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
; 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,

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

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

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

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

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

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

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

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

–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

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

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

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

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

(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.

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

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

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

)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

), 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

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

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

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

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

¨
p V (1997) Cytochrome cd
1
structure:
unusual haem environments in a nitrite reductase and
analysis of factors contributing to beta-propeller folds.
J Mol Biol 269, 440–455.
6Fu
¨
lo
¨
p V, Moir JWB, Ferguson SJ & Hajdu J (1995)
The anatomy of a bifunctional enzyme: structural basis
for reduction of oxygen to water and synthesis of nitric
oxide by cytochrome cd
1
. Cell 81, 369–377.
7 Chang CK, Timkovich R & Wu W (1986) Evidence
that heme d
1
is a 1,3-porphyrindione. Biochemistry 25,
8447–8453.
8 Matthews JC & Timkovich R (1993) Biosynthetic ori-
gins of the carbon skeleton of heme d
1
. Bioorg Chem
21, 71–82.
9 Kawasaki S, Arai H, Igarashi Y & Kodama T (1995)
Sequencing and characterisation of the downstream
region of the genes encoding nitrite reductase and cyto-

1
biosynthesis. J Bacteriol
179, 235–242.
14 Storbeck S, Walther J, Mu
¨
ller J, Parmar V, Schiebel
HM, Kemken D, Du
¨
lcks T, Warren MJ & Layer G
(2009) The Pseudomonas aeruginosa nirE gene encodes
the S-adenosyl-L-methionine-dependent uroporphyrino-
gen III methyltransferase required for heme d
1
biosyn-
thesis. FEBS J 276, 5973–5982.
15 Zajicek RS, Bali S, Arnold S, Brindley AA, Warren MJ
& Ferguson SJ (2009) d
1
haem biogenesis – assessing
the roles of three nir gene products. FEBS J 276, 6399–
6411.
16 Hasegawa N, Arai H & Igarashi Y (2001) Two c-type
cytochromes, NirM and NirC, encoded in the nir gene
cluster of Pseudomonas aeruginosa act as electron
donors for nitrite reductase. Biochem Biophys Res
Commun 288, 1223–1230.
17 Schubert HL, Raux E, Brindley AA, Leech HK,
Wilson KS, Hill CP & Warren MJ (2002) The struc-
ture of Saccharomyces cerevisiae Met8p, a bifunctional
dehydrogenase and ferrochelatase. EMBO J 21, 2068–

1
and other heme cofactors
from bacteria. Ciba Found Symp 180, 228–238.
24 Van Spanning RJ, Wancell CW, De Boer T, Hazelaar
MJ, Anazawa H, Harms N, Oltmann LF & Stouthamer
AH (1991) A method for introduction of unmarked
mutations in the genome of Paracoccus denitrificans –
construction of strains with multiple mutations in the
genes encoding periplasmic cytochrome-C
550
, cyto-
chrome-C
551
, and cytochrome-C
553
. J Bacteriol 173,
6962–6970.
25 Gordon EH, Sjo
¨
gren T, Lo
¨
fqvist M, Richter CD,
Allen JW, Higham CW, Hajdu J, Fu
¨
lo
¨
pV&
Ferguson SJ (2003) Structure and kinetic properties
of Paracoccus pantotrophus cytochrome cd
1

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,
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S. Bali et al. Periplasmic NirF binds d
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FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS 4955