Isolation and biochemical characterization of two soluble iron(III)
reductases from
Paracoccus denitrificans
Jir
ˇı
´Mazoch
1
, Radek Tesar
ˇı
´k
2
, Vojte
ˇch Sedla
´c
ˇek
1
, Igor Kuc
ˇera
1
and Jaroslav Tura
´nek
2
1
Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic;
2
Department of Immunology,
Veterinary Research Institute, Brno, Czech Republic
Two soluble enzymes (FerA and FerB) catalyzing the
reduction of a number of iron(III) complexes by NADH,
were purified to near homogeneity from the aerobically
grown iron-limited culture of Paracoccus denitrificans using
a combination of anion-exchange chromatography (Seph-
arose Q), chromatofocusing (Mono P), and gel permeation
chromatography (Superose 12). FerA is a monomer with
a molecular mass of 19 kDa, whereas FerB exhibited a
molecular mass of about 55 kDa and consists of probably
two identical subunits. FerA can be classified as an
NADH:flavin oxidoreductase with a sequential reaction
mechanism. It requires the addition of FMN or riboflavin
for activity on Fe(III) substrates. In these reactions, the
apparent substrate specificity of FerA seems to stem exclu-
sively from different chemical reactivities of Fe(III) com-
pounds with the free reduced flavin produced by the enzyme.
Observations on reducibility of Fe(III) chelated by vicinal
dihydroxy ligands support the view that FerA takes part in
releasing iron from the catechol type siderophores synthes-
ized by P. denitrificans. Contrary to FerA, the purified FerB
contains a noncovalently bound redox-active FAD coen-
zyme, can utilize NADPH in place of NADH, does not
reduce free FMN at an appreciable rate, and gives a ping-
pong type kinetic pattern with NADH and Fe(III)-nitrilo-
triacetate as substrates. FerB is able to reduce chromate, in
agreement with the fact that its N-terminus bears a homo-
logy to the previously described chromate reductase from
Pseudomonas putida. Besides this, it also readily reduces
quinones like ubiquinone-0 (Q
0
) or unsubstituted p-benzo-
quinone.
Keywords: iron(III) reductase; NADH:flavin oxidoreduc-
tase; Paracoccus denitrificans.
Ferric iron [Fe(III)] reductases, which catalyze the reduction
of various complexed forms of Fe(III), are of ubiquitous
occurrence in nature [1]. Conversion of Fe(III) to more
soluble Fe(II) generally increases the bioavailability of iron
and occurs either outside the cell [in conjunction with an
upward Fe(II) transport] or intracellularly (e.g. iron release
from the accumulated ferrisiderophores) [2]. In bacteria,
environmental Fe(III) can serve as a hydrogen sink,
allowing the regeneration of NAD
+
[3], or even as a
terminal electron acceptor in an anaerobic respiration
associated with translocation of H
+
and generation of a
transmembrane potential difference [4,5].
Fe(III) reductases constitute a heterogeneous group of
enzymes in respect to their biochemical properties. The
majority of the known bacterial Fe(III) reductases resides
in the cytoplasm, consists of one small polypeptide chain
lacking any distinguishable prosthetic group, reduces
Fe(III) at the expense of NADH or NADPH and requires
flavinasanelectrontransfermediator[6–13].Onthe
contrary, Fe(III) reductases of Fe(III) respirers are c-type
cytochromes excreted into the medium or exposed at the cell
surface where they can make direct contact with the solid
metal oxides [14,15]. A representative of eukaryotic type
enzymes is the FRE1 Fe(III) reductase of Saccharomyces
cerevisiae, a transmembrane flavocytochrome bwith prop-
erties similar to those of the NADPH oxidase of human
neutrophils [16]. In some instances the observed reduction
of Fe(III) possibly represents a side activity of an enzyme
that may catalyze a different reaction in vivo, as exemplified
by the enzymes flavin reductases [12], sulfite reductase [17]
or dihydropteridine reductase [18].
The facultative anaerobe Paracoccus denitrificans posses-
ses a flexible respiratory chain able to incorporate consid-
erable amounts of iron-containing proteins in response to
the changed growth conditions [19,20]. This ability depends
in part upon a capacity of the microbe to mobilize iron from
external and internal sources. Evidence for a high-affinity
iron-scavenging system involving low molecular weight,
Fe(III)-specific siderophores was first presented by Tait [21]
who isolated three iron-binding catechol compounds from
the culture supernatant of P. denitrificans and provided
initial data on their structure, metal-binding properties and
enzymes needed for their biosynthesis. Since then, several
Correspondence to I. Kuc
ˇera, Department of Biochemistry,
Faculty of Science, Masaryk University, Kotla
´r
ˇska
´2,
CZ-61137 Brno, Czech Republic.
