Tài liệu Báo cáo khoa học: NirF is a periplasmic protein that binds d1 heme as part of its essential role in d1 heme biogenesis pdf

NirF is a periplasmic protein that binds d
1
heme as part of
its essential role in d
1
heme biogenesis
Shilpa Bali
1
, Martin J. Warren
2
and Stuart J. Ferguson
1
1 Department of Biochemistry, University of Oxford, UK
2 Department of Biosciences, University of Kent, Canterbury, UK
Introduction
Denitrification is a four-step transformation of nitrate
to dinitrogen gas by various species of bacteria under
anaerobic conditions [1,2]. These four steps are cataly-
sed by complex metalloenzymes and involve stepwise
conversion of nitrate to nitrite, nitrite to nitric oxide,
nitric oxide to nitrous oxide and finally reduction of
nitrous oxide to nitrogen. In the denitrification path-
way, nitrite reduction is the key step, as it is the point
of divergence from assimilatory nitrogen metabolism in
which nitrite is reduced to ammonium [2,3]. There are
two types of respiratory nitrite reductase involved in
denitrification: one is copper-containing nitrite reduc-
tase (NirK), which is prevalent in, but not exclusive to,
alphaproteobacteria, the other being cytochrome cd
1
(NirS), which prevails in betaproteobacteria [4].

Cytochrome cd
1
nitrite reductase is a homodimeric
periplasmic enzyme with each subunit containing a
covalently attached c heme and noncovalently attached
d
1
heme, bound in a beta-propeller domain, as pros-
thetic groups [5,6]. Heme d
1
, which forms the active cen-
tre for the one electron reduction of nitrite to nitric
oxide, has a unique structure. The structure of this mod-
ified heme, a dioxoisobacteriochlorin to be more spe-
cific, has been known for more than two decades [7,8],
but quite how it is biosynthesized by denitrifying bacte-
ria under anaerobic conditions is not understood. Anal-
ysis of insertional mutagenesis and complementation
work in Pseudomonas aeruginosa, Pseudomonas fluores-
cens, Paracoccus denitrificans and Pseudomonas stutzeri
have shown that a set of several contiguous genes that
always follows the structural gene, nirS, for cytochrome
cd
1
, is necessary for the biogenesis of the d
1
cofactor
[9–13]. In P. denitrificans and closely related Para-
coccus pantotrophus, these genes are cotranscribed as
Keywords

cytochrome cd
1
; d
1
heme biosynthesis;
denitrification; nitrite reductase;
Paracoccus pantotrophus; tetrapyrrole
Correspondence
S. J. Ferguson, Department of
Biochemistry, University of Oxford, South
Parks Road, Oxford OX1 3QU, UK
Fax: +44 1865 613201
Tel: +44 1865 613299
E-mail:
(Received 24 June 2010, revised 27 August
2010, accepted 1 October 2010)
doi:10.1111/j.1742-4658.2010.07899.x
The cytochrome cd
1
nitrite reductase from Paracoccus pantotrophus catalyses
the one electron reduction of nitrite to nitric oxide using two heme cofactors.
The site of nitrite reduction is the d
1
heme, which is synthesized under anaer-
obic conditions by using nirECFD-LGHJN gene products. In vivo studies
with an unmarked deletion strain, DnirF, showed that this gene is essential
for cd
1
assembly and consequently for denitrification, which was restored
when the DnirF strain was complemented with wild-type, plasmid-borne,

nirF. Removal of a signal sequence and deletion of a conserved N-terminal
Gly-rich motif from the NirF coded on a plasmid resulted in loss of in vivo
NirF activity. We demonstrate here that the product of the nirF gene is a
periplasmic protein and, hence, must be involved in a late stage of the cofac-
tor biosynthesis. In vitro studies with purified NirF established that it could
bind d
1
heme. It is concluded that His41 of NirF, which aligns with His200
of the d
1
heme domain of cd
1
, is essential both for this binding and for the
production of d
1
heme; replacement of His41 by Ala, Cys, Lys and Met all
gave nonfunctional proteins. Potential functions of NirF are discussed.
Abbreviation
LB, Luria–Bertani.
4944 FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS
an operon in the following order nirECFD-LGHJN.It
has been proved that the biosynthesis of the d
1
heme proceeds via a common tetrapyrrole precursor
uroporphyrinogen III, which is transformed into pre-
corrin-2 by S-adenosyl-L-methionine-dependent methyl
transferase, NirE [14,15]. In addition, it has been dem-
onstrated that a Paracoccus derivative strain, in which
nirN is replaced with a kanamycin resistance cassette,
still makes holo-cd

1
, which suggests that this last gene
on the operon is dispensable for d
1
heme assembly [15].
Also, the nirC gene encodes a periplasmic c type cyto-
chrome that may have an electron transfer role in cyto-
chrome cd
1
activity [16] or maturation [15].
Conflicting evidence exists concerning the subcellular
location of NirF. NirF from P. pantotrophus shares
54% sequence identity and 72.3% sequence similarity
with the NirF from Ps. aeruginosa. However, the pro-
tein from Ps. aeruginosa lacks the apparent Sec-depen-
dent signal sequence for translocation to the periplasm,
which, in contrast, is readily identified in P. pantotro-
phus NirF. Information about NirF from the much-
studied Ps. aeruginosa has led to the widespread
assumption that NirF is cytoplasmic. Accordingly, we
wanted to determine the subcellular location of NirF in
P. pantotrophus, which produces larger amounts of cd
1
under denitrifying conditions than Ps. aeruginosa. NirF
also shares 34% sequence similarity with the beta-pro-
peller domain of cd
1
, indicating a scaffolding role for
an intermediate of heme d
1

