Heterologous synthesis of cytochrome c′ by Escherichia coli is not dependent on the System I cytochrome c biogenesis machinery

Heterologous synthesis of cytochrome c¢ byEscherichia coli is not dependent on the System Icytochrome c biogenesis machineryHiroki Inoue1, Satoshi Wakai1, Hirofumi Nishihara2 and Yoshihiro Sambongi1

1 Graduate School of Biosphere Science, Hiroshima University, Japan

2 Faculty of Agriculture, Ibaraki University, Japan

Introduction

Cytochromes c¢ are classified as class II cytochromes c

according to Ambler [1], and are found in the peri-

plasm of certain Gram-negative Alpha-, Beta- and

Gammaproteobacteria. Recent biochemical and genetic

analyses have demonstrated that cytochromes c¢mainly play roles in the cellular metabolism of nitric

oxide [2], which is an electron acceptor in denitrifying

bacteria and is also implicated as a signaling molecule

in a wide range of organisms.

The structure of cytochromes c¢ exhibits clear differ-

ences from that of the well-known Ambler’s class I

cytochromes c. The class I cytochromes c are spherical

proteins with a hexacoordinate heme covalently bound

near their N-termini. In contrast, cytochromes c¢, con-sisting of approximately 130 residues, contain a penta-

coordinate heme located towards the C-terminus of a

four-helix bundle protein. Escherichia coli cyto-

chrome b562 (EC b562), a 106-residue protein, also has

a four-helix bundle structure with a noncovalently

bound heme [3]. Despite the sequence difference

between cytochromes b562 and c¢, the four helices of

each nearly spatially coincide when the respective heme

groups are superimposed [4].

Although knowledge concerning the function

and structure of cytochromes c¢ has accumulated, their

biogenesis remains unclear. In general, covalent heme

Keywords

cytochrome c biogenesis; cytochrome c¢;Escherichia coli; heterologous synthesis;

System I

Correspondence

Y. Sambongi, Graduate School of Biosphere

Science, Hiroshima University, 1-4-4

Kagamiyama, Higashi-Hiroshima, Hiroshima

739-8528, Japan

Fax: +81 824 24 7924

Tel: +81 824 24 7924

E-mail: [email protected]

(Received 28 February 2011, revised 5 April

2011, accepted 27 April 2011)

doi:10.1111/j.1742-4658.2011.08155.x

Hydrogenophilus thermoluteolus cytochrome c¢ (PHCP) has typical spectral

properties previously observed for other cytochromes c¢, which comprise

Ambler’s class II cytochromes c. The PHCP protein sequence (135 amino

acids) deduced from the cloned gene is the most homologous (55% iden-

tity) to that of cytochrome c¢ from Allochromatium vinosum (AVCP). These

findings indicate that PHCP forms a four-helix bundle structure, similar to

AVCP. Strikingly, PHCP with a covalently bound heme was heterologously

synthesized in the periplasm of Escherichia coli strains deficient in the

DsbD protein, a component of the System I cytochrome c biogenesis

machinery. The heterologous synthesis of PHCP by aerobically growing

E. coli also occurred without a plasmid carrying the genes for Ccm pro-

teins, other components of the System I machinery. Unlike Ambler’s class I

general cytochromes c, the synthesis of PHCP is not dependent on the

System I machinery and exhibits similarity to that of E. coli periplasmic

cytochrome b562, a 106-residue four-helix bundle.

Database

The sequence data reported here have been deposited in the DDBJ database under accession

no. AB617519.

Abbreviations

AVCP, Allochromatium vinosum cytochrome c ¢; Ccm, cytochrome c maturation; Dsb, disulfide bond formation; EC b562, Escherichia coli

cytochrome b562; PHCP, Hydrogenophilus thermoluteolus cytochrome c ¢; PH c552, Hydrogenophilus thermoluteolus cytochrome c552.