Fax: + 420 541129623, Tel.: + 420 541129351,
E-mail: ikucera@chemi.muni.cz
Abbreviations: Bis/Tris, bis(2-hydroxyethyl)amino-tris(hydroxy-
methyl)methane; DHB, 2,3-dihydroxybenzoate; FNR, fumarate and
nitrate reductase regulator (Escherichia coli); FnrP, FNR homologue
from P. denitrificans; NfsB, oxygen-insensitive nitroreductase of
E. coli; PVDF, poly(vinylidene difluoride); pyrocat, pyrocatechol;
QR, quinone reductase.
(Received 30 September 2003, revised 27 November 2003,
accepted 5 December 2003)
Eur. J. Biochem. 271, 553–562 (2004) FEBS 2004 doi:10.1046/j.1432-1033.2003.03957.x
thorough investigations into the mechanism, stereospecific-
ity and kinetics of siderophore-mediated iron transport in
P. denitrificans have been carried out [22–24], but informa-
tion on the enzymology of Fe(III) reduction still remains
scarce and incomplete. Tait [21] described formation of
Fe(II) from the Fe(III)–siderophore complex upon incuba-
tion with NADH, succinate and an ultracentrifuged and
dialyzed cell-free extract while Dailey and Lascelles [25]
noticed reduction of Fe(III) citrate by NADH or succinate
as electron donors in the presence of crude cell extracts
containing membranes. More recently, Kucera and Match-
ova [26] succeeded in discriminating between the reduction
of Fe(III) sulfate by NADH catalyzed by cytosolic and
membrane fractions. The former reaction differed markedly
from the latter by about 10-times lower apparent K
m
value
for NADH, by a discernible stimulatory effect of FMN, and
by the inability of succinate to substitute for NADH.
The present report describes the results of studies leading
to the conclusion that at least two distinct soluble enzymes of
P. denitrificans exhibit an activity of Fe(III) reductase. These
enzymes were purified and their molecular and catalytic
properties were analyzed. The data obtained indicate that
one of them can be assigned to the NADH-dependent flavin
reductases while the other carries a bound flavin prosthetic
group and bears a resemblance to a previously described
enzyme capable of reducing chromate [27].
Materials and methods
Organism and cultivation
Paracoccus denitrificans, strain CCM 982 was obtained in
freeze-dried form from the Czech Collection of Micro-
organisms (CCM), Masaryk University, Brno, Czech
Republic. It was maintained on agar plates and stored at
4C. Growth was achieved via batch cultivation using
minimal succinate medium, containing (in m
M
) sodium
succinate, 50; KH
2
PO
4
, 33; Na
2
HPO
4
,17;NH
4
Cl, 50;
MgSO
4
, 1.1 and Fe(III) citrate, 0.5 l
M
. The final pH was
adjusted to 7.3 by dropwise addition of 0.1
M
NaOH.
Separate 750 mL aliquots were autoclaved in 3-L
Erlenmeyer flasks, inoculated with 20 mL of a pregrown
culture and incubated for 10–12 h at 30 Conareciprocal
shaker at 180 r.p.m. Cells were sedimented by centrifuga-
tion (6200 g,20minat4C), washed twice with 50 m
M
Tris/HCl buffer, pH 7.4, and then taken up in this buffer.
Preparation of the enzymes
For a typical purification 10 g wet cells harvested from
3-L culture were used. Chromatography was run on a
Pharmacia FPLC apparatus.
Step 1: Preparation of cell-free extract. Cell-free extracts
were made by passing the cell suspension twice through a
33-mL X-Press cell (AB Biox, Sweden), treating with 4 lgof
deoxyribonuclease (Sigma) per mL for 30 min at room
temperature to reduce the viscosity caused by DNA and
removing the cellular debris by centrifugation at 109 000 g
for 40 min in a rotor-type 45 TI (Beckman). The Fe(III)
reductase-containing extracts were stored at )15 C until
commencement of purification.
