Báo cáo khoa học: Characterization of a prokaryotic haemerythrin from the methanotrophic bacterium Methylococcus capsulatus (Bath) ppt

When analysing dif-ferential protein expression in the methane-oxidizing bacterium, Methylo-coccus capsulatusBath, grown at a high and low copper-to-biomass ratio, respectively, we ident

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

Haemerythrin proteins comprise a family of O2

-carrry-ing proteins mainly found in a few phyla of

marine invertebrates Members of this family differ

from haemoglobin and haemocyanin in that they

con-tain 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 haemeryth-rins also share a four-helix bundle fold which surrounds the diiron site Furthermore, the haemerythrins are divi-ded 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

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 6 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 dif-ferential protein expression in the methane-oxidizing bacterium, Methylo-coccus 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 hydropho-bic 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 identi-fied 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 Further-more, 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 signa-ture (PROSITE 00550), indicating that these putative haemerythrins may

be a conserved prokaryotic subfamily

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.

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

struc-ture 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

chemo-receptor H (DcrH), expressed in the anaerobic,

sul-fate-reducing bacterium Desulfovibrio vulgaris The

DcrH chemoreceptor was proposed to have a

mem-brane-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

charac-terize the encoded protein The gene was cloned

from the methanotrophic Gram-negative bacterium

Methylococcus capsulatus (Bath) and encodes a

pro-tein of 131 amino acid residues The prokaryotic

haemerythrin protein contains the haemerythrin

sig-nature typical for members of this family In vivo

expression of the putative M capsulatus haemerythrin

(McHr) was increased in cells grown at a high

cop-per-to-biomass ratio, indicating an important

physio-logical role under this growth condition The latter

observation was also reported by Kao et al [4]

McHr was expressed in Escherichia coli and

ana-lysed 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

pro-tein spots significantly affected by the growth

condi-tions (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

been annotated by us as a haemerythrin family pro-tein [5] MCA0715 was located to a single transcrip-tional unit, between the conserved hypothetical protein MCA0714 and the hypothetical protein MCA0716 (Fig 2) After the MS-based gene identifi-cation, 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 pro-tein 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 charac-teristic 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 gluta-mate⁄ 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 4 The regions of the McHr that contained the highest level of identical residues and conserva-tive 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 haem-erythrin subfamilies making McHr the largest known haemerythrin Thus, assuming that the corresponding

a helices are of equal length in the different haem-erythrins, the loops (random coils) between the heli-ces in McHr are longer than those of the eukaryotic haemerythrins Furthermore, a clustalx alignment-based 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

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

haem-erythrin-like domain (PROSITE PS00550) (Table 2)

[6] Interestingly, unlike the very restricted

distribu-tion 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

haem-erythrins are identified from Proteobacteria Until

now, none of these putative prokaryotic

haemeryth-rins has been characterized In fact, apart from the

McHr [4], there has been only one report of

micro-bial proteins containing a haemerythrin signature [3] This haemerythrin-like motif was found in the C-ter-minal 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 expres-sing 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 resi-dues 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

A

B

Fig 1 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.

Table 1 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 condi-tions +, indicates expression in given growth condition (+), indicate less abundant expression.

N-succinyltransferase

Fig 2 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 kcalÆmol )1

(http://www.genebee.msu.su/services/rna2_reduced.html)

(indica-ted as a hairpin in A).

Fig 3 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 trans-ferred to a nylon membrane The blot was hybridized at 42
C over-night, 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.

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

puri-fied, and the putative M capsulatus haemerythrin was

cleaved from the NusA by TEV-protease and purified

to homogeneity by chromatography (Fig 7) The rel-ative molecular mass of the McHr protein was deter-mined 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

Fig 4 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.

sequence and indicates that McHr is stable in a monomeric form under our experimental conditions

Structure predictions The tertiary structure of all characterized haemeryth-rins comprises a left-twisted four a-helix bundle which provides a hydrophobic pocket where dioxygen binds the two iron atoms Bioinformatical secondary struc-ture predictions using various software programs indi-cated 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, compu-tational 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 5 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 4 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 2 Prokaryotes which possess open reading frames encoding putative haemerythrins containing the haemerythrin signature (PROSITE 00550).

Accession no.

(SwissProt and

Amino acid length

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

struc-ture 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)

How-ever, 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

fur-ther 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

con-tent of a-helical structures determined by CD (58.0%)

agreed with the values obtained from model the

pre-diction (57%) The stability of the protein was

ana-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 tem-perature of 75
C

Spectrophotometric analysis and metal binding

In addition to the CD spectrum, UV⁄ vis spectrophoto-metric analysis of the purified protein showed a maxi-mum 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 con-tains conjugated iron ions However, in order to fur-ther 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 demon-strated 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 des-cribe difficulties in incorporation of iron when over-expressing 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 Further-more, we found that the haemerythrin motif is wide-spread 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 concen-trations 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

Fig 6 Sequence alignment of McHr and DcrH-Hr Dv: DcrH-Hr fragment of DcrH from

D vulgaris Mc: McHr of M capsulatus.