FEBS Journal 278 (2011) 2341–2348 ª 2011 The Authors Journal compilation ª 2011 FEBS 2341

http://www.ncbi.nlm.nih.gov/nuccore/AB617519

attachment to class I cytochromes c is catalyzed by the

cellular machinery, resulting in cytochrome c biogene-

sis [5]. For example, in some Gram-negative bacteria,

such as E. coli, the System I cytochrome c biogenesis

machinery, consisting of some disulfide bond forma-

tion (Dsb) and cytochrome c maturation (Ccm)

proteins, is responsible for the biogenesis of a wide

variety of both endogenous and exogenous class I cy-

tochromes c [6]. Successful heterologous synthesis of

several cytochromes c¢ has been reported using aerobi-

cally growing E. coli with co-expressed ccm genes from

a plasmid [7–9]. However, a variant of EC b562, which

has been mutated so as to bind heme covalently like

cytochromes c, can be formed as a holo-protein with-

out co-expressed ccm genes from a plasmid [10,11].

Although the heme-binding mode of the resulting

EC b562 variant differs from that with co-expressed

ccm genes from a plasmid, its holo-formation is obvi-

ous. This prompted us to re-examine the heterologous

synthesis of cytochromes c¢ with or without co-

expressed ccm genes from a plasmid. In addition, the

effects of Dsb proteins on cytochrome c¢ synthesis

have not been examined to date.

In this study, we examined the heterologous synthesis

of cytochrome c¢ proteins by E. coli strains deficient in

the DsbD protein and co-expressing or not co-express-

ing ccm genes from a plasmid. For this purpose, we first

purified and characterizedHydrogenophilus thermoluteo-

lus cytochrome c¢ (PHCP). Secondly, the PHCP gene

was cloned for sequence and expression analyses. Heter-

ologous synthesis of the PHCP protein by the E. coli

strains was investigated in direct comparison with that

of H. thermoluteolus cytochrome c552 (PH c552), which

is a typical class I cytochrome c that has been demon-

strated to be System I dependent with regard to its bio-

genesis in E. coli [12,13]. Our results provide

information on the biogenesis of cytochromes c¢, whichhas not been studied systematically.

Results

Purification of the PHCP protein

The PHCP protein was purified to homogeneity by col-

umn chromatography, as illustrated on an SDS ⁄PAGE

gel (Fig. 1). The estimated molecular weight of the

PHCP protein on the gel was 13 kDa, which was close

to that of other cytochromes c¢ isolated from various

bacteria. The N-terminal amino acid sequence of

PHCP was determined up to the 30th residue, as illus-

trated in Fig. 2. A blast search indicated that the pro-

tein sequence determined up to the 30th residue was

homologous to that of other cytochromes c¢ isolated

from other bacteria. Thus, at this stage of the present

work, we concluded that the purified PHCP protein

was a novel cytochrome c¢ isolated from H. thermolute-

olus.

Spectral properties of the authentic PHCP protein

Visible absorption spectra of the authentic PHCP pro-

tein purified from H. thermoluteolus were obtained to

examine the local heme environment in the protein

interior. The spectra of the oxidized and reduced

PHCP were essentially the same as those reported for

other cytochromes c¢ (Fig. 3A), indicating that the

heme environment in the PHCP protein was similar to

that in others. Specifically, a Soret band at 425 nm

was observed for the reduced form of PHCP, which is

characteristic of a pentacoordinate heme with a His

residue as an axial ligand [14]. Furthermore, a peak

around 630 nm was observed for the oxidized form of

PHCP, indicating that the position of the sixth ligand

to the heme iron is empty, as discussed for other cyto-

chromes c¢ [14]. In addition, the a-band in the pyridine

hemochrome spectrum of reduced PHCP corresponded

to 550 nm, which is indicative of the covalent bonding

of heme vinyl groups to the protein via two thioether

linkages.

