Báo cáo khoa học: In vivo production of catalase containing haem analogues pot

In vivo production of catalase containing haem analogues
Myriam Brugna*, Lena Tasse and Lars Hederstedt
Microbiology Group, Department of Biology, Lund University, Sweden
Introduction
Haem is present in most organisms as the prosthetic
group of proteins such as cytochromes, oxygenases,
haemoglobins, and catalases [1]. The versatile chemical
reactivity of the iron ion is expanded in the haem
molecule, giving the wide functional range of haem
proteins [2].
The physicochemical properties of the haem mole-
cule are, however, troublesome for cells [3]. At neutral
pH, haem is an amphiphilic, poorly water-soluble
molecule. In the reduced state, in the presence of
molecular oxygen, haem is also a potent catalyst for
reactive oxygen species formation. These toxic effects
must be avoided when haem is transported and stored
within cells. Haem, synthesized within the cell or
acquired from the environment, and destined to finally
be a prosthetic group of a haem protein, is therefore
Keywords
antibacterial agents; catalase;
Enterococcus faecalis; haem protein;
metal porphyrins
Correspondence
L. Hederstedt, Department of Biology,
Biology Building, Lund University,
So
¨
lvegatan 35, SE-22362 Lund, Sweden
Fax: +46 46 222 41 13

Tel: +46 46 222 86 22
E-mail:
Present address
*Laboratoire de Bioe
´
nerge
´
tique et Inge
´
nierie
des Prote
´
ines, Institut de la Me
´
diterrane
´
e,
CNRS, Marseille cedex 20, France; Univer-
site
´
de Provence, 3 Place Victor Hugo,
Marseille cedex 3, France

Laboratoire des Interactions Plantes-Micro-
organismes, UMR INRA-CNRS, Chemin de
Borde-Rouge BP 52627, Castanet-Tolosan,
France
(Received 4 March 2010, revised 31 March
2010, accepted 9 April 2010)
doi:10.1111/j.1742-4658.2010.07677.x

Haem (protohaem IX) analogues are toxic compounds and have been con-
sidered for use as antibacterial agents, but the primary mechanism behind
their toxicity has not been demonstrated. Using the haem protein catalase
in the Gram-positive bacterium Enterococcus faecalis as an experimental
system, we show that a variety of haem analogues can be taken up by bac-
terial cells and incorporated into haem-dependent enzymes. The resulting
cofactor-substituted proteins are dysfunctional, generally resulting in
arrested cell growth or death. This largely explains the cell toxicity of haem
analogues. In contrast to many other organisms, E. faecalis does not
depend on haem for growth, and therefore resists the toxicity of many
haem analogues. We have exploited this feature to establish a bacterial
in vivo system for the production of cofactor-substituted haem protein vari-
ants. As a pilot study, we produced, isolated and analysed novel catalase
variants in which the iron atom of the haem prosthetic group is replaced
by other metals, i.e. cobalt, gallium, tin, and zinc, and also variants con-
taining meso-protoheme IX, ruthenium meso-protoporphyrin IX and
(metal-free) protoporphyrin IX. Engineered haem proteins of this type are
of potential use within basic research and the biotechnical industry.
Structured digital abstract
l
MINT-7722358, MINT-7722368: katA (uniprotkb:Q834P5) and katA (uniprotkb:Q834P5)
physically interact (
MI:0915)bycopurification (MI:0025)
Abbreviations
Co-PP, cobalt protoporphyrin IX; Cu-PP, copper protoporphyrin IX; Fe-meso, iron meso-protoporphyrin; Fe-PP, protohaem IX; Ga-PP, gallium
protoporphyrin IX; Mg-PP, magnesium protoporphyrin IX; MIC, minimal inhibitory concentration; Ni-PP, nickel protoporphyrin IX; Pd-meso,
palladium meso-protoporphyrin IX; PP, protoporphyrin IX; Ru-meso, ruthenium meso-protoporphyrin IX; Sn-PP, tin protoporphyrin IX;
Zn-PP, zinc protoporphyrin IX.
FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 2663
most likely transiently bound to specific protein factors

in cells. Little is known about intracellular haem trans-
port and how haem is incorporated into proteins
in vivo to form haem proteins in various subcellular
locations [4].
Haem analogues, with a metal ion other than iron
or with structural alterations in the porphyrin macro-
cycle, are generally toxic to cells, and have been con-
sidered for use as antibacterial agents [5]. The
molecular basis for the toxicity of haem analogues and
the resistance of some bacteria against such com-
pounds have not been fully explained. In this work, we
have investigated the potential of the Gram-positive
bacterium Enterococcus faecalis to import different
haem analogues from the growth medium and incorpo-
rate these into catalase apo-protein in the cytoplasm to
form substituted catalase. This research was performed
to find the explanation for the general toxicity of haem
analogues, so that we can better judge these types of
compound as potential antimicrobial and antitumour
drugs. A second purpose was to increase our know-
ledge of the mechanisms of the uptake and intracellu-
lar transport of haem. A third aim was to establish an
in vivo system for the production of structurally
complex haem proteins containing synthetic metallo-
porphyrins.
Six features of E. faecalis make this bacterium very
suitable for our investigations: (a) the bacterium can
take up haem [6] and a variety of haem analogues
from the growth medium (this work); (b) as E. faecalis
is a Gram-positive bacterium, there is, unlike in Gram-