Step 2: Ion exchange chromatography. About 40 mL of
cell-free extract, prepared as described above, were loaded
onto an HP Sepharose Q (Pharmacia) column (XK26:
10 cm ·2.6 cm) pre-equilibrated with 20 m
M
Tris/HCl,
pH 7.4, and washed by the same buffer. The proteins were
eluted in two phases with 300 mL of linear gradient of
0–0.6
M
NaCl and 50 mL of linear gradient of 0.6–1
M
NaCl at a flow rate of 0.8 mLÆmin
)1
.Fractionsof5mL
were collected and those displaying Fe(III) complex reduc-
tase activity were pooled (each activity peak separately),
concentrated in an ultrafiltration cell (Amicon) through
YM-3 membrane (Millipore) and then dialyzed against
20 m
M
Tris/HCl, pH 7.4.
Step 3: Chromatofocusing. Two buffer systems in this
chromatography on MonoP HR 5/20 (Pharmacia) column
(20 ·0.5 cm) for two distinct enzymes were employed.
First, starting with 0.075
M
Tris/acetate, pH 9.3 used
Polybuffer 96, second one started with 0.025
M
Bis-Tris/
iminodiacetic acid, pH 7.1, and Polybuffer 74 (Pharmacia)
was applied here, according to instructions of the manu-
facturer. The flow rate was 0.3 mLÆmin
)1
during sample
loading and 0.5 mLÆmin
)1
during focusing. Fractions of
0.5 mL were collected. Pooled active fractions were desalted
on PD-10 columns (Supelco) equilibrated by the start buffer
for the next step.
Step 4 (optional): Chromatofocusing. The same condi-
tions as in the previous step with Polybuffer 74 were used.
Peak fractions were collected.
Step 5: Gel permeation chromatography. Maximum
0.5 mL of sample was loaded onto Superose 12 (Pharmacia)
analytical column (30 cm ·1 cm) equilibrated by 50 m
M
Tris/HCl, pH 7.4 containing 150 m
M
NaCl. The flow rate
after sample application was 0.5 mLÆmin
)1
. For determin-
ation of molecular masses, the same column was calibrated
by marker proteins including alcohol dehydrogenase
(141 kDa), albumin (67 kDa), ovalbumin (43 kDa), chy-
motrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa).
Step 6 (optional): Preparative native electrophoresis. In
some instances (e.g. for N-terminal sequenation), final
purification of the enzymes was performed from the activity
bands on native gels (see below). The proteins were
recovered by electroelution using electroelution tubes and
a Mini Protean Cell (Bio-Rad) with Tris/glycine buffer,
pH 8.3 [no sodium dodecyl sulfate (SDS)] at constant
current 8 mA per one electroelution tube for 5.5 h.
Determination of protein concentration
Protein concentration was determined by the method of
Lowry et al. [28] using bovine serum albumin as the
standard. Protein elution profiles from chromatographic
columns were also monitored by measuring fractional
absorbance at 280 nm.
Enzyme assays
Fe(III) reductase activity was determined according to
a variant of the method by Dailey and Lascelles [25]. The
554 J. Mazoch et al. (Eur. J. Biochem. 271)FEBS 2004
assay measures the formation of the Fe(II)(ferrozine)
3
complex at 562 nm (absorption coefficient ¼28 m
M
)1
Æ
cm
)1
)in25m
M
Tris/HCl buffer, pH 7.4, 30 C, with
0.8 m
M
ferrozine, 0.05 m
M
FMN and 0.2 m
M
Fe(III)
complex. The reaction was started by the addition of
0.15 m
M
NADH.
Chromate reductase activity was assayed at 30 Cin
0.5-mL reaction mixtures containing 25 m
M
Tris/HCl
buffer, pH 7.4, 0.045 m
M
K
2
CrO
4
,0.15m
M
NADH, and
5–30 lL of enzyme preparation. Chromate concentration
was quantified by adding samples to the reagent consisting
of 0.1
M
H
2
SO
4
and 0.01% 1,5-diphenyl carbazide and
measuring the absorbance at 540 nm [27].
Quinone reductase and flavin reductase activities were
monitored by following the disappearance of the NADH
absorbance at 340 nm in 25 m
M
Tris/HCl buffer, pH 7.4,
30 C. Concentrations of electron acceptors were 100 l
M
for quinones when measuring quinone reductase and
50 l
M
for FMN in flavin reductase assay. Stock solutions
of quinones were prepared as 5 or 10 m
M
in 20% acetone
(v/v).