synthesis. Roles for NirF in
ferrochelation or dehydrogenase activity have been pro-
posed [10,13]. Interestingly, NirF also shows some simi-
larity to a cobalamin decarboxylase, CobT [3,13]. The
physiological role of NirF would heavily rely on its cel-
lular location. For all these reasons we wanted to
develop an in vivo system to investigate its role by mak-
ing use of the generation and characterization of an
unmarked deletion in nirF. The unmarked gene deletion
mutant is expected to lose nitrite respiration and the
capacity to synthesize this tetrapyrrole derivative. More
importantly, an unmarked deletion mutant strain
should have denitrification restored on complementa-
tion with plasmid-borne nirF and, therefore, should
provide an in vivo system to further analyse the physio-
logical role of NirF and its variants.
Results
Construction of the DnirF strain and its in vivo
nitrite reductase activity
In P. pantotrophus, the operon associated with d
1
heme
biosynthesis has many overlapping genes. The nirF
gene overlaps four nucleotides with the preceding nirC
gene and it is also immediately followed by an overlap-
ping ORF for nirD-L. Previous studies in Ps. aerugin-
osa demonstrated that a marked mutation in the nirF
gene resulted in the formation of inactive nitrite reduc-
tase [9]. Similar results were also found for an nirF
mutant in P. stutzeri [10], but in this case a polar effect

of the mutation was not excluded. The present study
utilized an unmarked deletion in nirF where the entire
nirF ORF has been deleted from the chromosome.
When this unmarked deletion strain of nirF (i.e.
DnirF), named SBN11, was grown anaerobically in
minimal media supplemented with 20 mm nitrate, it
converted the entire available nitrate to nitrite within
10 h of growth and lost its nitrite reductase activity, as
shown by no consumption of nitrite to yield any gas-
eous products. The extracellular nitrite concentration
peaked at 20 mm and remained there even when the
cultures had reached the stationary phase. No brown
coloration from holo-cytochrome cd
1
or gas evolution
from nitrogen production was observed in the SBN11
cultures.
Reassuringly, the nitrite reductase activity of the
DnirF strain was restored within 10 h of anaerobic
growth on nitrate-supplemented minimal media, when
it was complemented with a plasmid-borne copy of
nirF (Fig. 1). Here, the extracellular nitrite concentra-
tion reached a maximum value of 14 mm, followed by
a rapid decline. This corresponds to a delay in the
expression or activation of functional cytochrome cd
1
,
but eventually a complete denitrification pathway was
established. As shown by the four independent growth
results, the extracellular nitrite concentration was a

function of cell density, rather than time, for the DnirF
strain expressing plasmid-encoded nirF. In addition,
expression of plasmid-encoded strep II-tagged nirF was
demonstrated by western blot analysis with alkaline
phosphatase-conjugated strep-tactin antibody (Fig. S1).
These results confirm the essential role of NirF in d
1
heme assembly.
Influence of deletion and replacement of the
signal sequence on NirF processing and
denitrification activity
The derived amino acid sequences of NirF from two
different denitrifiers, P. pantotrophus and Ps. aerugin-
osa, differ significantly at the N-termini. In P. panto-
trophus, nirF encodes a ( 42 kDa) protein that has an
N-terminal signal sequence suggestive of a location in
the periplasm. On the other hand, in Ps. aeruginosa
(PAO1), NirF has no apparent signal sequence and
therefore this protein should be located in the cyto-
plasm. In order to determine whether export of NirF
S. Bali et al. Periplasmic NirF binds d
1
heme
FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS 4945
to the periplasm of P. pantotrophus is essential for d
1
heme formation, and thus for the physiological func-
tion of NirF, we deleted the presumed signal sequence.
We also replaced the putative signal sequence of NirF
with the shorter signal sequence of a native periplasmic

protein, NirC, to see whether it could still perform its
physiological function. The replacement of the signal
sequence on the NirF coded for on a plasmid had no
effect on nitrite reductase activity as judged by the res-
toration of denitrification when this plasmid was used
to complement the DnirF strain (Fig. 2). This result
also ruled out the need for a specific signal sequence
for the function of NirF. On the other hand, a plasmid
carrying an nirF gene lacking the native signal
sequence failed to restore denitrification upon
attempted complementation of the DnirF strain
(Fig. 2).
A C-terminally strep II-tagged version of NirF was
produced anaerobically from a pEG276-based plasmid
in the DnirF strain using minimal media supplemented
with nitrate as the terminal electron acceptor. When
the cells of this derivative strain producing tagged
22
Time (h)
2
6
10
12
22
24
20
18
16
14
12

10
8
6
Nitrate (mM)
4
2
0
GB-17 SBN11 SBN13-1 SBN13-2
Strain
SBN13-3 SBN13-4 SBN14
Fig. 1. Restoration of nitrite reduction in a Paracoccus pantotrophus strain carrying an unmarked gene deletion of nirF (DnirF) with plasmid-
borne nirF. GB17 is the parental wild-type P. pantotrophus in which nitrite does not accumulate following reduction of added nitrate; SBN11
is the DnirF strain that does not synthesize d
1
heme and hence cannot turnover nitrite to nitric oxide. SBN13 is SBN11 complemented with
nirF on pEG276 (four replicas are shown) and SBN14 is a control with the SBN11 strain containing the empty expression vector pEG276
only. Replicas of SBN13 indicate that the concentration of extracellular nitrite is dependent on the cell density at any given time. The code
for the times of analysis is shown on the right.
22
SBN20 (ΔnirF + NirF (no signal sequence))
20
18
16
14
12
10
8
6
Nitrate (mM)
4

2
0
2.2
2
1.8
1.6
1.4
1.2
0.8
0.6
0.4
0.2
0
1
0510
Time (h)
2015 25
SBN28 (ΔnirF + NirF (NirC signal sequence))
22
2
1.8
1.6
1.4
1.2
0.8
A
600
0.6
0.4
0.2