negative bacteria such as Escherichia coli, no outer
membrane constituting a possible barrier for the
uptake of porphyrin compounds; (c) E. faecalis does
not synthesize haem; (d) E. faecalis does not depend
on haem for growth, and grows well also without
haem; (e) if supplied with haem, the bacterium is
capable of aerobic respiration [7–10] and produces
two haem proteins – a membrane-bound cytochrome
bd respiratory quinol oxidase [7], and a cytoplasmic
monofunctional catalase [6]; and (f) the growth of
many bacteria is inhibited by noniron metalloporphy-
rins, but E. faecalis strains are generally resistant to
this type of compound ([11] and this work).
Catalase (EC 1.11.1.6) is present in most aerobic
organisms, and serves in part to protect the cells from
the toxic effects of hydrogen peroxide by catalysing its
decomposition into O
2
and H
2
O [12,13]. E. faecalis
catalase is a typical monofunctional catalase [14]. It is
a homotetrameric enzyme containing one proto-
haem IX (Fe-PP) molecule per KatA polypeptide of
478 amino acids. The structure of the E. faecalis
enzyme is rather complex (Fig. S1). Each monomer
has an extended arm-like N-terminal domain and a
major globular C-terminal domain, with the haem-con-
taining active site deeply buried in a b-barrel structure.
The four subunits are tightly associated with the N-ter-

minal domain of one monomer woven into the globu-
lar domain of the neighbouring subunit. Attempts to
accomplish the assembly of catalase in vitro from its
constituents, (polypeptide and Fe-PP) have so far
failed [15].
Results
Susceptibility of E. faecalis to haem analogues
The recombinant E. faecalis strain V583 ⁄ pLUF15 used
in this work carries, on a plasmid, a variant of the
E. faecalis katA gene, resulting in overproduction of
hexahistidyl-tagged KatA polypeptide [6]. The tag
allows catalase to be purified from disrupted cells in a
single affinity chromatographic step.
Production of catalase in E. faecalis requires haem
in the growth medium. In a previous study, we found
that the maximal amount of catalase in E. faecalis
V583 ⁄ pLUF15 is obtained in the presence of about
10 lm haemin [6]. Stojiljkovic et al. [11] reported that
growth of E. faecalis cells is resistant to 17 different
tested noniron porphyrins, but minimal inhibitory con-
centrations (MICs) for the different compounds were
not provided. To determine the relative toxicities of
haem analogues and find the concentrations at which
E. faecalis would grow well, we cultivated E. faecal-
is V583 ⁄ pLUF15 in TSBG (a haem-free medium) sup-
plemented with different concentrations of the
compounds of interest. The MIC value obtained
for the metal-substituted haem analogues cobalt proto-
porphyrin IX (Co-PP), copper protoporphyrin IX
(Cu-PP), Fe-PP, gallium protoporphyrin IX (Ga-PP),

magnesium protoporphyrin IX (Mg-PP), nickel proto-
porphyrin IX (Ni-PP), and zinc protoporphyrin IX
(Zn-PP), and for protoporphyrin IX (PP), was
> 150 lm, and for tin protoporphyrin IX (Sn-PP)
it was > 130 lm, i.e., these compounds are not
toxic to E. faecalis. Ruthenium meso-protoporphyrin
IX (Ru-meso) and iron meso-protoporphyrin IX
(Fe-meso) were found to be somewhat toxic, and palla-
dium meso-protoporphyrin IX (Pd-meso) very toxic
(Table 1).
In vivo synthesis of gallium-substituted catalase
Ga
3+
and Fe
3+
are similar in size and form a very
stable complex with PP. In contrast to iron, the gallium
ion is not oxidized or reduced under physiological
Cofactor-substituted catalase M. Brugna et al.
2664 FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS
conditions. Ga-PP is very toxic to Gram-negative
bacteria (MIC < 2 lm) and some Gram-positive bacte-
ria such as Staphylococcus species (MIC < 3 lm) [11].
Ga-PP seems to be transported into Gram-negative
bacteria via haem uptake systems [11]. The toxicity of
Ga-PP has been suggested to result from the incorpora-
tion of this haem analogue into haem proteins, which
thereby become nonfunctional, but this has not been
experimentally demonstrated [5]. Reasons for the
resistance of E. faecalis to Ga-PP (Table 1) could be