Stopped-flow experiments
A JASCO J-810 stopped-flow spectrophotometer with a
dead time of 5.2 ms and a cell path length of 1.5 mm was
used to measure redox reactions of Fe(III) complexes with
chemically reduced FMN. A solution of 1.25 m
M
FMN in
25 m
M
Tris/HCl, pH 7.4, containing PtO
2
(0.1 mgÆmL
)1
)as
a catalyst [29] was made anaerobic with argon bubbling and
then a stream of hydrogen gas was introduced to convert
FMN into FMNH
2
. Following the sedimentation of the
catalyst, the yellowish supernatant was placed in one
syringe, and the second syringe was filled with a buffered
solution of 1.6 m
M
ferrozine and 0.4 m
M
Fe(III) complexed
with an appropriate ligand. After 1 : 1 mixing, changes in
absorbance at 562 nm were recorded. Data from at least six
repetitions were averaged and processed with the software
package supplied with the instrument, giving the desired
initial velocity values.
Mass spectrometry
The mass spectra of proteins were recorded on a Bruker
Reflex IV mass spectrometer. Prior analysis, the samples
were dialyzed against 10 m
M
Tris/HCl, pH 7.4, and then
concentrated to a final value of about 1–10 lgofproteinper
lL using a Speed-Vac concentrator (Savant, Holbrook,
USA). One microliter each of sample was mixed with 2 lLof
matrix solution (ferulic acid, saturated in 50% acetonitrile,
1% trifluoroacetic acid). Aliquots (0.6 lL) were placed on
the target and left to dry. The spectra were taken in positive-
ion linear mode. External mass calibration was performed
using the molecular ions from the bovine insulin at
5734.5 Da and the horse myoglobin at 16952.6 Da 100
single shot spectra were accumulated in each mass spectrum.
Gel electrophoresis
One-dimensional SDS gel electrophoresis was carried out in
10–15% polyacrylamide gels [30], containing 0.1% SDS,
using a Mini Protean II (Bio-Rad) vertical electrophoretic
system. Staining was carried out using Coomassie brilliant
blue R-250. Standard molecular mass markers were from
Fluka. Non-denaturing electrophoresis was carried out in
the same manner, but with omission of SDS from the gel
running and loading buffers, and the sample was not
pretreated under denaturing conditions. Staining was car-
ried out using Coomassie blue R-250 or by zymogram
analysis. The latter entailed overlay of the gel with 0.15 m
M
NADH and 0.8 m
M
ferrozine in 50 m
M
Tris/HCl buffer,
pH 7.4, containing 0.05 m
M
FMN. Fe(III)nitrilotriacetic
acid solution in the same buffer was added to the resulting
concentration of 0.2 m
M
. Bands of Fe(III) reductase activity
were detected as red zones against a slightly reddish
background.
Spectroscopic measurements
Absorption spectra of the enzymes were taken with an
Ultrospec 2000 UV/visible spectrophotometer (Pharmacia)
using a 1-cm light path cell. Fluorescence spectra were
obtained using an LS 50B spectrofluorimeter (Perkin Elmer).
Thin-layer chromatography analysis of flavins
About 0.4 mg of the purified flavoprotein, dissolved in
0.1 mL of buffer, was mixed with 0.9 mL of methanol and
the mixture was heated at 95 C for 15 min. After cooling
on ice and centrifugation at 12 000 gfor 5 min, the
supernatant was dried with a Speed-Vac concentrator.
Water (10 lL) and methanol (10 lL) were added to the
residue, and the solution was analyzed by TLC employing a
cellulose gel plate (DC-Alufolien Cellulose, 0.1 mm thick-
ness; Merck AG, Darmstadt, Germany) and the upper layer
of an n-butyl alcohol/glacial acetic acid/water (4 : 1 : 5)
mixture as the solvent phase. Methanol/water (1 : 1)
solution of riboflavin, FMN and FAD were spotted as
reference and the migration of the compounds were
visualized with UV light.
N-Terminal sequence analysis
The highly purified enzymes were subjected to SDS/PAGE
(15% polyacrylamide) and then electroblotted onto a
poly(vinylidene difluoride) membrane (Bio-Rad). Selected
bands were cut out of the Coomassie Blue-stained blots
and analyzed by the Edman method with a 491 protein
sequencer (Perkin-Elmer Applied Biosystems) according to
the standard program (PL PVDF Protein).