0
1
20
18
16
14
12
10
8
6
4
2
0
0510
Time (h)
2015 25 30
Fig. 2. A periplasmic targeting sequence is essential for Paracoccus pantotrophus NirF function. Growth plots and time courses of nitrite
appearance and disappearance for the SBN11 (DnirF) strain complemented with a plasmid coding for NirF from which the putative periplas-
mic targeting sequence had been deleted (to give SBN20). Also shown is the effect of providing the DnirF strain with a plasmid coding for
NirF where its native signal sequence had been replaced by the proven periplasmic targeting sequence of NirC (to give SBN28). Cell density
was determined at A
600
and is depicted by grey diamonds, whereas the extracellular nitrite concentration was determined using a colorimet-
ric method (for more details, see Experimental procedures) and is shown by black triangles. The data shown here are the average of four
different experiments.
Periplasmic NirF binds d
1
heme S. Bali et al.
4946 FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS
NirF were fractionated and run on the SDS ⁄ PAGE

for western analysis by using the alkaline phosphatase
conjugate of strep-tactin antibody, we found that both
membrane and cytoplasmic fractions were free of NirF
protein and it was present only in the periplasmic frac-
tion (Fig. 3). These results, together with the outcome
of the complementation analysis, prove that NirF is a
periplasmic protein in P. pantotrophus.
Influence of variations of conserved residues on
the in vivo activity of NirF
Interestingly, like NirN, NirF shares sequence similar-
ity with the C-terminal d
1
heme-containing domain of
cytochrome cd
1
. Strikingly, the axial ligand of iron in
d
1
heme in P. pantotrophus cd
1
(His200) is conserved in
NirF (His41); this conservation applies to all other
NirF sequences known in the database (Fig. S2). How-
ever, the other catalytic site histidines (His 345 and
His388) of NirS are not conserved in NirF.
Restoration of denitrification upon complementation
of the unmarked nirF deletion strain of P. pantotrophus
with plasmid-borne nirF provided a good in vivo sys-
tem for testing the molecular basis for the NirF activ-
ity (Fig. 4). Replacement of the aforementioned His41

with Ala completely abolished the in vivo nitrite reduc-
tase activity as seen by the accumulation of large
amounts of extracellular nitrite in the DnirF strain
complemented with nirF (H41A), when growing under
denitrifying conditions (Fig. 5). We were also curious
whether denitrification could be rescued to any extent
if this His were replaced with some of the other heme
iron-binding residues, such as Met, Cys or Lys. All
plasmids bearing nirF with this residue changed to any
of these three potential heme ligands failed to rescue
denitrification in the DnirF strain (Fig. 5). These results
indicate that His41 is important for NirF function.
It has been reported that NirF shows 21% sequence
similarity to the first 100 amino acids of NirE [13].
There is also a highly conserved N-terminal Gly-rich
(GXGX
2
GX
7
G) motif in all NirF sequences, which is
suggestive of a binding to a nucleotide-containing
cofactor. This motif has also been found in several
other dehydrogenases involved in tetrapyrrole biosyn-
thesis pathways, including CysG
A
and SirC in the
siroheme biogenesis pathway [17]. Furthermore, when
a pairwise alignment of NirF was performed with
Met8P (a bifunctional dehydrogenase-ferrochelatase
from Saccharomyces cerevisiae), we found that the

two proteins had 24% sequence similarity. A crystal
structure of Met8P has shown that this protein has
an aspartate residue (Asp141), which is important
for both chelatase and dehydrogenase function [17];
interestingly, this aspartate, Asp129, is also conserved
98
kDa
M Wt
Insoluble
Total cell lysate
Periplasm
Membrane
Cytoplasm
kDa
M Wt
Insoluble
Total cell lysate
Periplasm
Membrane
Cytoplasm
62
49
38
28
17
14
98
62
49
38

28
17
14
AB
Fig. 3. Distribution of NirF between different cell fractions of P. pantotrophus. (A) Western blot assay with the different fractions of the cells
expressing plasmid-borne and strep-tagged NirF from SBN13 strain, using an alkaline phosphatase conjugate of strep-tactin antibody (for
more information see Experimental procedures). NirF ( 42 kDa) with a C-terminal strep II tag could be found in the total cell lysate and the
periplasmic fraction, but was absent from the membrane and cytoplasmic fractions. (B) The same cell fractions as shown in (A) when sub-
jected to SDS ⁄ PAGE analysis and stained with Coomassie blue for proteins.
S. Bali et al. Periplasmic NirF binds d
1
heme
FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS 4947
in NirF sequences from P. pantotrophus and other den-
itrifiers (Fig. S3). When plasmids carrying the variation
in NirF of Asp129 to Ala or to Gln were used to
complement the DnirF strain, they showed the same
phenotype as when complementation was done with
wild-type NirF. There were no growth defects and nei-
ther of these strains showed a large accumulation of
extracellular nitrite during the log phase, when grown
on 20 mm nitrate under denitrifying conditions
(Fig. 5). This result demonstrated that Asp129 of NirF
could not be essential for any function similar to that
in Asp141 of Met8P.
The idea of NirF being a dehydrogenase is appealing
because of the presence of a putative nucleotide-bind-
ing motif in the N-terminal of the protein sequence
and also because there is a need for oxidation in the d
1