that this haem analogue is not taken up by the bacte-
rium, or that Ga-PP is incorporated into haem proteins
but has no drastic effect because E. faecalis cells are
not dependent on haem proteins for growth.
To investigate whether E. faecalis cells can take up
haem analogues from the growth medium and incorpo-
rate them into protein in the cytoplasm, we first tested
whether catalase substituted with Ga-PP can be pro-
duced. E. faecalis V583 ⁄ pLUF15 was grown in the
presence of 8 lm Ga-PP, and in parallel in the absence
of any porphyrin compound and in the presence of
8 lm Fe-PP, respectively, as controls. The cytoplasmic
fraction was isolated from the cells and analysed for
KatA polypeptide by immunoblot (Fig. 1). We have
previously shown that KatA protein is only found in
cytoplasmic extracts of E. faecalis if the prosthetic
group has been incorporated [6]. In its absence, the
KatA polypeptide is probably not completely folded
and is therefore degraded. The cytoplasmic fraction
from E. faecalis V583 ⁄ pLUF15 grown in the absence
of a porphyrin compound completely lacked KatA
antigen, as expected (Fig. 1, lane B). Extracts from
cells grown in the presence of Fe-PP (Fig. 1, lane C)
or Ga-PP (lane I) contained KatA. These results sug-
gested that Ga-PP is incorporated into the KatA poly-
peptide to form a gallium-substituted catalase protein
(Ga-PP-KatA).
Purification and characterization of Ga-PP-KatA
The His
6

-tagged iron-containing (Fe-PP-KatA) and
Ga-PP-KatA catalases were purified from cell-free
extracts by using metal affinity chromatography. The
purity of preparations was evaluated by SDS ⁄ PAGE,
which, for both Fe-PP-KatA and Ga-PP-KatA,
showed one polypeptide band corresponding to KatA
with an apparent molecular mass of 54 kDa (Fig. 2).
In the case of Ga-PP-KatA, an additional protein
band of about 110 kDa was observed. This band cor-
responds to KatA dimer, as determined by immuno-
blot analysis (Fig. 1, lane I).
Amino acid analysis of preparations of isolated
Fe-PP-KatA and Ga-PP-KatA confirmed the composi-
tion of KatA polypeptide as deduced from the katA
Table 1. Toxicity of porphyrins, porphyrin concentrations used for
growth and presence of catalase protein in E. faecalis V583 ⁄
pLUF15. ND, not done.
Porphyrin added
to the growth
medium MIC (l
M)
Porphyrin
concentration
used in the
growth medium
(lM)
a
KatA
polypeptide
present

b
Co-PP > 150 8 +
Cu-PP > 150 8 +
Fe-PP > 150 8 +
Ga-PP > 150 8 +
Mg-PP > 150 2 +
Ni-PP > 150 8 )
PP > 150 9 +
Sn-PP > 130 7 +
Zn-PP > 150 8 +
Fe-meso < 15 8 +
Pd-meso < 1.5 ND ND
Ru-meso 4 1 +
a
Concentration used in the growth medium for production of KatA.
b
As determined by immunoblot with cell extracts (see Fig. 1).
Fig. 1. KatA immunoblot of cytoplasmic fraction from E. faecal-
is V583 ⁄ pLUF15 grown in TSBG medium (lane B) or TSBG medium
supplemented with the indicated porphyrin compounds
(lanes C–M). The concentrations of porphyrin used in the growth
medium are provided in Table 1. Lanes A and N each contained
80 ng of purified haem-containing E. faecalis catalase (Fe-PP-KatA).
Lanes B–M each contained 2 lg of total cytoplasmic protein.
Fig. 2. SDS ⁄ PAGE of preparations of isolated E. faecalis normal
and gallium-substituted catalase. Lane A: molecular mass markers
(kDa). Lane B: 2 lg of Fe-PP-KatA. Lane C: 1 lg of Ga-PP-KatA.
The gel was stained for protein with Coomassie brilliant blue.
M. Brugna et al. Cofactor-substituted catalase
FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 2665

sequence, and was used for quantitative determination
of KatA protein. Metal analysis combined with the
protein analysis showed the presence of 1.12 mol of
iron atoms per mol of KatA polypeptide in Fe-PP-
KatA. Isolated Ga-PP-KatA contained 0.96 mol of
gallium per mol of KatA polypeptide, and only trace
amounts of iron (Table 2).
Porphyrin present in Fe-PP-KatA and Ga-PP-KatA
was extracted from the isolated catalase proteins by
using acid ⁄ acetone, and analysed by RP-HPLC. The
porphyrin of Ga-PP-KatA eluted as a single peak at
7.0 min. Reference Ga-PP had the same retention time.
Fe-PP and the porphyrin extracted from Fe-PP-KatA
both eluted at 8.5 min. This showed that Fe-PP and
Ga-PP are present in the isolated proteins, and
excluded the possibility that these metalloporphyrins
added to the culture are modified during transport into
E. faecalis or after being incorporated into KatA poly-
peptide, as is the case for catalase HPII of E. coli [16].
Enzymatic and spectroscopic properties of
Ga-PP-KatA
Isolated Ga-PP-KatA showed less than 1% catalase
activity as compared with Fe-PP-KatA (Table 2).
Enzyme activity measurement with the cytoplasmic cell
fraction from E. faecalis V583 ⁄ pLUF15 containing
Ga-PP-KatA as compared with that containing Fe-PP-
KatA showed the same relative results (data not
shown). This confirmed that Ga-PP-KatA is essentially
inactive, and that this is not due to inactivation during
isolation of the protein.