Homology searching and sequence alignments
Homology searching against the databases was performed
with the Basic Logical Alignment Search Tool (
BLAST
)on
NCBI Server (http://www.ncbi.nih.nlm.gov). Alignments of
the sequences were produced using
CLUSTAL W
Multiple
Sequence Alignment software on NPS@Server (http://
npsa-pbil.ibcp.fr).
Kinetic data analysis
Input data represented the mean values from at least three
replicates. Nonlinear regression analysis was performed
FEBS 2004 P. denitrificans Fe(III) reductases (Eur. J. Biochem. 271) 555
with the kinetic software package
EZ
-
FIT
developed by
Perrella [31].
Results
Choice of a Fe(III) substrate for the enzyme assay
Most of Fe(III) reductases have no known native substrates,
but they exhibit a fairly weak specificity and can reduce
many artificial Fe(III) complexes. Therefore, we explored
a series of five compounds (Fe(III)EDTA, Fe(III)EGTA,
Fe(III)nitrilotriacetic acid, Fe(maltol)
3
and FeCl
3
) as poten-
tial artificial substrates for measuring the activity in cell-free
extracts by the ferrozine assay. When no flavin was added
exogenously, we found the following specific activities [nmol
Fe(II)(ferrozine)
3
Æs
)1
Ægprotein
)1
, means of three replicates]:
2, 151, 146, 16, 2, whereas with 50 l
M
FMN the respective
values were: 5, 210, 392, 323, 83. These results told us that
the Fe(III)nitrilotriacetic acid substrate together with FMN
would best serve the purpose of activity measuring during
enzymes purification. Contrary to some [8,13], but in
accordance with other [10] previous research, our assay
for the enzymes from P. denitrificans was not significantly
sensitive to oxygen, as air removal from the reaction mixture
with a stream of argon increased the rate of Fe(II)(ferro-
zine)
3
production by 10% maximum. Based on this finding,
all further measurements could be performed under ambient
oxygen pressure.
Purification of the Fe(III) reductases
Preliminary experiments established that cultivation of
P. denitrificans in a growth medium with limiting initial
levels of Fe(III) citrate (0.5 l
M
instead of the normally used
concentration of 30 l
M
) increased the specific activity of
Fe(III) reductase in the cell-free extract about 1.5-fold.
Accordingly, the enzyme was isolated from the iron-
deficient cells that had reached the stationary phase after
aerobic growth. A summary of the data of the purification
is given in Table 1 for a typical preparation. Aspects
deserving comment are as follows:
(i) Assay of the fractions in the HP-Sepharose Q elution
profile identified two peaks of Fe(III) reductase activity,
eluting at approximately 50–150 m
M
and 250–400 m
M
NaCl. They were termed FerAand FerB, respectively.
Peak fractions were pooled separately and purification was
continued for each pooled peak of activity.
(ii) The presence of two Fe(III) reductases in cell-free
exacts could also be directly demonstrated by native gel
electrophoresis of the cell-free extracts followed by activity
staining, which produced two bands with the intensity ratio
of about 10 : 1. The more intense band corresponded to the
enzyme with a higher electrophoretic mobility towards the
anode.
(iii) Preparative chromatofocusing proved to be an
efficient purification step, which could be used twice for
FerB, but not for FerA due to less resistance to pH changes.
Chromatofocusing enabled us to determine isoelectric
points of FerA and FerB as 6.9 and 5.5, respectively. These
values are in accord with the observed chromatographic
behavior of the enzymes at the Sepharose Q step.
(iv) During optimization of the purification protocol the
possibility of employing a hydrophobic interaction chro-
matography was also noticed. After loading the test sample
on a Phenyl Superose column (Pharmacia) or on a Hema-
Bio 1000 Phenyl column (Tessek, Prague) and applying a
1.5–0
M
ammonium sulfate gradient, FerA was typically
eluted at 80–90% of the gradient compared to FerB eluted
at 45–50%. FerA thus seems to be considerably more
hydrophobic than FerB.
Determination of molecular mass
SDS/PAGE analysis gave single Coomassie blue staining
bands at a position of an approximate M
r
of 18 000 for both
the purified FerA and FerB (Fig. 1). These values were
further refined by applying a MALDI-MS analysis, which
revealed peaks at m/z18 814 and 18 917 for FerA and at
m/z20 196 for FerB. We are currently unable to identify
unequivocally the reason for the presence of two peaks in
the spectrum of FerA.