heme biosynthetic pathway, for example, oxidation of
C17 propionate to give an acrylate side chain. This
type of step would normally require FAD-based chem-
istry. Another potential dehydrogenation is NAD ⁄
NADP-dependent oxidation of precorrin-2 to sirohy-
drochlorin that might be a shared intermediate in the
d
1
heme and cobalamin biosynthesis pathway. Some,
but not all, flavoproteins have tightly bound flavin
when overexpressed and thus are yellow on extraction.
However, no such coloration was observed for the
NirF when it was overproduced in either Escherichia
coli or in P. pantotrophus under either aerobic or
anaerobic conditions. We also did not observe any
interaction between the purified NirF and a range of
nucleotide-containing cofactors by using a variety of
biophysical methods (data not shown). Nonetheless,
we still decided to test the effect of the deletion of the
entire GXGX
2
GX
7
G motif on the in vivo NirF and
nitrite reductase activity. Although deletion of the
entire Gly-rich region resulted in an inactive NirF,
analysis of variant NirF species with one or more of
the individual Gly residues changed to Ala did not
result in loss of NirF function. Thus, we conclude that
although a significant stretch of the N-terminus is

important for the formation of a functional protein,
we have no evidence that this functionality relates to
the Gly residues; thus, the important residues may lie
elsewhere within this N-terminal region.
Purification and in vitro characterization of NirF
and its variants
NirF was recombinantly produced in E. coli and puri-
fied from the periplasmic fraction to near homogeneity
22
20
18
16
12
14
10
8
6
4
2
0
0 5 10 20 2515
22
20
18
16
12
14
10
8
6

4
2
0
22
20
18
16
12
14
10
8
6
4
2
0
20
18
16
12
14
10
8
6
4
2
0
2.2
2
1.8
1.4

1.2
1.6
1
0.8
0.6
0.4
0.2
0
2
1.8
1.4
1.2
1.6
1
0.8
0.6
0.4
0.2
0
2.2
1.2
1.7
0.7
0.2
–0.3
2.2
1.2
1.7
0.7
0.2

–0.3
GB17 SBN03 (nirF : :Kan
R
)
SBN13 (ΔnirF + NirF)
SBN11 (ΔnirF )
Nitrite (mM)Nitrite (mM)
A
600
A
600
A
600
A
600
Time (h)
0 5 10 20 2515
Time (h)
0 5 10 20 2515
Time (h)
0 5 10 20 2515
Time (h)
Fig. 4. Time courses of nitrite accumulation
and consumption in Paracoccus pantotro-
phus strains. Starter cultures were grown
aerobically in LB with shaking before inocu-
lation of mineral salt medium containing
20 m
M nitrate in a 1% v ⁄ v dilution and
appropriate antibiotics. These cultures were

incubated without shaking at 37 °C. Growth
of wild-type P. pantotrophus GB17 strain is
shown in the upper left panel and of the
kanamycin insertion mutant nirF strain
(SBN03) in the upper right panel. Growth of
SBN11 (DnirF) is shown in the bottom left
panel and of SBN13 (DnirF) containing
pEG276-nirF in the bottom right panel. Cell
density was determined at A
600
and is
depicted by grey diamonds, whereas the
extracellular nitrite concentration was deter-
mined using a colorimetric method and is
shown by black triangles. The data shown
here are the averages of four different
experiments.
Periplasmic NirF binds d
1
heme S. Bali et al.
4948 FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS
in a single affinity chromatography step, with a yield
of  3.0 mg protein per litre of culture. All the NirF
variants were also produced and purified in a similar
manner with a yield ranging from 1.5 to 4.0 mgÆL
)1
of
culture. SDS ⁄ PAGE gels for all proteins are shown in
Fig. S4. Size exclusion chromatography demonstrated
that NirF is monomeric. Dynamic light scattering

showed that the protein was well folded with a hydro-
dynamic radius fitting with the molecular weight of the
mature protein. CD of the protein in potassium phos-
phate buffer at pH 7.5 displayed a predominantly
beta-sheet structure (data not shown). This is consis-
tent with the sequence similarity of this protein with
the C-terminal beta-propeller domain of NirS (cyto-
chrome cd
1
) that houses d
1
heme. MS confirmed the
molecular mass of the protein to be 41.937 kDa, which
is expected after processing and cleavage of the signal
peptide. Surprisingly, a D129A mutant of NirF failed
to give any soluble protein when overexpressed in
E. coli, although this variant rescued denitrification
when it complemented the Paracoccus DnirF strain
under denitrifying conditions. This observation sug-
gests that the conserved Asp129 is important for fold-
ing of the recombinant protein.
SBN23 (ΔnirF + NirF (H41M))SBN19 (ΔnirF + NirF (H41A))
SBN21 (ΔnirF + NirF (H41K)) SBN22 (ΔNirF + NirF (H41C))
SBN25 (ΔnirF + NirF (D129Q))SBN24 (ΔnirF + NirF (D129A))
22
20
18
16
12
14

10
8
6
4
2
0
0102030
2.2
2
1.8
1.4
1.2
1.6
1
0.8
0.6
0.4
0.2
0
Nitrite (mM)Nitrite (m
M)
Time (h)
22
20
18
16
12
14
10
8

6
4
2
0
0 5 10 2015
25
2.2
2
1.8
1.4
1.2
1.6
1
0.8
0.6
0.4
0.2
0
2.2
2
1.8
1.4
1.2
1.6
1
0.8
0.6
0.4
0.2
0

Nitrite (mM)
Time (h)
0
0
5
5
10
10
20
20
15
15
25
Time (h)
22
20
18
16
12
14
10
8
6
4
2
0
0 5 10 2015 25
2.2
2
1.8

1.4
1.2
1.6
1
0.8
0.6
0.4
0.2
0
Time (h)
22
20
18
16
12
14
10
8
6
4
2
0
0 5 10 2015
25 30 35 40
2.2
2
1.8
1.4
1.2
1.6

1
0.8
0.6
0.4
0.2
0
Time (h)
22
20
18
16
12
14
10
8
6
4
2
0
05 1510 20 25
2.2
2
1.8
1.4
1.2
1.6
1
0.8
0.6
0.4