A far-UV CD spectrum (190–260 nm) was obtained

to examine the secondary structure of the PHCP

Mw (kDa) 1 2 3 4

75

2520

15

10

5

5

Fig. 1. Purification of the Hydrogenophilus thermoluteolus cyto-

chrome c¢ (PHCP) protein. Lane 1, total soluble extract of H. therm-

oluteolus cells; lane 2, HiTrap Q batch elution with 0.2 M NaCl; lane

3, HiTrap Q linear gradient elution with 0–0.2 M NaCl; lane 4,

HiTrap SP flow-through elution; lane 5, Sephadex 75 elution. The

arrow indicates the position of PHCP. One to ten micrograms of

protein were loaded per lane, and the gel was stained with Coo-

massie Brilliant Blue.

Biogenesis of cytochrome c¢ H. Inoue et al.

2342 FEBS Journal 278 (2011) 2341–2348 ª 2011 The Authors Journal compilation ª 2011 FEBS

protein. From the ellipticity peak height of the PHCP

protein at 222 nm (Fig. 3B), its helical content was cal-

culated to be 60.3% [15]. This value is close to the

a-helical content of Allochromatium vinosum cyto-

chrome c¢ (AVCP), i.e. 63.0%, which was calculated

directly from its primary (Fig. 2) and three-dimen-

sional [16] structures.

Cloning of the PHCP gene

PCR with mixed primers PHcp01fw and PHcp01rv,

using H. thermoluteolus chromosomal DNA as a tem-

plate, gave a DNA fragment of approximately 360 bp,

which was then cloned into the pUC19 vector. At least

five independent clones were sequenced, and the amino

acid sequence (25th to 125th residues, Fig. 2) deduced

from the DNA was homologous to the sequences of

cytochromes c¢ deposited previously in the database.

Using the inverse PCR method, we obtained a single

6.5-kbp DNA fragment from an SphI-digested

H. thermoluteolus chromosomal DNA library. DNA

sequencing of the fragment revealed that the product

contained the 5¢ and 3¢ ends of the PHCP gene plus

putative promoter, Shine–Dalgarno and transcriptional

terminator sequences. From the deduced sequence, the

mature PHCP was found to consist of 135 amino

acids, and the N-terminal Asp was preceded by a Sec-

dependent periplasmic targeting signal peptide of 19

amino acid residues (Fig. 2). This indicates that the

PHCP protein is synthesized as a precursor, and that

its signal peptide is cleaved off during translocation to

the periplasm of H. thermoluteolus cells.

From the amino acid sequence deduced from the

cloned PHCP gene, the heme-binding motif observed

in general cytochromes c, Cys–X–X–Cys–His, was

found to be located close to the C-terminus of the

PHCP protein, which is conserved in other biochemi-

cally characterized cytochromes c¢ (Fig. 2). The mature

PHCP protein exhibited overall sequence identity of

54.8% to AVCP, this being the highest identity among

the homologs in the genome database.

Heterologous synthesis of the PHCP and PH c552

proteins by E. coli

The cloned PHCP gene, together with the typical

class I PH c552 gene as a reference control, was exam-

ined with regard to its heterologous expression in

various E. coli strains by means of heme-specific stain-

ing of SDS ⁄PAGE gels. On such gels, when stained

materials are observed at positions coinciding with

those of PHCP and PH c552, the proteins each have a

covalently attached heme, which is defined here as

completion of cytochrome c synthesis.

The PHCP protein was heterologously synthesized

in the periplasm of anaerobically growing E. coli dsbD

20 30 40 50 60 70

80 90 100 110 120 130

1 10 mkriamitaltlcaaaahaDALKPEDKVKFRQAS mkhvlastaaglmalgl-assaiaAGLSPEEQIETRQAGmkklstlaalacmtvgsll-atsaqaQFAKPEDAVKYRQSA mrrvllatlmaalpaaaMAADAEHVVEARKGY