Light absorption spectra of the purified iron-con-
taining and gallium-containing catalases are presented
in Fig. 3A,B. Fe-PP-KatA showed a Soret peak at
406 nm and weak absorption bands at 504, 541 and
625 nm. These features are characteristic for haem-
containing catalases [17]. Ga-PP-KatA showed a very
different spectrum, with the Soret peak at 422 nm and
distinct absorption maxima at 548 and 588 nm.
Screening for in vivo production of
cofactor-substituted catalase
The results obtained with Ga-PP and the properties of
Ga-PP-KatA demonstrated that E. faecalis cells can
take up a haem analogue from the growth medium
and incorporate it into KatA polypeptide to form
cofactor-substituted catalase. To determine whether
this is a general property, we grew E. faecalis
V583 ⁄ pLUF15 cells in the presence of Co-PP, Cu-PP,
Mg-PP, Ni-PP, Sn-PP, and Zn-PP. The bacteria were
also grown in the presence of Fe-meso, Ru-meso, and
PP. The concentrations of porphyrins used in the
growth medium are given in Table 1. In the case of
Ru-meso, a low concentration had to be used because
of the toxicity of this compound.
Production of catalase was determined by immuno-
blot analysis of cytoplasmic fractions (Fig. 1; Table 1).
KatA protein was obtained with all of the porphyrins
tested, except for Ni-PP, indicating that various haem
analogues can be inserted into the protein.
The catalase proteins were isolated by the same
procedure as used for Fe-PP-KatA and Ga-PP-KatA.

The resulting preparations were pure or contai-
ned some contaminating proteins (in the cases of
Co-PP-KatA, Sn-PP-KatA, and Fe-meso-KatA) as
evaluated by SDS ⁄ PAGE (gel not shown). The cata-
lase activities of purified proteins are presented in
Table 2. Fe-meso-KatA showed 35% activity as
Table 2. Properties of isolated normal and cofactor-substituted catalases. ND, not done.
Variant
Porphyrin
present
a
Metal content
(mol ⁄ mol KatA)
PP content
b
(mol ⁄ mol KatA)
Relative
activity
c
(%)
Co-PP-KatA ? Co, 0.70; Fe, 0.001 ND < 1
Cu-PP-KatA PP Cu, 0.04; Fe, < 0.01 1.1 2
Fe-PP-KatA Fe-PP Fe, 1.12 ND 100
Ga-PP-KatA Ga-PP Ga, 0.96; Fe, < 0.01 ND < 1
Mg-PP-KatA PP Mg, 0.02; Fe, 0.08 0.7 < 1
d
PP-KatA PP Fe, 0.02 1.2 2
Sn-PP-KatA Sn-PP Sn, 0.76; Fe, 0.001 ND < 1
Zn-PP-KatA ? and PP Zn, 0.58; Fe, 0.02 0.075 3
Fe-meso-KatA Fe-meso Fe, 0.65 ND 35

Ru-meso-KatA ? Ru, 0.49; Fe, 0.05 ND < 1
a
Porphyrin found in isolated catalase protein as determined by HPLC, light absorption spectroscopy, and fluorometry. A question mark indi-
cates that the identity of the porphyrin(s) has not been established.
b
Protoporphyrin content determined by fluorescence measurements.
ND, not done.
c
Specific enzyme activity with hydrogen peroxide as substrate relative to that of Fe-PP-KatA.
d
For some preparations, we
found higher activity (up to 7%).
Cofactor-substituted catalase M. Brugna et al.
2666 FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS
compared with Fe-PP-KatA. The other proteins lacked
detectable activity or showed very low activity.
Covalently bound KatA dimers (in addition to
monomeric KatA) were found in preparations of iso-
lated cofactor-substituted catalases (Fig. 2) and also in
those of isolated Fe-PP-KatA that had been stored at
4 °C for several weeks. In most cases, the dimers did
not constitute more than 20% of the total KatA, but
for Ga-PP-KatA and Sn-PP-KatA, they could repre-
sent a major form of the protein. Dimers were not
observed in the case of Co-PP-KatA and Fe-meso-
KatA (Fig. 1, lanes E and G). PP-KatA also formed
dimers, indicating that their formation is not
porphyrin metal-dependent. It has been reported that
lyophilization or storage of catalase in solution
enhances dimer formation [18]. Intermolecular disulfide