0.2
0
A
600
A
600
A
600
Time (h)
Fig. 5. His41 is essential for Paracoc-
cus pantotrophus NirF, but Asp129 is dis-
pensable. Growth plots and time courses of
nitrite appearance and disappearance for
P. pantotrophus SBN11 (DnirF) strain com-
plemented with plasmid carrying a gene
coding for NirF(H41A) (upper left),
NirF(H41M) (upper right), NirF(H41K) (middle
left), NirF(H41C) (middle right), NirF(D129A)
(lower left) and NirF(D129Q) (lower right).
Cell density was determined at A
600
and is
depicted by grey diamonds, whereas the
extracellular nitrite concentration was deter-
mined using a colorimetric method and is
shown by black triangles. The data shown
here are the averages of four different
experiments.
S. Bali et al. Periplasmic NirF binds d
1

heme
FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS 4949
In vitro binding of d
1
heme to NirF
As explained above, there are sequence similarities
(Fig. S2) between NirF and the d
1
heme-binding
domain of cytochrome cd
1
. Therefore, we tested for
the binding of d
1
heme to purified NirF. The addition
of d
1
heme to the NirF resulted in the appearance of a
distinctive visible absorption spectrum (Fig. 6). The d
1
heme peak shifted from 681 to 630 nm. Considering
that NirF is colourless and has no absorbance in the
UV–visible region, this shift of 50 nm in the spectrum
is due to extreme changes in the d
1
heme environment.
This binding of d
1
heme with NirF was stoichiometric,
i.e. 1 mol of heme was taken up by 1 mol of NirF

(concentrations of heme and protein were calculated
by using the extinction coefficient mentioned in the
experimental section and the standard Bradford assay,
respectively). To test whether the binding of d
1
heme
to NirF was specific, and thus physiologically signifi-
cant, we added heme to NirF and found that there
were no shifts in the visible absorption spectrum and
thus no interaction. It is already known for other peri-
plasmic proteins that the d
1
heme-binding region is
very sensitive to proton concentration and prefers a
lower pH for d
1
heme addition, consistent with the
periplasm probably having a pH lower than 7 [15].
Similarly, the process of d
1
heme addition to NirF was
also pH dependent. At relatively high pH values (8 or
higher) the spectral change described above for adding
d
1
to apo-protein, did not occur; however, when the
pH was lowered to neutral pH the uptake of d
1
heme
proceeded.

As NirF could have at least two other interacting
partners in the periplasm for the d
1
heme assembly,
namely NirC and NirN, we wanted to test if the com-
plex of NirF.d
1
could transfer d
1
heme to NirN, which
was recently also shown to bind d
1
heme [15]. This
binding would be difficult to judge, as the NirN.d
1
heme complex shows a peak at 627 nm. Unfortunately,
we could not observe any significant peak shifts when
NirN was added to the NirF.d
1
heme complex in slight
molar excess under anaerobic conditions (data not
shown). His200 of P. pantotrophus NirS is conserved
between NirF and NirS; it is the His residue that in
NirS is the proximal axial ligand to the d
1
heme.
Replacement of an equivalent His, His41, in NirF by
Ala abolished binding of the heme to the protein.
Known distal heme-binding residues, such as Met, Lys
or Cys [18,19], were substituted for His41 in NirF. No

changes in the visible spectra were observed when
all three variants, NirF(H41K), NirF(H41M) and
NirF(H41C), were added individually to the d
1
heme
in slight molar excess. There was no equivalent peak at
630 nm, which was observed for the NirF.d
1
complex.
These results, when taken together with in vivo comple-
mentation analysis of NirF(H41) variants, suggest that
interaction of NirF with d
1
heme is very specific for
His41. This His41 residue must play a part in both
structural and functional roles of NirF.
Discussion
On the basis of several criteria, including cell fraction-
ation and the consequences of either deleting the
putative signal sequence or replacing it by a proven
signal sequence from nirC, it can be concluded that
NirF is a periplasmic protein in P. pantotrophus. This
has an important implication as the only other known
d
1
biogenesis proteins with a periplasmic location are
NirC and NirN, both of which are not essential for
d
1
heme synthesis [15,16]. It follows that, unless there

are other unrecognized d
1
biogenesis proteins,
then NirF must catalyse the last step(s) in d
1
heme
synthesis.
The nature of these synthesis steps is conjectural at
this stage, but the NirF.d
1
heme complex might reflect
a product complex. The failure of the D129A mutation
to prevent d
1
heme biogenesis suggests that the activity
of NirF cannot be similar to that of Met8P activity
where a comparable mutation is inhibitory. A puzzle
is that some NirF sequences, notably for two strains
of Ps. aeruginosa, PA7 and PAO1, but also that in
Magnetospirillum magneticum, do not have any readily
recognizable, i.e. N-terminal positive charges, central
hydrophobic core (or h-region) of seven to 15 amino
acid residues, followed by a peptidase recognized
‘c-region’ [20], signal sequences. These sequences are in
0.21
0.18
0.15
0.12
0.09
0.06