As the enzymes could be separated in a column of
Superose 12, the same column was used to estimate their
molecular sizes. By calibration of the column with standard
proteins of known molecular masses, the molecular masses
of native FerA and FerB were estimated as 25 kDa and
Table 1. Purification of P. denitrificans Fe(III) reductases. The activity was determined following absorbance of Fe(II)(ferrozine)
3
complex at
562 nm. Specific activity is expressed as enzyme activity per mg of total protein.
Treatment
Volume
(mL)
Total protein
(mg)
Total activity
(nkat)
Specific activity
(nkatÆmg
)1
)
Purification
factor (-fold)
Yield
(%)
FerA
Cytosolic extract 25.5 517.4 120.3 0.23 1 100
Sepharose Q 2.8 7.81 59.1 7.57 32.9 49.13
Polybuffer exchanger 96 2.8 0.155 15.9 102.58 446 13.22
Superose 12 HR 10/30 4.0 0.028 9.17 327.50 1424 7.62
FerB
Cytosolic extract 25.5 517.4 85.50 0.17 1 100
Sepharose Q 2.85 23.73 38.37 1.62 9.8 44.9
Polybuffer exchanger 74 1.5 0.434 16.47 37.93 230 19.3
Polybuffer exchanger 74 1.0 0.069 5.22 75.23 457 6.1
Superose 12 HR 10/30 0.6 0.024 2.25 93.89 568 2.6
556 J. Mazoch et al. (Eur. J. Biochem. 271)FEBS 2004
55 kDa, respectively. From these results, we concluded that
FerA exists in a monomeric form in the native state and that
FerB has an oligomeric structure (a homodimer or, less
probable, a homotrimer).
N-Terminal sequence comparison
The N-terminal amino acid sequences of FerA and FerB
were determined and are presented in Fig. 2(A). A BLAST
search revealed sequence similarity of 73% and 65%
(sequence identity of 50% in both cases) of this region in
FerB with chromate reductase from Pseudomonas putida
andflavinreductaseformPseudomonas syringae (Fig. 2B),
respectively. On the contrary, no significant similarity was
found between the N-terminus of FerA and any known
oxidoreductase.
Spectral properties
Measurement of the UV-visible absorption spectrum of
FerA (about 95% pure, 0.04 mgÆmL
)1
) showed no chro-
mophore uniquely attributable to this enzyme. On the other
hand, the solution of FerB was yellowish in color and
exhibited absorption bands at 380 and 448 nm (Fig. 3),
diagnostic for the presence of a flavin group. When 0.4 m
M
NADH was added, the absorption peak at 448 nm
disappeared, indicating a reducibility of the bound flavin
by the physiological electron donor. The possession by FerB
of a flavin cofactor was also apparent from the fluorescence
Fig. 1. SDS/PAGE followed by silver staining. Lane 1, 10 lgproteinof
the starting cell-free extract; lane 2, 0.3 lgofpurifiedFerA;lane3,
1lg of purified FerB; lane 4, molecular mass markers indicated (in
kDa) on the right.
Fig. 2. N-Terminal amino acid sequences of P. denitrificans FerA and FerB (A) and comparison of the N-terminal amino acid sequence of FerB with the
sequences of other oxidoreductases (B). N-Terminal sequences were determined by Edman degradation. Multiple alignment of chosen sequences was
performed using
CLUSTAL W
programme. Fre, flavin reductases of Pseudomonas syringae pv. syringae B728a (gi:23469150) and of P. syringae pv.
tomato DC3000 (gi:28870863); CR, chromate reductase of Pseudomonas putida (gi:14209680); ?, a hypothetical protein (sequence derived from the
known DNA sequence). Identical (w), strongly similar (:) and weakly similar (.) residues are indicated.
Fig. 3. Absorption spectra of as isolated (—) and NADH reduced (- - -)
FerB. The spectrum of FerB, 0.2 mg proteinÆmL
)1
, were measured
under aerobic conditions in 25 m
M
Tris/HCl buffer (pH 7.4),
employing the buffer solution as a reference, with a light path of 1 cm.
The reduced spectrum was obtained 5 min after the addition of
0.4 m
M
NADH.
FEBS 2004 P. denitrificans Fe(III) reductases (Eur. J. Biochem. 271) 557