crosslinks are formed in porcine erythrocyte catalase
when the enzyme is stored at 4 °C for more than
1 week [19]. Disulfide bond formation can be excluded
in the case of E. faecalis catalase, because this protein
does not contain any cysteine, and proteins were
reduced before SDS ⁄ PAGE.
All isolated catalase variants were analysed for
cobalt, copper, iron, gallium, magnesium, ruthenium,
tin and zinc content. The major metal found in each
preparation was generally the same as that contained
in the metalloporphyrin added to the growth medium
(Table 2). Notable exceptions, however, were the prep-
arations of isolated Cu-PP-KatA and Mg-PP-KatA,
which contained only low amounts of metals; Cu-PP-
KatA contained 0.04 mol of copper per mol of KatA
polypeptide, and Mg-PP-KatA contained 0.02 mol of
magnesium per mol of KatA. Similarly, the prepara-
tion of isolated PP-KatA contained little metal,
< 0.08 mol of metal per mol of KatA polypeptide,
except for zinc, which was found at 0.13 mol per mol
of KatA. In the following, we deal separately with the
metal-substituted (Co-PP-KatA, Sn-PP-KatA, Zn-PP-
KatA, Fe-meso-KatA, and Ru-meso-KatA) and the
porphyrin-containing but metal-deprived (Cu-PP-
KatA, Mg-PP-KatA, and PP-KatA) catalases.
Characterization of metal-substituted catalases
Porphyrins present in the different preparations with
metal-substituted catalase were analysed by RP-HPLC.
For Fe-meso-KatA and Sn-PP-KatA, the chromato-
gram of the extracted porphyrin completely agreed

with that of the reference compound, i.e. Fe-meso
(retention time of 5.5 min) and Sn-PP (retention time
of 13 min), respectively. In the cases of Co-PP-KatA,
Zn-PP-KatA, and Ru-meso-KatA, the chromatograms
of the extracted porphyrins showed complex patterns
that did not entirely correspond to those of the refer-
ence compounds (data not shown). Some metallopor-
phyrins, e.g. Zn-PP, lose the metal ion under acidic
conditions, but this was not the reason for the com-
plexity observed in the chromatograms.
Light absorption spectra of purified Co-PP-KatA,
Sn-PP-KatA, Zn-PP-KatA and Ru-meso-KatA were
all different and distinct from that of Fe-PP-KatA
(Fig. 3A,B) (maxima at 430, 544 and 577 nm for
Co-PP-KatA, at 420, 551 and 590 nm for Sn-PP-KatA,
at 421, 554, 574, 629 and 670 nm for Zn-PP-KatA,
and at 404, 526, 559 and 677 nm for Ru-meso-KatA).
The spectrum of Fe-meso-KatA was similar to that of
Fe-PP-KatA, but with slightly shifted absorption
Fig. 3. Light absorption spectra of isolated normal and cofactor-
substituted catalases. Porphyrins added to the growth medium to
produce the various catalases are indicated. (A) The Soret band
region (380–470 nm). (B) The region from 500 to 700 nm. (C)
Spectra of catalases produced in the presence of Cu-PP, Mg-PP,
and PP. The intensities of these spectra have been normalized
with respect to the Soret band absorption. The inset in (C)
shows a magnified view of the spectra between 460 and
720 nm. The absorption scale in each panel is indicated by a
vertical bar. The proteins were in 50 m
M potassium phosphate

buffer (pH 8.0).
M. Brugna et al. Cofactor-substituted catalase
FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 2667
maxima (396, 499, 531 and 619 nm as compared with
406, 504, 541 and 625 nm).
The fluorescence emission spectrum of the porphyrin
contained in Zn-PP-KatA, in a pyridine⁄ water ⁄
NaOH ⁄ Tween-80 mixture, was not similar to that of
the reference solution of Zn-PP in the same solvent,
indicating that the bound compound is not Zn-PP
(Fig. 4). Moreover, fluorescence spectral analysis
showed that isolated Zn-PP-KatA contained a small
amount of PP (0.075 mol of PP per mol of KatA poly-
peptide) (Fig. 4; Table 2). Zn-PP-KatA contained
0.58 mol of zinc per mol of KatA polypeptide
(Table 2). These results, taken together, indicate that
zinc in Zn-PP-KatA is bound to a nonfluorescent,
unidentified porphyrin compound. This conclusion is
consistent with the light absorption spectrum of
Zn-PP-KatA, which presents features reminiscent of
the spectrum of PP-KatA (maxima at 554, 574 and
629 nm) (see next section) and features of the unidenti-
fied zinc porphyrin (maxima at 421 and 670 nm)
(Fig. 3A–C).
Characterization of catalase obtained with PP in
the growth medium
The HPLC chromatogram of porphyrin extracted from
PP-KatA showed one major species, with the same
retention time (20 min) as PP (data not shown). To
further characterize and analyse the amount of