0.03
0
580 600 620 640 660 680 700 720
Wavelength (nm)
Absorbance
Fig. 6. Visible absorption spectra of oxidized d
1
heme, 0.060 mM,
before (
) and after the addition of NirF ( ) in slight molar
excess. The flat trace at the bottom is the visible absorption
spectrum of NirF at 0.041 m
M. All spectra were taken in 50 mM
phosphate buffer, pH 7, at room temperature.
Periplasmic NirF binds d
1
heme S. Bali et al.
4950 FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS
contrast to many other sequences for NirF proteins
where the signal sequence is readily recognizable. It is
possible that the function of NirF can be realized in
the cytoplasm of Ps. aeruginosa, with the resulting d
1
heme then being translocated to the periplasm. In the
case of P. pantotrophus it would be the substrate for
NirF that is translocated. In either case the transport
process is enigmatic as none of the Nir proteins codes
for a transmembrane protein that could be a candidate
for moving d
1

heme, or a precursor, across the mem-
brane. Alternatively, as suggested by Suzuki et al. [21],
NirF in some organisms might be periplasmic but with
an N-terminal transmembrane helix anchoring the pro-
tein to the membrane. However, our bioinformatics
analysis of the N-terminal sequences for NirF for
Ps. aeruginosa and M. magneticum does not agree with
this suggestion. A function of NirF related to binding
nucleotide in a putative Rossman fold now appears
unlikely, as the putative Gly of such a fold are not
essential. This result also correlates well with the
export of NirF via the sec system; a periplasmic pro-
tein with a bound nucleotide would be exported via
the Tat system in a folded conformation along with
the cofactor.
Heme d
1
differs from other tetrapyrrole derivatives
in that it is a dioxoisobacteriochlorin, as opposed to
porphyrin, characterized by the presence of two oxo
groups at C3 and C8 and methyl groups at positions
C2 and C7 [7,22]. Also, its synthesis is mediated via a
separate branch of the tetrapyrrole biosynthetic path-
way from uroporphyrinogen III [14,15,23]. Recently
we showed that methylation at C2 and C7 is catalysed
by NirE to give another tetrapyrrole intermediate pre-
corrin-2 [14,15]. Further modifications would include:
(a) decarboxylations of the acetate side chain at posi-
tions C12 and C18, (b) dehydrogenation of the C17
side chain to give an acrylate moiety, (c) introduction

of oxo groups at positions C3 and C8, (d) ferrochela-
tion and (f) transport to the periplasm. Not only the
enzymes and chemistry of all these steps are unknown,
but even the order in which the modifications occur
remains mostly unknown. Our result that NirF is a
periplasmic enzyme indicates that this protein catalyses
the chemistry required for the last stages of d
1
heme
biosynthesis. However, defining the substrate for NirF
will not be an easy task. Possibilities include the d
1
heme lacking iron and ⁄ or with the side chain satu-
rated, but accessing these putative substrates is not
trivial. An alternative approach would be to seek accu-
mulation of the substrate of NirF in a mutant that
lacks NirF; this too is not trivial as the DnirF strain
does not accumulate readily detectable amounts of an
intermediate of d
1
synthesis.
Experimental procedures
DNA manipulations
DNA manipulations were performed by standard methods.
Primers were synthesized by Sigma–Genosys (Haverhill,
UK). Amplifications by PCR using KOD DNA polymerase
(from Thermococcus kodakaraensis) were according to sup-
plier’s instructions (Novagen, now Merck Biosciences, Not-
tingham, UK). All constructs generated by PCR were
confirmed to be correct by sequencing. All the primers used

in this study are shown in Table S1.
Construction of bacterial strains
Initially, an unmarked deletion was generated in nirF in a
wild-type GB17 P. pantotrophus strain. This was performed
in a two-step process. First, the 5¢ and 3¢ flanking regions of
nirF were cloned and the kan
R
cassette inserted between
them. This was cloned into pJQ200ks (gentamicin-resistant),
which is incapable of replication in P. pantotrophus. Chro-
mosomal integrants in which double crossover events had
replaced the nirF ORF with the kanamycin-resistance cas-
sette, but lost the pJQ200ks backbone, were selected as
kanamycin-resistant gentamicin-sensitive strains. Correct
integration of the cassette was confirmed by PCR screening.
Second, the deletion was made unmarked using a con-
struct in which the nirF flanks were cloned into the pRVS2
vector (gentamicin resistance), which was modified from
pRVS1 [24]. This vector is also incapable of replication in
P. pantotrophus. Single crossover events were selected via
resistance to streptomycin (P. pantotrophus), gentamicin
(pRVS2) and kanamycin (nirF::kan
R
). This strain was then
screened for a second crossover event in which the kan
R
cassette was removed via homologous recombination. This
strain was selected essentially as described in [24] and iden-
tified by the growth of kanamycin-sensitive white colonies
in the presence of 200 mgÆmL

)1
X-gal (5-bromo-4-chloro-3-
indolyl-b-galactoside). Putative strains were confirmed to be
correct by PCR screening (D nirF). Full details of the con-
struct generation and strategy employed can be found in
supporting information (Doc. S1, S2 and S3).
Cloning of P. pantotrophus nirF and nirF variants
The nirF ORF was amplified from P. pantotrophus genomic
DNA using SB5 and SB6, digested with NcoI and XhoI
and ligated into NcoI ⁄ XhoI-digested pET22b (for overex-
pression in E. coli). A C-terminal strep II tag was intro-
duced in the pET22b-based construct by inverse PCR using
primers SB45 and SB46, and by self-ligating the purified
PCR product after phosphorylation with T4 PNK. The
native P. pantotrophus signal sequence of nirF was removed
by inverse PCR with SB62 and SB63 to generate a new
construct that had the PelB signal sequence in frame with
S. Bali et al. Periplasmic NirF binds d
1
heme
FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS 4951
downstream nirF, for recombinant production of NirF in
E. coli. The internal EcoRI site within the nirF gene was
silently mutated using the primers SB30 and SB31 and the
product of this PCR was used to amplify EcoRI and Hin-
dIII flanked nirF to clone into EcoRI ⁄ HindIII digested
pEG276 (for expression in P. pantotrophus strains) [25].
Inverse PCR was used to generate a number of mutations
using the following primer combinations on both the
pET22b- and pEG276-based clones: H41A – SB82 and

