA2374) atpA-1 (MCA0010) M capsulatus outer membrane protein E (MopE) Methane monooxygenase, subunit A, a-chain Methane monooxygenase, subunit A, b-chain Methane monooxygenase, subunit A, c-chain Methane monooxygenase, subunit B Methane monooxygenase, subunit C Chaperonin, 60 kDa subunit (mmoG) Chaperonin, 60 kDa subunit Chaperonin, 10 kDa subunit Haemerythrin family protein Methanol dehydrogenase, large subunit Methanol dehydrogenase, small subunit MxaD protein, putative Nucleoside diphosphate kinase 2, 3, 4, 5-Tetrahydropyridine-2,6 dicarboxylate N-succinyltransferase Ribulose-phosphate 3-epimerase Antioxidant, AhpC ⁄ Tsa family protein Transketolase Translation elongation factor Tu ATP synthase F1, a-subunit Fig Genomic orientation (A), nucleotide and amino acid sequence (B) of MCA0715 (A) MCA0715 is located between the genes MCA0716 and MCA0714, putatively encoding a conserved hypothetical protein and a hypothetical protein, respectively (B) The amino acids are indicated below the nucleotide sequence The underlined promoter region is predicted using the Neural Network Promoter Prediction (http://www.fruitfly.org/seq_tools/ promoter.html) and has a probability of 0.98 (indicated by a red arrow in A) Enlarged (T) indicates possible transcription start Putative ribosomal binding site is enlarged in bold The predicted termination loop (black arrows) has a calculated energy of )23.0 kcalỈmol)1 (http://www.genebee.msu.su/services/rna2_reduced.html) (indicated as a hairpin in A) FEBS Journal 272 (2005) 2428–2440 ª 2005 FEBS +Cu –Cu + + + + + + + + + (+) (+) + + + + + + + + + + + + + + + + + + + + + Fig Northern blot of MCA0715 Total RNA (5 lg) isolated from high- (+ Cu) and low-copper (– Cu) cultures of M capsulatus were electrophoresed through an agarose ⁄ formaldehyde gel and transferred to a nylon membrane The blot was hybridized at 42 °C overnight, with a radioactive probe made from the PCR fragment amplified with the primers used in the cloning of MCA0715 The arrowhead indicates the 540 bp transcript, corresponding to the predicted length of the MCA0715 transcript The molecular sizes are indicated as given by the Invitrogen 0.24–9.5 kb RNA ladder 2431 Characterization of prokaryotic haemerythrin O A Karlsen et al Fig Sequence comparisons between McHr and members of the haemerythrin (A) and myohaemerythrin (B) subfamily with CLUSTALX software CLUSTALX was used in secondary structure-based penalty mode The haemerythrin sequences are obtained from the spinculids P gouldii (Pg) [39], T zostericola (Tz) [40], T dyscritum (Td) [41], S cumanense (Sc) [42], and from the brachiopods L unguis (LuA and LuB) [43,44], L reevii (LrA and LrB) [45] The myohaemerythrin sequences are from the sipunculids P gouldii (Pg) [46], T zostericola (Tz) [47], the achaete annelid T tessulatum (Tt) [48], and the polychaete annelid N diversicolor (Nd, MPII) [49,50] McHr of M capsulatus is indicated as Mc in both (A) and (B) The helical regions based on the X-ray crystal structures of P gouldii [20] (A) and T zostericola [19] (B) are indicated with (a) on top of each sequence block (I, II, III and IV) The seven amino acids involved as iron ligands are indicated by (#) (*) indicates the five hydrophobic pocket-forming amino acids To study this protein in more detail, the mchr gene was cloned and expressed in E coli as a NusA– (McHr) fusion protein The fusion protein was purified, and the putative M capsulatus haemerythrin was cleaved from the NusA by TEV-protease and purified 2432 to homogeneity by chromatography (Fig 7) The relative molecular mass of the McHr protein was determined in calibrated gel filtration chromatography to be  15 kDa (data not shown) This is close to a molecular mass of 14.7 predicted from its amino acid FEBS Journal 272 (2005) 2428–2440 ª 2005 FEBS O A Karlsen et al Characterization of prokaryotic haemerythrin sequence and indicates that McHr is stable in a monomeric form under our experimental conditions Structure predictions The tertiary structure of all characterized haemerythrins