porphyrin present in PP-KatA, the isolated protein
was diluted into a pyridine ⁄ water ⁄ NaOH ⁄ Tween-80
mixture to denature the protein and dissolve the por-
phyrin. The fluorescence emission spectrum of the
solution was identical to that of PP in the same solvent
(maximum at 632 nm) (Fig. 4). This confirmed the
HPLC data showing that PP-KatA contained PP. On
the basis of fluorescence measurements and compari-
son with standard solutions of PP, a stoichiometry of
approximately 1 mol of PP per mol of KatA polypep-
tide was found (Table 2). These results demonstrated
that PP (metal-free porphyrin) is taken up by the bac-
terial cell and incorporated into catalase protein.
The light absorption spectrum of the isolated PP-
KatA showed absorption maxima at 416, 517, 554, 574
and 628 nm (Fig. 3C). This spectrum has features very
similar to those described for Proteus mirabilis catalase
produced in E. coli, which contains a mixture of Fe-PP
and PP [20]. The X-ray crystal structure of this cata-
lase shows that PP can replace haem, and that this has
essentially no effect on the architecture of the active
site. PP has also been found bound to the chlorophyll
biosynthetic protein BchH of Rhodobacter capsulatus
expressed in E. coli [21].
Catalase obtained with Cu-PP and Mg-PP in the
growth medium
Unexpectedly, catalases produced during growth of
E. faecalis V583 ⁄ pLUF15 in the presence of Cu-PP or
Mg-PP (Fig. 1, lanes D and M) contained only trace
amounts of copper and magnesium (Table 2). Light

absorbance spectroscopy (Fig. 3C) and fluorescence
spectroscopy (Fig. 4) showed that PP was the major
porphyrin in the two proteins. Approximately 1 mol of
PP and 0.7 mol of PP per mol of KatA polypeptide
were found in Cu-PP-KatA and Mg-PP-KatA, respec-
tively (Table 2).
These findings suggested that the metal ion of the
porphyrin is removed during transport of the porphy-
rin from the medium to the catalase protein in the
cytoplasm. Alternatively, but less likely, the metal-
loporphyrin is incorporated into catalase and the metal
is subsequently lost from the protein, leaving the PP
bound to the protein.
Discussion
Our findings demonstrate, first, that the haem pros-
thetic group of catalase can be replaced by various
haem analogues. Moreover, they explain the general
cellular toxicity of noniron metalloporphyrins. They
also show the potential of the bacterium E. faecalis as
Fig. 4. Fluorescence emission spectra of PP, Mg-PP, Cu-PP, and
Zn-PP, and of the porphyrins contained in isolated PP-KatA, Mg-PP-
KatA, Cu-PP-KatA, and Zn-PP-KatA. The excitation wavelengths
used are indicated in Experimental procedures. Emission spectra
peak maxima are indicated by dotted lines. The solvent was pyri-
dine ⁄ water ⁄ NaOH ⁄ Tween-80 (see Experimental procedures for
details).
Cofactor-substituted catalase M. Brugna et al.
2668 FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS
an in vivo system for the production of cofactor-substi-
tuted haem proteins.

It is sometimes desirable to replace the metal of a
metalloporphyrin in a specific protein. For example,
investigations on the reaction mechanisms of haem-
containing enzymes and electron transfer in proteins
are greatly aided if the metal of the normal prosthetic
group can be substituted. The photosensitivity of, for
example, zinc, tin and magnesium porphyrins provides
a convenient way of initiating a reaction very quickly
[22]. Other reasons to modify the prosthetic group are
to search for proteins with novel properties suitable
for various biotechnical applications, e.g. the design of
sensors, and for structural analysis by, for example,
NMR, where Fe
3+
can strongly interfere with the
analysis through being paramagnetic [23].
The haem group of some water-soluble proteins can,
in vitro, be removed and reinserted, or substituted for
another metalloporphyrin, such as Zn-PP or Co-PP
[24]. The assembly of more complex haem proteins,
such as membrane-bound respiratory enzymes or cata-
lase, however, can, at present, generally not be accom-
plished in vitro. The lack of knowledge concerning the
biogenesis of haem proteins makes it difficult to design
experimental conditions under which haem in a com-
plex haem protein can be substituted in vitro. Various
approaches have been used to construct artificial haem
enzymes [25]. Woodward et al. [26] recently presented
a method for the incorporation of haem analogues into
protein using a haem-permeable E. coli strain that is