SB83, H41K – SB114 and SB115, H41C – SB116 and SB117,
H41M – SB118 and SB119, D129A – SB101 and SB102,
D129Q – SB103 and SB104. The Gly-rich region in the
N-terminus of the nirF was deleted by inverse PCR using
primers SB110 and SB111. Similarly, the native signal
sequence of the nirF was deleted from the pEG276-based
plasmid using the primers SB112 and SB113. A hybrid NirF
was made by introduction of the NirC signal sequence in
front of NirF by two sequential inverse PCRs using primers
SB121, SB122, SB123 and SB124. The mutants generated in
this study are detailed in Table 1.
Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids used in this study are
detailed in Table 1. Paracoccus pantotrophus strains were
grown in Luria–Bertani (LB) medium or in a defined mini-
mal medium [26] supplemented with 20 mm succinate as a
carbon and energy source. Overnight aerobic growth was
achieved in 5 mL growth medium in 50 mL universals, which
were incubated in a shaker at 250 rpm at 37 °C. Anaerobic
growth was conducted in 600 mL growth medium in com-
pletely filled bottles, with stationary incubation at 37 °C.
For anaerobic growth, cultures were supplemented with
20 mm sodium nitrate. Anaerobic cultures were inoculated
with 1% v ⁄ v freshly grown aerobic overnight culture in LB
and cell density determined at A
600
. Antibiotic-resistant
strains were supplemented with antibiotics at the following
concentrations: streptomycin (100 mgÆmL
)1

), kanamycin
(50 mgÆmL
)1
), carbenicillin (100 mgÆmL
)1
) and gentamicin
(20 mgÆmL
)1
). Growth on solid media used liquid growth
medium supplemented with 1.5% bacteriological agar.
Analysis of extracellular nitrite
Cells were pelleted from 1 mL anaerobic culture via centri-
fugation at 14 000 g for 1 min. The nitrite concentration in
the medium was estimated colorimetrically using the
method in [27].
Fractionation of P. pantotrophus extracts and
western blotting
Paracoccus pantotrophus strains were grown in 2 L cultures
of minimal media supplemented with 20 mm sodium nitrate
and 20 mm sodium succinate and harvested at 6000 g for
20 min. Cell pellets were resuspended in 10 mL SET buffer
(100 mm Tris ⁄ HCl pH 7.5, 3 mm EDTA and 0.5 m sucrose)
to which 1 mgÆmL
)1
lysozyme, 75 mg DNaseI and 1 ⁄ 5ofa
protease inhibitor tablet were added. This suspension was
incubated at 37 °C for 40 min and spun at 26 000 g for
40 min to collect the periplasmic fraction. The pellet from
the last step was resuspended in 20 mm Tris ⁄ HCl pH 7.5,
and French-pressed three times at 1000 psi. Cell debris and

the insoluble fraction were removed by centrifugation at
12 000 g for 30 min. The supernatant was centrifuged at
150 000 g for 2 h to collect the membranes, which were
resuspended in 5 mL 20 mm Tris ⁄ HCl pH 7.5 and stored at
)80 °C. The supernatant from the 150 000 g step was kept
as cytoplasm and stored at )20 °C.
Paracoccus pantotrophus strains were grown anaerobically
in 50 mL minimal salt medium supplemented with 20 mm
sodium nitrate, to an A
600
of  1 before harvesting at
6000 g for 10 min. Pellets were resuspended in BugBuster
(Novagen, now Merck) at 0.2 g dry pelletÆmL
)1
and incu-
bated at room temperature with rocking for 30 min. Ten
millilitre samples of lysate or 3 mL membrane extracts con-
taining equal protein concentrations (total 30 mg) were run
on an SDS ⁄ PAGE gel for analysis. Western blots to detect
strep II tags were performed using an alkaline phos-
phatase conjugate of strep-tactin antibody (IBA, Go
¨
ttingen,
Germany) according to the manufacturer’s instructions.
For all SDS ⁄ PAGE, the markers used were SeeBlue Plus 2
(Invitrogen, Paisley, UK).
Recombinant production and purification of NirF
and its variants
Overexpression was performed in the E. coli strain BL21
codonplus (RIPL) (Stratagene, Leicester, UK). All cells

expressing protein were grown at 37 °C in 500 mL volumes
of LB broth in 2 L flasks from overnight starter cultures to
an A
600
of 0.6–0.7 and transferred to 16 °C before induc-
tion with 0.2 mm isopropyl thio-b-d-galactoside. After fur-
ther incubation for 16 h, the cells from the 2 L culture were
harvested and resuspended in 6 mL 50 mm Tris ⁄ HCl pH
7.5, containing a trace amount of DNaseI and protease
inhibitor tablet. Periplasmic fractions were obtained by
incubating the resuspended cells with 1 mgÆmL
)1
polymyxin
B sulphate at 4 °C for 45 min and removing the insoluble
material by centrifuging at 15 000 g for 40 min. The peri-
plasmic fraction was applied to 5 mL of Strep-Tactin-
Sepharose (IBA) equilibrated with 50 mm Tris ⁄ HCl,
250 mm NaCl (pH 7.5). The column was washed with six
column volumes of 50 mm Tris ⁄ HCl, 250 mm NaCl (pH
7.5) and the protein was eluted with 50 mm Tris ⁄ HCl (pH
7.5), 150 mm NaCl, 2.5 mm desthiobiotin (IBA) according
to the manufacturer’s instructions. All the NirF variants
were also produced in the same manner. The purity of the
samples was checked by running SDS ⁄ PAGE 10% Bis ⁄ Tris
NuPAGE gels (Invitrogen).
Periplasmic NirF binds d
1
heme S. Bali et al.
4952 FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS
MS with the purified NirF