comprises a left-twisted four a-helix bundle which provides a hydrophobic pocket where dioxygen binds the two iron atoms Bioinformatical secondary structure predictions using various software programs indicated predominant a-helix content in McHr We were able to model a three-dimensional structure of McHr in swiss-model [10], using the multiple alignments and the crystallized myohaemerythrin from Themiste zostericola as a template (Fig 8A) [11] Hence, computational analysis modelled the putative haemerythrin to a four a-helix bundle, maintaining both the conserved haemerythrin positioning of the amino acid residues coordinating the metal ions, and the hydrophobic pocket in which dioxygen is situated (Fig 8B,C) Furthermore, McHr contains two cysteins, Cys88 and Cys128 In our model, Cys88 was placed near the Fig Unrooted phylogenetic tree of the haemerythrin sequences of eukaryotic members of the haemerythrin family and McHr A bootstrapped phylogenetic tree was constructed from the multiple alignments of Fig The tree was displayed using TREEVIEW The abbreviations of organism names correspond to those given in the legend to Fig 4, and the myohaemerythrins are indicated by (MHr) Table Prokaryotes which possess open reading frames encoding putative haemerythrins containing the haemerythrin signature (PROSITE 00550) Accession no (SwissProt and TrEMBL) Gene name Phylogeny Organism Amino acid length Q67PL5 O67614 Q6NE67 Q62LM6 Q63SE6 Q84IH8 Q8Y1B3 Q9I352 Q8GI12 Q9AQM2 Q8P892 Q8PJP7 Q6MHK9 Q6AMA9 Q748S2 Q74G47 Q74GJ0 Q7MRA7 Q9PIQ3 Q7VK87 Q7VK88 Q891L2 Q97MX1 Q8RCZ5 Q73N54 Q73NY7 STH1393 AQ_1719 ORF13 BMA0605 BPSL2377 ORF33 RSc0777 PA1673 ORF33 ORF33 XCC2351 XAC2483 Bd3532 DP1787 GSU2929 GSU0402 GSU0256 WS1527 Cj0241c HH0005 HH0004 CTC02359 CAC0069 TTE0259 TDE1302 TDE1013 Actinobacteria Aquificae Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Firmicutes Firmicutes Firmicutes Spirochaetes Spirochaetes Symbiobacterium termophilum Aquifex aeolicus Magnetospirillum gryphiswaldense Burkholderia mallei Burkholderia pseudomallei Janthinobacterium sp J3 Ralstonia solanacearum Pseudomonas aeruginosa Pseudomonas resinovorans Pseudomonas resinovorans Xanthomonas campestris Xanthomonas axonopodis Bdellovibrio bacteriovorus Desulfotalea psychrophila Geobacter sulfurreducens Geobacter sulfurreducens Geobacter sulfurreducens Wolinella succinogenes Campylobacter jejuni Helicobacter hepaticus Helicobacter hepaticus Clostridium tetani Clostridium acetobutylicum Thermoanaerobacter tengcongensis Treponema denticola Treponema denticola 147 131 149 149 141 149 137 153 146 165 153 153 136 156 131 135 145 168 133 187 184 137 129 133 143 137 FEBS Journal 272 (2005) 2428–2440 ª 2005 FEBS (a) (b) (b) (b) (b) (c) (c) (c) (c) (c) (d) (d) (d) (d) (d) (e) (e) (e) (e) 2433 Characterization of prokaryotic haemerythrin O A Karlsen et al Fig Sequence alignment of McHr and DcrH-Hr Dv: DcrH-Hr fragment of DcrH from D vulgaris Mc: McHr of M capsulatus A B lysed by the decrease in elipticity at 222 nm for temperature scans (25–90 °C) (Fig 9B) The protein was very stable with an approximate denaturing temperature of 75 °C Spectrophotometric analysis and metal binding Fig SDS ⁄ PAGE analysis of proteins during purification of McHr Samples from each step in the purification procedure were collected and analysed (A) 10% polyacrylamide gel Lane 1, crude extract of E coli BL21 StarTM (DE3)[pETM60] prior to IPTG induction Lane 2, as lane 1, after IPTG induction Lane 3, purified His-tagged NusA–(McHr) from the induction in lane Lane 4, gel filtrated His-tagged NusA–(McHr) fusion protein (lane 3) (B) 15% polyacrylamide gel Lane 1, NusA–(McHr) before cleavage with TEV-protease Lane 2, as lane 1, after TEV treatment Lane 3, purified McHr after gel filtration of TEV cleaved NusA–(McHr) Molecular size markers are indicated for both subfigures C-terminal end of helix III, pointing towards the inside of the bundle, and Cys128 was located to the random coil of the C-terminal end of the protein (C-terminal to helix IV) (not shown) The long