(1) H. thermoluteolus(2) A. vinosum(3) A. xylosoxidans(4) R. sphaeroides

YTTMAWNMGKIKAMVVDGTMPFSQTQVSAAANVIAAIANSGMGALYSPDTLGVVGFKKSRYEFMGWNMGKIKA-NLEGE--YNAAQVEAAANVIAAIANSGMGALYGPGTDKNVGDVKTRLTLMASHFGRMTP-VVKGQAPYDAAQIKANVEVLKTLSAL-PWAAFGPGTEGG-D-----FSLVALEFGPLAAM-AKGEMPYDAAAAKAHASDLVTLTKYDPSDLYAPGTSAD-DVKGTA

LKENFFQEQDEVRKIATNFVEQANKLAEVAAMGDKDEIKAQFGEVGKACKACHEKFREEEVKPEFFQNMEDVGKIAREFVGAANTLAEVAATGEAEAVKTAFGDVGAACKSCHEKYRAK-ARPEIWSDAASFKQKQQAFQDNIVKLSAAADAGDLDKLRAAFGDVGASCKACHDAYRKKKAKAAIWQDADGFQAKGMAFFEAVAALEPAAGAGQKE-LAAAVGKVGGTCKSCHDDFRVKR

* ** * *

* * * *

Fig. 2. Multiple sequence analysis of biochemically characterized cytochrome c¢ proteins. Experimentally determined signal peptides are

depicted in lower case letters. The numbering on the Hydrogenophilus thermoluteolus cytochrome c¢ (PHCP) sequence is that of the mature

protein. The sequence of PHCP was chemically determined up to the 30th residue in this study and confirmed by the protein sequence

deduced from the cloned gene. The stretches of the PHCP amino acid sequence used for the design of the PCR primers are underlined with

arrows indicating the 5¢ to 3¢ direction. The sequences of biochemically characterized cytochromes c¢ were obtained from a database: (2)

locus tag of Alvin_2765 of Allochromatium vinosum DSM180; (3) accession number P00138 of Achromobacter xylosoxidans NCIMB11015;

(4) locus tag of RSP_0474 of Rhodobacter sphaeroides 2.4.1. The consensus cytochrome c Cys–X–X–Cys–His heme-binding motif is close to

the C-terminus of each protein. Gaps in the alignment are indicated by dashes. Identical residues to those in PHCP are highlighted in gray.

Helical regions determined from the crystal structure of A. vinosum cytochrome c¢ (AVCP) are underlined. A residue occupying the empty

sixth ligand to the heme iron and hydrophobic residues in contact with the heme in the AVCP structure are indicated by asterisks above the

sequence (see details in the Discussion section).

H. Inoue et al. Biogenesis of cytochrome c¢


null mutant strain RI242, whereas the PH c552 protein

was not (Fig. 4). The isogenic wild-type E. coli RI89

strain with the intact DsbD protein was able to heter-

ologously synthesize the PHCP and PH c552 proteins,

confirming that the observed difference between

the two proteins in the RI242 strain is a result of the

absence of the DsbD protein.

PHCP was also synthesized as a holo-protein in the

periplasm of aerobically growing E. coli JCB387 cells

not harboring the pEC86 plasmid carrying the ccm

genes (Fig. 4). In contrast, when it did not harbor the

plasmid, the E. coli JCB387 strain was not able to

produce PH c552 aerobically. These results indicate that

the present growth conditions in the absence of pEC86

do not confer the cytochrome c biogenesis ability to

the PH c552 protein. This is possibly a result of the

shortage of Ccm proteins, because the expression of

ccm genes is repressed under aerobic growth condi-

tions.

In the presence of the pEC86 plasmid, both the

PHCP and PH c552 proteins were heterologously syn-

thesized in the periplasm of E. coli JCB387 cells

(Fig. 4). Judging from the staining intensity, the level

of production of the PHCP protein in the presence of

the pEC86 plasmid was significantly lower than that

without the plasmid, indicating that the co-expression

of plasmid-borne ccm genes represses PHCP overpro-

duction by aerobically growing E. coli cells. A similar

difference in the PHCP production level was observed

in the early and late logarithmic and stationary phases

of E. coli JCB387 cells with and without the pEC86

plasmid.