Fig 7 SDS ⁄ PAGE analysis of proteins during purification of McHr.

Samples from each step in the purification procedure were

collec-ted 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 2 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,

puri-fied McHr after gel filtration of TEV cleaved NusA–(McHr)

Mole-cular size markers are indicated for both subfigures.

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

morpholo-gical and physiolomorpholo-gical 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

concentra-tions [4]

Our 2DE analysis revealed a strongly

copper-regula-ted protein Initial sequence analysis indicacopper-regula-ted that this

protein was a homologue of the eukaryotic

haemeryth-rins In further sequence analysis, the resolved

struc-tures of Hr and MHr isolated from Phascolopsis

gouldii and T zostericola [19,20] opened up the

possi-bility of using secondary structure-based penalties in

multiple alignments Such an approach has been shown to improve their accuracy and reliability The clustalxalignments of McHr vs Hr and MHr identi-fied the candidate residues which are known to coordi-nate 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 simi-larity 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 haemeryth-rins are amino acid residues each located within the four helices, and by diiron binding generating an intramolecular cross-linking of these helices, which

A

B

C

Fig 8 SWISS - MODEL generated

three-dimen-sional structure of McHr SWISS - MODEL was

used in the alignment interface mode The

multiple alignment of McHr vs

myohaem-erythrins (Fig 2B), was subjected for

model-ling using crystallized T zostericola (PDB

Accession no 1a7d) as the template and

McHr as the target sequence The graphical

presentation was prepared in

Deep-View ⁄ 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,

candi-date residues of (1) in McHr; 3, Overlay of

(1) and (2).

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

analysis, which estimated  58% a-helices in McHr Further, computational analysis modelled McHr to a four a-helix bundle, maintaining the conserved haem-erythrin 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 sipun-culids, brachipods and priapulids [2,22] It is not unlikely that McHr serves a similar function in

M capsulatus by reversibly binding oxygen The con-servation of both iron-binding ligands and O2-binding pocket residues strongly suggests that interaction with this molecule is part of its biological function Sev-eral enzymes (i.e oxygenases and oxidases) require a supply of O2and may be recipients for a McHr-bound dioxygen molecule Furthermore, at a high copper-to-biomass 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 con-centration in the medium it is suggested that it plays an important physiological role under high copper-to-bio-mass growth conditions, possibly in methane oxidation

Experimental procedures

Growth of M capsulatus (Bath)

M capsulatus NCIMB 11132 was grown in batch cultures

at 45
C while shaking in an atmosphere of CH4, CO2and

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 cop-per-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

cellsÆmL)1before harvesting

Fig 10 UV ⁄ vis absorption spectra of recombinant McHr The

spec-trum was recorded in gel filtration buffer (20 m M Tris ⁄ HCl, pH 8.0,

400 m M NaCl, 0.4 m M AEBSF-hydrochloride).

Fig 9 CD spectra and thermal scan of McHr (A) Far-UV CD

spec-trum at 25
C of the purified McHr obtained in 20 m M KH 2 PO 4 ,

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 m M Tris ⁄ HCl, pH 8.0, 400 m M NaCl, 0.4 m M

AEBSF-hydrochloride).

Preparation of the soluble fraction

Cells were harvested by centrifugation at 5000 g for

10 min 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)

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

precipi-tated in 80% (v⁄ v) acetone, and the pellet was solubilized

in an isoelectric focusing buffer containing 7 m urea, 2 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

per-formed 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

manu-facturers’ procedure Prior to the second dimension

SDS⁄ PAGE, the IPG strip was equilibrated twice for

15 min in buffer containing 6 m urea, 30% (v⁄ v) glycerol,

50 mm Tris⁄ HCl (pH 8.8), 2% SDS, 2.5 mgÆmL)1

dithio-threitol and traces of bromophenol blue In the second

equilibration step, dithiothreitol was omitted and replaced

by 45 mgÆmL)1iodoacetamide The second dimension

elec-trophoresis was carried out in the Protean II XI

(Bio-Rad) apparatus at 20
C and using a 12.5%

poly-acrylamide gel The power programme consisted of two

phases; 5 mA per gel for 2 h, followed by 8 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

Cloning, expression and purification of McHr mchr (MCA0715 of the M capsulatus genome) was ampli-fied 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 lin-ker 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 (Appli-chem), 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 col-umn (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 filtra-tion 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 concen-tration 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 frac-tions A final gel filtration of McHr was performed on a Superdex 75 16⁄ 60 column to remove traces of NusA and imidazole

Spectrophotometric analysis

UV⁄ vis spectrophotometric absorption data was obtained

in 1 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