distance between these two residues in the predicted tertiary structure would prevent the formation of an intramolecular disulfide bridge The major difference in tertiary structure of this model compared with haemerythrins, is the increased random coiled structure between helices I and II, and between helices III and IV (Fig 8A) However, because swiss-model transposes only a target sequence to a model based on a template structure and does not predict additional regular motifs, it is interesting that the secondary structure programs (scratch, phdsec and psipred) utilized predict longer helices, thereby shortening the modelled loops To further establish the secondary structure of McHr, the protein was analysed using circular dichroism (CD) (Fig 9A) The recorded CD spectrum of McHr revealed two negative maxima at 208 and 222 nm, and one positive maximum at 195 nm, typical of proteins with extensive a-helical secondary structures The content of a-helical structures determined by CD (58.0%) agreed with the values obtained from model the prediction (57%) The stability of the protein was ana2434 In addition to the CD spectrum, UV ⁄ vis spectrophotometric analysis of the purified protein showed a maximum at 280 nm (Fig 10) Most importantly, two local maxima of  330 and 380 nm were also revealed These maxima are typical of diiron-centre absorbance, and thus characteristic for all haemerythrins [12] This strongly indicates that our protein preparation contains conjugated iron ions However, in order to further confirm the presence of iron in the purified protein, McHr was subjected to inductively coupled plasma atomic emission-mass spectrometry (ICP-MS) analyses after extensive removal of free metal ions from the protein solution The analyses clearly demonstrated the presence of iron, and we estimated that  33% of the McHr contained two irons ⁄ molecule The finding that not all McHr molecules contained iron was in agreement with previous reports that describe difficulties in incorporation of iron when overexpressing heterologous diiron proteins in E coli [13] Discussion Haemerythrins have been considered to be important O2-handling proteins for some marine invertebrates and for a long time were believed to be restricted to only a few phyla of such eukaryotes However, we identified a putative prokaryotic haemerythrin expressed in high-copper content cultures of the methane-oxidizing bacterium M capsulatus Furthermore, we found that the haemerythrin motif is widespread in the prokaryotic kingdom, and, to date, we have located putative haemerythrins in 21 different bacteria of 174 sequenced bacterial genomes Owing to the importance of copper in the biology of M capsulatus, it is of great interest to study protein expression in cells cultured at different copper concentrations Copper ions regulate the expression of the two types of methane monooxygenase (MMO) of M capsulatus [14–16] At high copper-to-biomass ratios, the particulate membrane-associated pMMO is FEBS Journal 272 (2005) 2428–2440 ª 2005 FEBS O A Karlsen et al Characterization of prokaryotic haemerythrin A Fig SWISS-MODEL generated three-dimensional structure of McHr SWISS-MODEL was used in the alignment interface mode The multiple alignment of McHr vs myohaemerythrins (Fig 2B), was subjected for modelling using crystallized T zostericola (PDB Accession no 1a7d) as the template and McHr as the target sequence The graphical presentation was prepared in DeepView ⁄ Swiss-PdbViewer (A) 1, X-ray crystal structure of T zostericola myohaemerythrin ˚ at 1.8 A resolution [11]; 2, modelled 3D structure of McHr (B) 1, iron-binding ligands of T zostericola; 2, candidate iron-binding residues of McHr; 3, overlay of (1) and (2) (C) 1, amino acids of T zostericola forming the O2-binding hydrophobic pocket; 2, candidate residues of (1) in McHr; 3, Overlay of (1) and (2) B C produced pMMO expression is accompanied by the formation of a complex intracytoplasmic membrane system, in which the pMMO enzyme activity resides [17] At a low copper-to-biomass