Spectral properties of PHCP heterologously

synthesized by E. coli

The visible absorption spectra of periplasmic extracts

containing the PHCP protein heterologously synthe-

sized by E. coli RI242 and JCB387 without pEC86

were the same as those observed for the authentic

Wavelength (nm)

Oxidized PHCP

Reduced PHCPA

bsor

banc

e (A

.U.)

300 400

425

630

500 600 7000.0

0.1

0.2

0.3

0.4

0.5

0.6

200 220 240 260

–20

–10

0

10

20

30

40

[θ] ×

10–3

(deg

·cm

2 ·dm

ol–1

)

Wavelength (nm)

222

B

A

Fig. 3. Spectral analysis of the authentic Hydrogenophilus thermo-

luteolus cytochrome c¢ (PHCP) protein: (A) visible absorption spec-

tra; (B) CD spectra. Specific wavelengths referred to in the text are

indicated by arrows in (A) and (B).

20

–pEC86

c' c'c552 c552 c' c552 c' c552

Mw (kDa)

RI242 JCB387

15

10

*

5

RI89

+pEC86

Fig. 4. Heterologous synthesis of cytochromes c by Escherichia

coli strains. Periplasmic extracts (equivalent to 5 · 108 cells) of the

E. coli RI242 and RI89 strains, and the JCB387 strain without (indi-

cated by –) or with (indicated by +) the pEC86 plasmid carrying the

ccm genes, were analyzed by heme staining after SDS ⁄ PAGE. In

each lane of the gel, periplasmic extracts from the E. coli cells

transformed with the Hydrogenophilus thermoluteolus cyto-

chrome c¢ (PHCP) and H. thermoluteolus cytochrome c552 (PH c552)

genes are indicated as c¢ and c552, respectively. The arrow and

arrowhead indicate the positions of the PHCP and PH c552 proteins,

respectively. The band denoted by the asterisk on the right-hand

side is the result of nonspecific staining of the extracts containing

the PH c552 protein.



purified protein in both the oxidized and reduced

states (Fig. 3A). These findings indicate that the heme

is correctly incorporated into the apo-form of PHCP

heterologously synthesized by E. coli, even without the

DsbD protein and without co-expression of the ccm

genes from the pEC86 plasmid. In addition, the

a-band in the pyridine hemochrome spectra of the

same periplasmic extracts with dithionite corresponded

to 550 nm, as observed for the authentic PHCP pro-

tein, indicating the covalent attachment of the heme to

the protein through two thioether bonds.

Discussion

In this study, we attempted to determine whether or

not Ambler’s class II cytochromes c¢ are synthesized

by the System I cytochrome c biogenesis machinery.

For this purpose, we first performed spectral analysis

of the authentic PHCP protein, aiming at the predic-

tion of its structure, which is the final state of biogene-

sis. Secondly, the PHCP gene was cloned to gain

sequence information and to examine its heterologous

expression in E. coli strains with reference to PH c552,

which has been characterized as a System I-dependent

cytochrome c.

Spectral properties of the authentic PHCP protein

The visible absorption and CD spectral features of the

PHCP protein indicate that its local heme environment

and helical content are similar to those found in typi-

cal cytochromes c¢. In the four-helix bundle structure

of general cytochromes c¢, access to the sixth ligand

position with regard to the heme iron is hindered

primarily by the side-chains of aromatic or nonaromat-

ic hydrophobic residues. Such a responsible residue is

Tyr16 in the crystal structure of the AVCP protein

[16]. The same residue is also conserved in the PHCP

protein (Fig. 2).