For MALDI analysis, the purified protein was desalted
on a C18 column. Approximately 10 lm of the purified
solution was premixed with the matrix: a-cyano-4-hy-
droxycinnamic acid (10 mm in 35% aqueous acetonitrile,
0.1% trifluoroacetic acid) at a 1 : 1 ratio and 1 lLof
mixture applied directly to the sample plate. The droplet
was air-dried before analysis in the MS. MALDI spectra
were obtained in reflectron mode and a nitrogen laser,
emitting 337 nm light in a 3 ns pulse, was the ionization
source. The accelerating voltage in the ion source was
30 kV.
Acknowledgements
This work was funded by research grant BBE0229441
from the Biotechnology and Biological Sciences
Table 1. Strains and plasmids used in the present study.
Strain Genotype and description Reference
Escherichia coli DH5a supE44 DlacU169 (f80 lacZDM15) hsdR17 recA1
endA1 gyrA96 thi1 relA1 (general cloning vehicle)
Gibco BRL
S17-1 Sm
r
pro r
)
m
+
RP4-2 integrated (Tc::Mu) (Km::Tn7) [28]
Paracoccus pantotrophus GB17 wild-type P. pantotrophus strain, Strep
R
[26]
nirF::kan

R
or SBN3 Chromosomally disrupted copy of nirF This work
DnirF or SBN11 Unmarked deletion in nirF This work
SBN13 DnirF derivative with pEG276-NirF-strepII This work
SBN15 DnirF derivative with pEG276 This work
SBN19 DnirF derivative with pEG276-NirF
H41A
This work
SBN20 DnirF derivative with pEG276-NirF
(no signal sequence)
This work
SBN21 DnirF derivative with pEG276-NirF
H41K
This work
SBN22 DnirF derivative with pEG276-NirF
H41C
This work
SBN23 DnirF derivative with pEG276-NirF
H41M
This work
SBN24 DnirF derivative with pEG276-NirF
D129A
This work
SBN25 DnirF derivative with pEG276-NirF
D129Q
This work
SBN26 DnirF derivative with pEG276-NirF
(D4-17)
This work
SBN28 DnirF derivative with pEG276-NirF

(nirC signal sequence)
This work
Plasmids Description Reference
pTZ19R Amp
R
, general cloning vector Fermentas
pEG276 Gent
R
, expression vector [25]
pUC4K Amp
R
, source of kan
R
cassette Pharmacia
pJQ200ks Gent
R
, suicide vector [29]
pRVS1 Strep
R
, suicide vector [24]
pRVS2 Strep
R
, Gent
R
, suicide vector This work
pJQ200ks-nirF::kan
R
Contains cassette for nirF disruption This work
pRVS2-DnirF Contains cassette for unmarked deletion of nirF This work
pEG276-NirF-strepII P. pantotrophus nirF cloned into pEG276 with strep II tag This work

pEG276-NirF
H41A
P. pantotrophus nirF
H41A
cloned into pEG276 This work
pEG276-NirF
H41K
P. pantotrophus nirF
H41K
cloned into pEG276 This work
pEG276-NirF
H41M
P. pantotrophus nirF
H41M
cloned into pEG276 This work
pEG276-NirF
H41C
P. pantotrophus nirF
H41C
cloned into pEG276 This work
pEG276-NirF
D129A
P. pantotrophus nirF
D129A
cloned into pEG276 This work
pEG276-NirF
D129Q
P. pantotrophus nirF
D129Q
cloned into pEG276 This work

pEG276-NirF
(NirC signal sequence)
P. pantotrophus nirF
(NirC signal sequence)
cloned into pEG276 This work
pEG276-NirF
(no signal sequence)
P. pantotrophus nirF
(no signal sequence)
cloned into pEG276 This work
pEG276-NirF
(D4-17)
P. pantotrophus nirF
(D4-17)
cloned into pEG276 where
(D4-17) is a deletion of N-terminal GXGX
2
GX
7
G motif
This work
pET-22b Expression vector, Amp
R
, Novagen
pET-22b-NirF-strepII P. pantotrophus nirF cloned into pEG276 with strep II tag This work
pET-22b -NirF
H41A
P. pantotrophus nirF
H41A
cloned into pET-22b This work

pET-22b -NirF
H41K
P. pantotrophus nirF
H41K
cloned into pET-22b This work
pET-22b -NirF
H41M
P. pantotrophus nirF
H41M
cloned into pET-22b This work
pET-22b -NirF
H41C
P. pantotrophus nirF
H41C
cloned into pET-22b This work
pET-22b -NirF
D129A
P. pantotrophus nirF
D129A
cloned into pET-22b This work
pET-22b -NirF
D129Q
P. pantotrophus nirF
D129Q
cloned into pET-22b This work
S. Bali et al. Periplasmic NirF binds d
1
heme
FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS 4953
Research Council to S. J. F and M. J. W. Dr Andreas

Schlueter and Alfred Phu
¨
ler at the University of Biele-
feld are thanked for their kind gift of plasmid pMS255.
Dr Katalin di Gleria at The Weatherall Institute of
Molecular Medicine, University of Oxford is thanked
for her assistance with the MS of the proteins. Amy
Varney and Christopher Greening are thanked for con-
structing some of the NirF mutants.
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Supporting information
The following supplementary material is available:
Fig. S1. Expression of NirF in Paracoccus pantotro-
phus DnirF strain.
Fig. S2. Multiple sequence alignment of NirF with d
1
domain of cd
1
, NirS.
Fig. S3. Sequence alignment of NirF with Met8p and
CysG.
Fig. S4. Purification of recombinant NirF and its vari-

ants.
Doc. S1. Construction of nirF::kan
R
disruption cas-
sette.
Doc. S2. Construction of DnirF cassette for generating
unmarked nirF deletion and modification of suicidal
vector pRVS1.
Doc. S3. Construction of marked and unmarked dele-
tion in nirF.
Table S1. Oligonucleotides used in this work.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
S. Bali et al. Periplasmic NirF binds d
1
heme
FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS 4955