ratio, the soluble cytoplasmic version of MMO (sMMO) is expressed The underlying mechanisms for these major morphological and physiological changes are not known, but copper-mediated gene activation or repression has been implicated [18] Recently, MS technology was used to study protein expression at different copper concentrations [4] Our 2DE analysis revealed a strongly copper-regulated protein Initial sequence analysis indicated that this protein was a homologue of the eukaryotic haemerythrins In further sequence analysis, the resolved structures of Hr and MHr isolated from Phascolopsis gouldii and T zostericola [19,20] opened up the possibility of using secondary structure-based penalties in FEBS Journal 272 (2005) 2428–2440 ª 2005 FEBS multiple alignments Such an approach has been shown to improve their accuracy and reliability The clustalx alignments of McHr vs Hr and MHr identified the candidate residues which are known to coordinate two iron atoms, in addition to those forming the O2-binding hydrophobic pocket The presence of a corresponding iron-binding motif was also supported by the UV ⁄ vis spectrum of McHr, which demonstrated specific iron centre absorbance peaks between 320 and 380 nm, and ICP-MS analyses which confirmed the presence of iron In addition, the strong sequence similarity to the bacterial haemerythrin-like domain of DcrH [3] also supports the presence of an oxo-bridged diiron(III) site in McHr The seven ligands of the diiron site in haemerythrins are amino acid residues each located within the four helices, and by diiron binding generating an intramolecular cross-linking of these helices, which 2435 Characterization of prokaryotic haemerythrin Fig CD spectra and thermal scan of McHr (A) Far-UV CD spectrum at 25 °C of the purified McHr obtained in 20 mM KH2PO4, pH 8.0 (B) CD-monitored thermal scan following the changes in ellipticity at 210 nm The temperature scan was recorded in the gel filtration buffer (20 mM Tris ⁄ HCl, pH 8.0, 400 mM NaCl, 0.4 mM AEBSF-hydrochloride) O A Karlsen et al structure was also determined experimentally by CD analysis, which estimated  58% a-helices in McHr Further, computational analysis modelled McHr to a four a-helix bundle, maintaining the conserved haemerythrin positioning of all the amino acids involved in the active site However, the validity of this model must be explored further by structural analyses as NMR and X-ray crystallography Most haemerythrin proteins have been assigned a function in handling oxygen, like the multisubunit haemerythrins found for the haemerythrocytes of sipunculids, brachipods and priapulids [2,22] It is not unlikely that McHr serves a similar function in M capsulatus by reversibly binding oxygen The conservation of both iron-binding ligands and O2-binding pocket residues strongly suggests that interaction with this molecule is part of its biological function Several enzymes (i.e oxygenases and oxidases) require a supply of O2 and may be recipients for a McHr-bound dioxygen molecule Furthermore, at a high copper-tobiomass ratio, pMMO is the enzyme responsible for oxidizing methane in M capsulatus [16] This enzyme uses oxygen as an oxidant in the process [23] and therefore pMMO could also be an important receiver of McHr delivered O2 In conclusion, we describe the cloning of the first prokaryotic haemerythrin and demonstrate that the protein has a high a-helix content and binds iron in its native form We also provide novel evidence for the presence of haemerythrin gene homologues in a large group of prokaryotes The exact biological function of McHr in M capsulatus is currently not known, but because its expression is regulated by copper-ion concentration in the medium it is suggested that it plays an important physiological role under high copper-to-biomass growth conditions, possibly in methane oxidation Experimental procedures Growth of M capsulatus (Bath) Fig 10 UV ⁄ vis absorption spectra of recombinant McHr The spectrum was recorded in gel filtration buffer (20 mM Tris ⁄ HCl, pH 