Other residues responsible for the maintenance of

the hydrophobic environment around the heme in

AVCP are Met19, Gly20, Met23, Tyr61, Val76,

Phe80, Val87, Val95 and Val120 (PHCP numbering,

Fig. 2), which directly face the heme group [16]. Of

these nine residues, seven are identical in PHCP, the

other two, Gly20 and Val76, in AVCP being homolo-

gously replaced by Ala20 and Leu76, respectively, in

PHCP. These sequence similarities, together with the

spectral properties observed for the PHCP and AVCP

proteins, indicate that the former has a three-dimen-

sional structure comprising a four-helix bundle, as

demonstrated for other cytochromes c¢, including the

latter.

Heterologous synthesis of PHCP by E. coli

The E. coli System I cytochrome c biogenesis machin-

ery, consisting of the Dsb and Ccm proteins, is respon-

sible for the synthesis of class I cytochromes c even

from various exogenous sources [6]. Normally, the

E. coli chromosomal ccm genes are not aerobically

expressed. Therefore, through co-expression of the ccm

genes in the pEC86 plasmid, together with various

class I cytochrome c genes, holo-cytochromes c can be

successfully overproduced by aerobically growing

E. coli cells. In previous studies, it has been shown that

co-expression of the ccm genes in the pEC86 plasmid

is required for the heterologous expression of class II

cytochromes c¢ by E. coli [7–9], predicting that cyto-

chrome c¢ biogenesis is System I dependent. However,

systematic studies on the effects of the ccm and dsb

genes with reference controls have not been performed.

It is clear from our results that the co-expression of

the ccm genes in the pEC86 plasmid and the presence

of the DsbD protein are not necessarily required for

the heterologous synthesis of the PHCP protein by

E. coli, unlike that of class I cytochromes c, including

the PH c552 protein.

Similarity to and differences from periplasmic

EC b562

Previously, the c-type heme-binding Cys–X–X–Cys–

His motif was introduced into periplasmic EC b562 in

order to determine whether or not the resulting variant

is synthesized as a holo-protein with a covalently

bound heme [10]. Even without the DsbD protein or

without co-expression of the ccm genes in a plasmid,

the EC b562 variant can be formed as a holo-protein

with a covalently bound heme [11]. Although the pro-

duction level and heme-binding mode of the EC b562variant under these conditions differ from those with

the dsbD gene product or with the co-expression of the

ccm genes in a plasmid, holo-protein synthesis clearly

occurs with such an imperfect System I cytochrome c

biogenesis machinery. Therefore, the EC b562 variant

resembles the PHCP protein in terms of biogene-

sis, which is different from System I cytochrome c

biogenesis.

The above EC b562 variant with the c-type heme-bind-

ing Cys–X–X–Cys–His motif was further modified so as

to add extra Cys residues around the motif. The result-

ing variants were examined for heterologous synthesis

by E. coli JCB387 with or without the pEC86 plasmid, it

being shown that co-expression of the ccm genes in the

plasmid caused enhanced levels of production of the

variants [17]. These observations are not consistent with



those in the present study, in which the co-expression of

the ccm genes in the pEC86 plasmid was found to result

in a low level of production of PHCP (Fig. 4). There is

presently no explanation as to why the production levels

differ between the PHCP protein and the EC b562variant. Further experiments on the two proteins with

the same growth medium and aerobicity are required for

a clear comparison, which will provide information on

the function of Ccm proteins.

Structural implication for PHCP synthesis

Although the sequence identity is low between the

PHCP protein and the EC b562 variant, they may have

the same architecture, comprising a four-helix bundle

structure, indicating that their folding mechanisms,

including heme attachment, are conserved, as sug-

gested previously [18]. A large portion of the EC b562protein can fold in the absence of heme to yield its

apo-form with an empty heme-binding site [19]. Should

such a folding process in apo-EC b562 also occur in

apo-PHCP, the latter protein may incorporate free

heme, which is then spontaneously bound in a

System I-independent manner. Although no direct evi-

dence for this is available, hydrophobic interactions

within apo-PHCP may facilitate protein folding in the

absence of heme, as observed for Aquifex aeolicus

class I cytochrome c555, whose apo-form is exception-

ally folded [20,21]. It would be of interest to investi-

gate further the biogenesis mechanism for PHCP with

regard to the relation to its structural features in

conjunction with a mutagenesis study.