unable to biosynthesize haem. The usefulness of this
method is, however, restricted to compounds that are
not toxic to E. coli, thus excluding many metal-substi-
tuted porphyrins.
For the in vivo production of catalase containing
haem analogues, we grew E. faecalis strain V583 ⁄ -
pLUF15, which overproduces catalase polypeptide, in
the presence of the respective haem analogue in the
growth medium. Noniron metalloporphyrin com-
pounds are generally toxic to bacteria [11], but, with
the notable exception of Pd-meso, they do not inhibit
growth of E. faecalis (Table 1). This allowed us to pro-
duce and isolate catalases in which the normal iron
atom is replaced by other metals, i.e. cobalt, gallium,
tin, and zinc. We could also produce catalase proteins
containing Fe-meso and Ru-meso. Supplementation of
the haem-free growth medium with PP resulted, much
to our surprise, in PP-containing catalase, i.e. protein
containing a metal-free porphyrin group.
The hemH gene (Ef1989) of E. faecalis V583 appar-
ently encodes a ferrochelatase. Ferrochelatases catalyse
the last step in haem synthesis, i.e. the insertion of
ferrous iron into PP. As growth of bacteria in the
presence of PP did not result in catalase containing
Fe-PP, it appears that the hemH gene is not expressed,
or that the HemH protein lacks ferrochelatase activity
with PP. Lactococcus lactis and E. faecalis are closely
related bacteria. L. lactis contains a hemH gene (called
hemZ), and can apparently take up Fe-PP from the
growth medium. Indirect evidence with a HemZ-defi-

cient strain suggests that HemZ is a ferrochelatase [27].
Thus, an alternative explanation for our results
obtained with E. faecalis is that the hemH gene does
encode a functional and expressed ferrochelatase, but
that iron is not available in sufficient amounts in the
cell to allow Fe-PP synthesis. Supplementation of the
TSBG growth medium with both PP and iron chloride,
however, did not result in Fe-PP-KatA being found in
E. faecalis.
The molecular machinery responsible for staphylo-
coccal haem acquisition is encoded by the genes of two
distinct membrane-associated transport systems, the
iron-regulated surface determinant system, and the
haem transport system [28]. Iron-regulated surface
determinant system-like proteins are present in Bacillus
anthracis and Listeria monocytogenes, suggesting that
they function in haem uptake in these Gram-positive
pathogens [29]. The Gram-positive bacteria Streptococ-
cus pyogenes and Corynebacterium diphtheriae contain
the HmuTUV proteins for haem acquisition [30]. Hmu-
TUV proteins are similar to the proteins involved in
haem transport in Gram-negative bacteria, and are pro-
posed to be components of an ATP-binding cassette-
type transporter. E. faecalis V583 contains HmuTUV,
as indicated by the genome sequence, and possibly, in
parallel with other, as yet unknown, carriers, it might
transport both haem and haem analogues. If so, these
transport systems are rather promiscuous, much like
the E. faecalis catalase protein, in binding porphyrins.
It is not known how haem is transported intracellularly

in bacteria, and the mechanisms by which haem is
incorporated into soluble and membrane-bound pro-
teins inside cells have not been determined [4]. It is evi-
dent from our results that these putative transport
components also work with haem analogues. The toxic-
ity of noniron metallo-PPs for bacteria has been sug-
gested, but not previously demonstrated, to result from
these compounds being taken up into the cell and
incorporated into vital haem proteins instead of haem
[5]. We show here that haem analogues are indeed
taken up and can be incorporated into haem proteins,
resulting in their inactivation. Pd-meso and, to a lesser
extent, Ru-meso were found to be toxic also for
E. faecalis (Table 1). This toxicity might be connected
to meso-protoporphyrin IX, as the bacterium tolerated
Fe-PP better than Fe-meso.
M. Brugna et al. Cofactor-substituted catalase
FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS 2669
The semisynthetic catalases that we have produced
in the present investigation are metalloproteins of a
type hitherto not described. Our results show that the
E. faecalis cell can be used for the in vivo production
of novel haem proteins. Following the same general
approach, one may be able to generate a great variety
of complex soluble and membrane-bound substituted
haem proteins.
Experimental procedures
Bacterial strains and growth conditions
E. faecalis V583 cells containing plasmid pLUF15 (a deriv-
ative of plasmid pAM401 containing the katA–his

6
gene
under control of the native promoter) [6] was grown in
TSBG [15 gÆL
)1
tryptone, 5 gÆL
)1
soytone peptone (both
from Lab M, Bury, UK), 5 gÆL
)1
NaCl, 1% (w ⁄ v) glucose,
30 mm Mops (pH 7.4), and 5 mm potassium phosphate
buffer, pH 7.0]. TSBG contains less than 0.05 lM Fe-PP
[6]. Chloramphenicol was added to a final concentration of
20 mg ⁄ L. For the production of normal and metal-substi-
tuted His
6
-tagged catalases, the TSBG medium was supple-
mented with various porphyrins. Co-PP, Cu-PP, Ga-PP,
Mg-PP, Ni-PP, Sn-PP, Zn-PP, Fe-meso and Ru-meso were
purchased from Porphyrin Products (Logan, UT, USA)
and were considered to be pure. Haemin and PP were
obtained from Sigma Chem Co. Pd-meso was a kind
gift from S. Vinogradov (University of Pennsylvania,
Philadelphia). Porphyrins were dissolved in dimethylsulfox-
ide in the cases of Co-PP, Cu-PP, Ga-PP, Ni-PP, Sn-PP,
Zn-PP, Fe-meso, and Ru-meso, in Tween-80 [1.5% (w ⁄ v) in
water] in the cases of Mg-PP and PP, or in Tween-20
[12.5% (v ⁄ v) in an alkaline solution] in the case of Fe-PP.
Bacteria were grown at 37 °C under oxic conditions.