8.0, 400 mM NaCl, 0.4 mM AEBSF-hydrochloride) will stabilize the overall protein structure [21] The conservation of all seven ligand amino acid residues in McHr constitutes strong evidence for a four-helix bundle structure of this protein A helical secondary 2436 M capsulatus NCIMB 11132 was grown in batch cultures at 45 °C while shaking in an atmosphere of CH4, CO2 and O2 (45 : 10 : 45, v ⁄ v ⁄ v) in NMS medium as described by Whittenbury et al [24] Cells were grown either at a high copper-to-biomass ratio by including a final concentration of 0.8 lm copper in the growth medium, or at a low copper-to-biomass ratio in ‘copper-free’ medium (no copper added) Low-copper cultures were screened for sMMO activity using the naphthalene assay described by Brusseau et al [25] to ensure that a low copper-to-biomass ratio was achieved Batch cultures of M capsulatus were grown to a cell density of  108 cellsỈmL)1 before harvesting FEBS Journal 272 (2005) 2428–2440 ª 2005 FEBS O A Karlsen et al Characterization of prokaryotic haemerythrin Preparation of the soluble fraction Cloning, expression and purification of McHr Cells were harvested by centrifugation at 5000 g for 10 Enriched fractions of the soluble proteins were obtained as described by Fjellbirkeland et al [26] Protein concentrations were determined by using the Protein DC-kit from Bio-Rad (Oslo, Norway) mchr (MCA0715 of the M capsulatus genome) was amplified by PCR (primers: McHr_F_NcoI, 5¢-CCATGGCATT AATGACGTGG-3¢; McHr_R_XhoI, 5¢-GCTCGAGTTAT GCGCTCAGG-3¢) and cloned into the pETM60 vector using XhoI and NcoI restriction sites, thereby fusing mchr to the nusA gene separated by a linker region [29] The linker region contains a His-tag and the sequence for the TEV-protease cleavage site Positive clones were verified by sequencing Large-scale protein expression was performed using E coli BL21 StarTM (DE3) containing the pETM60 expression vector grown at 37 °C Expression was induced with addition of 0.7 mm isopropyl thio-b-d-galactoside (IPTG) at A600  0.6, and cultured for additional 4–5 h Pelleted bacteria were resuspended and sonicated in NaCl ⁄ Pi, pH 8.0, 0.4 mm AEBSF-hydrochloride (Applichem), 10 mm imidazole After centrifugation at 39 000 g for 20 min, the supernatant was filtered (0.22 lm) before loaded onto a pre-equilibrated HisTrapTM chelating column (Amersham) Bound proteins were eluted by stepwise addition of increasing concentrations of imidazole in NaCl ⁄ Pi Fractions containing the NusA–(McHr) fusion protein were pooled and applied onto a Superdex 75 16 ⁄ 60 gel filtration column for removal of imidazole (gel filtration buffer: 20 mm Tris ⁄ HCl, pH 8.0, 400 mm NaCl, 0.4 mm AEBSF-hydrochloride) Further, TEV-protease (Invitrogen, Carlsbad, CA, USA) was added to a concentration of 10 units for 20 lg substrate and incubated at room temperature overnight His-tagged TEV and NusA were selectively removed by reloading the solution onto the HisTrapTM chelating column where they were tightly bound, whereas McHr was recovered in the unbound fractions A final gel filtration of McHr was performed on a Superdex 75 16 ⁄ 60 column to remove traces of NusA and imidazole SDS/PAGE SDS ⁄ PAGE was performed as described by Laemmli et al [27] using either 15 or 10% (w ⁄ v) running gels and 3% (w ⁄ v) stacking gels Two-dimensional gel electrophoresis 2DE was performed essentially as described by Rabilloud et al [28] An appropriate volume of sample was precipitated in 80% (v ⁄ v) acetone, and the pellet was solubilized in an isoelectric focusing buffer containing m urea, m thiourea, 4% (w ⁄ v) CHAPS, 0.5% (v ⁄ v) Triton X-100, 20 mm dithiothreitol, 0.5% (v ⁄ v) carrier ampholytes pH 3.5–10 (Amersham, Uppsala, Sweden) and trace amounts of bromphenol blue The sample was applied into an immobilized pH gradient (IPG) strip by ‘in-gel’ rehydration over night, using the Immobiline Drystrip Reswelling Tray (Amersham) Isoelectric focusing was performed in the Multiphor II system (Amersham) at 20 °C using the Pharmacia (Uppsala, Sweden) EPS 3500 XL