Materials and methods

Purification of PHCP from H. thermoluteolus

Hydrogenophilus thermoluteolus TH-1 [22] was cultured at

45 �C in an inorganic medium under H2 : O2 : CO2

(75 : 15 : 10). The constituents of this medium have been

given previously [23]. The H. thermoluteolus cells (30 g wet

weight) were resuspended in 210 mL of 10 mm Tris ⁄HCl

(pH 8.0). The cells were then disrupted with a French pres-

sure cell, followed by centrifugation (200 000 g) to obtain a

total soluble extract.

The resulting soluble extract was dialyzed against 10 mm

Tris ⁄HCl (pH 8.0) at 4 �C, and then loaded onto a Hi-

Trap Q anion-exchange column (diameter, 1.4 cm; height,

3 cm; GE Healthcare, Tokyo, Japan) that had been equili-

brated with 10 mm Tris ⁄HCl (pH 8.0). Batch elution was

carried out with 50 mL of the same buffer containing 0, 0.2

or 1.0 m NaCl, a red-colored fraction containing the PHCP

protein being eluted with 0.2 m NaCl. The red-colored

fraction was further dialyzed against 10 mm Tris ⁄HCl

(pH 8.0), and then loaded onto the same column that had

been equilibrated with the same buffer. Proteins were eluted

with a linear gradient of NaCl (0–0.2 m). The resulting red

fraction was dialyzed against 25 mm sodium acetate

(pH 5.5), and then loaded onto a HiTrap SP cation-

exchange column (diameter, 1.4 cm; height, 3 cm; GE

Healthcare) that had been equilibrated with the same buf-

fer. The fraction containing the PHCP protein flowed

through, and was finally separated by gel filtration on a

column of Sephadex 75 (diameter, 1.6 cm; height, 60 cm;

GE Healthcare) that had been equilibrated with 25 mm

sodium acetate (pH 5.5).

Characterization of the purified PHCP protein

Protein purity during the column chromatography steps

was checked by SDS ⁄PAGE and staining with Coomassie

Brilliant Blue. The gels were also subjected to heme stain-

ing, proteins with covalently bound heme being stained to

detect cytochrome c specifically [24]. The band correspond-

ing to the PHCP protein on a gel was blotted onto a polyv-

inylidene fluoride membrane (Millipore, Tokyo, Japan) for

direct protein sequencing analysis with an automatic

protein sequencer (Applied Biosystems, Tokyo, Japan). The

protein concentrations of the crude extracts were deter-

mined with a protein assay kit (Bio-Rad, Tokyo, Japan)

with bovine serum albumin as a standard. For the purified

PHCP protein, the concentrations were determined spectro-

photometrically using the extinction coefficient at 205 nm

caused by the peptide bond [25].

Visible absorption and CD spectra of the purified

authentic PHCP protein in 10 mm potassium phosphate

buffer (pH 7.0) were obtained with JASCO V-530 and

JASCO J-820 spectrometers, respectively, at 25 �C. The

PHCP protein was air oxidized or reduced with a grain of

sodium dithionite. The protein concentrations were 6 and

20 lm for visible absorption and CD spectral analysis,

respectively. Pyridine hemochrome spectra were obtained

according to the method described by Bartsch [26].

Isolation of full-length DNA encoding the PHCP

protein

In order to clone the PHCP gene and to determine the

complete DNA sequence, we used the PCR method. From

N-terminal sequence information on the PHCP protein up

to the 30th residue, we designed 512 mixed forward primers

(PHcp01fw) corresponding to the resulting PHCP protein

sequence Glu-Asp-Lys-Val-Lys-Phe-Arg-Glu-Ala (5th to

14th residues of the mature PHCP sequence, see Fig. 2),

and 18 432 mixed reverse primers (PHcp01rv) correspond-

ing to the well-conserved cytochrome c¢ sequence Cys-Lys-

Ala-Cys-His-Asp-X-Tyr-Arg (124th to 132nd residues in

the case of PHCP, Fig. 2; X denotes any residue), and used



them to amplify H. thermoluteolus chromosomal DNA with

Ex Taq polymerase (Takara, Shiga, Japan). The DNA frag-

ment obtained from PCR was sequenced and found to code

a part of the PHCP protein.