Preparative cultures were grown in 1 L portions in 5 L baf-
fled Erlenmeyer flasks at 200 r.p.m. in a rotary incubator.
Cultures with porphyrins added were protected from light
to avoid possible photoeffects. The cells were harvested by
centrifugation, at 5000 g for 30 min, when the cultures
reached late exponential growth phase as determined from
the attenuance measured at 600 nm (D600 nm).
Isolation of cytoplasmic cell fraction and
purification of catalase
The cytoplasmic fraction of E. faecalis cells was prepared
essentially as described previously [6], except in the case of
large-scale preparations. The cells from 3 L cultures were
suspended in 50 mm potassium phosphate buffer (pH 8.0),
incubated with 1 mgÆmL
)1
lysozyme at 37 °C with shaking
for 1 h, and then broken in a French pressure cell operated
at 16 000 p.s.i. The His
6
-tagged catalases were isolated from
the cytoplasmic cell fraction, corresponding to a 3 L culture,
with a 1 mL HiTrap chelating column (Pharmacia Bio-
tech), loaded with nickel ions, according to the manufac-
turer’s instructions. The cytoplasmic fraction,
supplemented with 300 mm NaCl and 1 mm histidine, was
loaded onto the affinity column, equilibrated in 50 mm
potassium phosphate buffer (pH 8.0) containing 300 mm
NaCl and 1 mm histidine. This buffer was used to wash
the column, and His
6

-tagged catalase was eluted from the
matrix by raising the histidine content of the buffer to
50 mm. The purified catalases were dialysed against 50 mm
potassium phosphate buffer (pH 8.0) and stored on ice.
We have previously shown that the His
6
-tag at the C-ter-
minal end of KatA does not interfere with the function of
the protein [6].
MIC determinations
Bacteria were grown in TSBG medium supplemented with
20 mgÆL
)1
chloramphenicol and different concentrations of
the porphyrin to be tested. The media were inoculated to a
D600 nm of 0.15 with a fresh culture grown in unsupple-
mented TSBG. The cultures (3 mL) were incubated at
37 °C for 8 h, under oxic conditions, in the dark. The MIC
was defined as the lowest concentration of porphyrin that
prevented growth. All experiments were carried out in trip-
licate. The amount of dimethylsulfoxide, Tween-80 or
Tween-20 added to the medium as solvent for the porphy-
rin compound did not affect bacterial growth.
Fluorescence spectroscopy
Purified substituted catalase proteins were denatured in a
mixture of 2.1 m pyridine, 0.075 m NaOH and
0.0075% (w ⁄ v) Tween-80 in water. After centrifugation at
10 000 g for 10 min to remove the denatured proteins, the
fluorescence emission spectra between 530 and 750 nm of
the solutions were recorded on a Shimadzu RF 5301 PC

spectrofluorophotometer, using excitation wavelengths of
406 nm for PP and Cu-PP, 424 nm for Zn-PP, and 418 nm
for Mg-PP. The excitation and emission slits were 3 and
10 nm, respectively. Known concentrations of PP dissolved
in the same solvent as the samples were used for calibration
of the fluorometer readings.
Other methods
Extraction of porphyrins from purified catalase protein and
analysis by RP-HPLC were performed as described by Sone
and Fujiwara [31]. Protein concentrations were determined
using the bicinchoninic acid assay (Pierce Chem Co.), with
BSA as the standard. Concentrations of KatA were
determined by the combined use of quantitative amino acid
analysis (performed at the Department of Laboratory Medi-
cine, Malmo
¨
University Hospital), pyridine haemochromogen
Cofactor-substituted catalase M. Brugna et al.
2670 FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS
analysis [32], and rocket immunoelectrophoresis. Immuno-
blot and rocket immunoelectrophoresis were performed
with rabbit anti-KatA serum as previously described [6].
Catalase activity was assayed as described previously [6].
Light absorption spectra were recorded with a Shima-
dzu UV-2101PC spectrophotometer. Metal content analysis
was performed by using inductively coupled plasma MS.
Acknowledgements
This work was supported by grant 621-2007-6094 from
the Swedish Research Council and a grant from the
Crafoord Foundation. M. Brugna was the recipient of

a EU Marie Curie long-term fellowship (contract
HPMF-CT-2000-00918). We are grateful to S. Vinog-
radov (University of Pennsylvania, Philadelphia, PA)
for the generous gift of Pd-meso and to T. Olsson
(Lund University, Sweden) for the metal content anal-
ysis. We thank I. Sta
˚
l for expert technical assistance.
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Supporting information
The following supplementary material is available:
Fig. S1. Crystal structure of E. faecalis catalase.
This supplementary material can be found in the
online version of this article.
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2672 FEBS Journal 277 (2010) 2663–2672 ª 2010 The Authors Journal compilation ª 2010 FEBS