power supply in gradient mode according to the manufacturers’ procedure Prior to the second dimension SDS ⁄ PAGE, the IPG strip was equilibrated twice for 15 in buffer containing m urea, 30% (v ⁄ v) glycerol, 50 mm Tris ⁄ HCl (pH 8.8), 2% SDS, 2.5 mgỈmL)1 dithiothreitol and traces of bromophenol blue In the second equilibration step, dithiothreitol was omitted and replaced by 45 mgỈmL)1 iodoacetamide The second dimension electrophoresis was carried out in the Protean II XI (Bio-Rad) apparatus at 20 °C and using a 12.5% polyacrylamide gel The power programme consisted of two phases; mA per gel for h, followed by mA per gel until the tracing dye reached the end of the gel Proteins were visualized by SYPRO RUBYTM fluorescence staining (Bio-Rad), and gels were scanned with a Fuji (Tokyo, Japan) FLA-2000 phosphoimager Identification of proteins by MS and N-terminal sequencing MS analyses were performed at the Mass Spectrometry Facility at the University of Warwick (Coventry, UK), and at the PROBE facility at the University of Bergen, Norway N-Terminal amino acid sequencing was carried out at the University of Oslo, Norway FEBS Journal 272 (2005) 2428–2440 ª 2005 FEBS Spectrophotometric analysis UV ⁄ vis spectrophotometric absorption data was obtained in cm path-length quartz cuvettes on a UNICAM UV ⁄ VIS UV2 spectrometer CD analysis was performed using a Jasco (Cremella, Italy) J-810 spectropolarimeter equipped with a Jasco 423S Peltier element for temperature control Protein samples were prepared in different buffers as indicated The standard analysis program provided with the instrument was used for analysing the data Secondary structure elements were estimated by the cdnn program that utilizes a neural network procedure [30] Metal determination ICP-MS analyses were used to determine the metal content in McHr, and were carried out at the Department of Earth 2437 Characterization of prokaryotic haemerythrin and Science, University of Bergen, Norway Prior to analyses, the McHr preparation was passed twice through a PD-10 desalting column (Amersham) pre-equilibrated with H2O for removal of contaminating metal ions present in the gel filtration buffer Furthermore, McHr was diluted with concentrated nitric acid [6% (v ⁄ v) final concentration] and digested for 24 h at 110 °C After cooling, the samples were diluted with H2O to a final concentration of 2% (v ⁄ v) nitric acid and analysed for iron content RNA isolation using the hot phenol extraction method M capsulatus flask cultures were harvested by centrifugation at 16 300 g for Pellets were resuspended in Tris ⁄ EDTA buffer (0.1 mm Tris ⁄ HCl, 0.1 mm EDTA, pH 8.0) and submerged directly into liquid nitrogen and stored at )80 °C until used RNA isolation and DNase treatment were carried out as described at http://www microarrays.org/pdfs/Total_RNA_from_Ecoli.pdf The RNA was dissolved in 50 lL of RNase-free water and the A260 and A280 values were determined O A Karlsen et al checked using verify 3d [34] and prosa [35] which both showed that the model has a plausible overall fold; in particular, the predicted helical regions have scores in the same range as the template structure Secondary structure predictions were made by submitting the McHr sequence to the following prediction programs; scratch (ssPRO) [36], phdsec [37] and psipred [38] Acknowledgements This work was supported by grants from the Norwegian Research Council and the Meltzer Foundation, University of Bergen We thank Professor J C Murrel for mass spectrometry analyses performed at the University of Warwick (Coventry, UK) and PROBE at the University of Bergen for peptide ⁄ protein analyses We thank D Straume for help with the northern blot analyses, and Rolf-Birger Pedersen and Ole Tumyr at the Department of Earth and Science at the University of Bergen, Norway for performing the ICP-MS analyses We also thank the Computational Biology Unit (CBU) at the University of Bergen for assessment of our structural model Northern blotting Blotting and hybridization experiments were