We next used the inverse PCR method to obtain the entire

PHCP gene. DNA fragments that had been prepared by

digestion of H. thermoluteolus chromosomal DNA with sev-

eral restriction enzymes separately were self-ligated and then

used as the first PCR templates with a gene-specific reverse

primer, PHcp03rv, corresponding to the PHCP protein

sequence of the 31st to 40th residues (Fig. 2), and a gene-spe-

cific forward primer, PHcp03fw, corresponding to the

sequence of the 45th to 53rd residues. The resulting PCR

products were then used as the second PCR templates with a

gene-specific nested reverse primer, PHcp04rv, correspond-

ing to the protein sequence of the 27th to 31st residues, and

a gene-specific nested forward primer, PHcp04fw, corre-

sponding to the sequence of the 106th to 111th residues.

Heterologous synthesis of the PHCP and PH c552

proteins by E. coli

Escherichia coli DH5a was used for the maintenance and

propagation of all plasmids. The E. coli RI89, RI242 and

JCB387 strains were examined with regard to the synthesis

of exogenous PHCP and PH c552 proteins. The RI89 strain

is a parental strain of RI242, which is a dsbD null mutant

[27], and the JCB387 strain is usually used for heterologous

synthesis of cytochromes c in our laboratory [28]. These

strains were transformed with pKK223-3 derivatives carry-

ing the PHCP or PH c552 gene (ampicillin resistance). The

original signal sequence of PHCP was replaced with that of

Pseudomonas aeruginosa cytochrome c551 to target the

PHCP apo-protein to the E. coli periplasm by the PCR

method described previously for PH c552 [12]. The resulting

PHCP gene was flanked by artificially introduced restriction

sites (EcoRI, 5¢ and SalI, 3¢), and then inserted into the

corresponding sites of pKK223-3. The E. coli JCB387 strain

was further co-transformed with pEC86 [29], which carries

the E. coli cytochrome c maturation genes ccmABCDEFGH

(chloramphenicol resistance).

The transformed E. coli RI89, RI242 and JCB387 cells

were grown in LB liquid medium containing appropriate

antibiotics overnight at 37 �C. The resulting precultures of

RI89 and RI242 cells were each inoculated into 50 mL of

minimal medium supplemented with 0.4% (v ⁄ v) glycerol asa carbon source, and with nitrite and fumarate as

substrates for respiration, in a screw capped bottle, which

was then incubated anaerobically for 24 h at 37 �C [30].

The preculture of the JCB387 strain was inoculated into

20 mL of the same minimal medium supplemented with

0.4% (v ⁄ v) glycerol in a 50-mL flask, which was then incu-

bated aerobically for 16 h at 37 �C [28]. The growing

E. coli cells at the late logarithmic phase were harvested.

Periplasmic extracts of these cells were obtained by the cold

osmotic shock method [31], and then subjected to

SDS ⁄PAGE, followed by heme staining of the gels in order

to detect holo-cytochromes c [24]. The same extracts were

subjected to visible absorption spectral analysis, as carried

out for the purified PHCP protein.

Reagents

Restriction enzymes, T4 DNA ligase and other reagents for

DNA handling were purchased from Takara. All other

chemicals used were of the highest grade commercially

available.

Acknowledgements

We wish to thank D. Miyake, R. Sano and S. Fujii

(Hiroshima University) for technical assistance. This

work was partly supported by a Grant-in-Aid for

Scientific Research on Innovative Areas (No.

20118005) from the Ministry of Education, Culture,

Sports, Science and Technology of Japan.

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