performed as described by Sambrook et al [31] The gene probe of mchr (MCA0715) was generated by PCR amplification using DyNAzymeEXT polymerase (Finnzymes, Espoo, Finnland) and the oligonucleotides used in the cloning of mchr Bioinformatic tools Scanprosite [6] and the Conserved Domain Database [7,8] were used when analysing the McHr primary sequence for conserved patterns Multiple alignments were constructed by clustalx (v 1.83) [9] using secondary structure-based penalties obtained from the solved haemerythrins of P gouldii (Hr, pdb 1i4y) and T zostericola (MHr, pdb 1a7d), respectively Standard alignment parameters of clustalx were utilized and the resulting multiple alignments were visualized in genedoc Bootstrapped phylogenetic trees based on the multiple alignments were made in clustalx and viewed in treeview The 3D model of McHr was built using swissmodel [10] in multiple alignment mode, using the multiple alignment of McHr with the myohaemerythrins (Fig 4B) and the X-ray structure of T zostericola MHr (pdb 1a7d) as a template The quality of the model was first assessed using procheck [32,33] The vast majority of residues (89.7%) fall into the most favoured regions of the Ramachandran plot, 9.4% in the allowed regions and only one residue (Ala100) into the disallowed region These results are consistent with the values obtained for the template structure (94.3, 5.7, 0%, respectively) Furthermore, the 3D)1D compatibility was 2438 References Stenkamp RE (1994) Dioxygen and Hemerythrin Chem Rev 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Tedeschi G & Bonomi F (1992) Myohemerythrin from the sipunculid, Phascolopsis gouldii: purification, properties and amino acid sequence Biochim Biophys Acta 1122, 136–142 Klippenstein GL, Cote JL & Ludlam SE (1976) The primary structure of myohemerythrin Biochemistry 15, 1128–1136 2440 O A Karlsen et al 48 Coutte L, Slomianny MC, Malecha J & Baert JL (2001) Cloning and expression analysis of a cDNA that encodes a leech hemerythrin Biochim Biophys Acta 1518, 282–286 49 Demuynck S, Li KW, Van der Schors R & DhainautCourtois N (1993) Amino acid sequence of the small cadmium-binding protein (MP II) from Nereis diversicolor (annelida, polychaeta) Evidence for a myohemerythrin structure Eur J Biochem 217, 151–156 50 Takagi T & Cox JA (1991) Primary structure of myohemerythrin from the annelid Nereis diversicolor FEBS Lett 285, 25–27 Supplementary material The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4663/EJB4663sm.htm Fig S1 Multiple alignment of McHr and the bacterial sequences containing the hemerythrin signature (PROSITE 00550) (Table 2) clustalx was used with standard alignment parameters The sequences were obtained by searching with the PROSITE 00550 pattern in the SWISSPROT and TrEMBL databases Accession nos for sequences and abbreviations for organism names are given in the alignment; Geobacter sulfurreducens (Gs), Xanthomonas campestris (Xc), Xanthomonas axonopodis (Xa), Phseudomonas aeruginosa (Pa), Symbiobacterium termophilum (St), Thermoanaerobacter tengcongensis (Tt), Treponema denticola (Td), Helicobacter hepaticus (Hh), Clostridium acetobutylicum (Ca), Clostridium tetani (Ct), Magnetospirillum gryphiswaldense (Mg), Burkholderia mallei (Bm), Burkholderia pseudomallei (Bp), Ralstonia solanacearum (Rs), Pseudomonas resinovorans (Pr), Janthinobacterium sp J3 (Js), Wolinella succinogenes (Ws), Desulfotalea psychrophila (Dp), Bdellovibrio bacteriovorus (Bb), Campylobacter jejuni (Cj), Aquifex aeolicus (Aa) FEBS Journal 272 (2005) 2428–2440 ª 2005 FEBS ... The gene was cloned from the methanotrophic Gram-negative bacterium Methylococcus capsulatus (Bath) and encodes a protein of 131 amino acid residues The prokaryotic haemerythrin protein contains... Actinobacteria Aquificae Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria... generating an intramolecular cross-linking of these helices, which 2435 Characterization of prokaryotic haemerythrin Fig CD spectra and thermal scan of McHr (A) Far-UV CD spectrum at 25 °C of the