Báo cáo khóa học: Isolation and biochemical characterization of two soluble iron(III) reductases from Paracoccus denitrificans docx

Isolation and biochemical characterization of two soluble iron(III)
reductases from
Paracoccus denitrificans
Jir
ˇ
ı
´
Mazoch
1
, Radek Tesar
ˇ
ı
´
k
2
, Vojte
ˇ
ch Sedla
´
c
ˇ
ek
1
, Igor Kuc
ˇ
era
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 b with 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
ˇ
era, Department of Biochemistry,
Faculty of Science, Masaryk University, Kotla

´
r
ˇ
ska
´
2,
CZ-61137 Brno, Czech Republic.
Fax: + 420 541129623, Tel.: + 420 541129351,
E-mail:
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) Ó 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
4 °C. 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,20minat4°C), 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) Ó FEBS 2004
assay measures the formation of the Fe(II)(ferrozine)
3
complex at 562 nm (absorption coefficient ¼ 28 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 g for 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 (). 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
Ó 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 ÔFerAÕ and ÔFerBÕ, 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/z 18 814 and 18 917 for FerA and at
m/z 20 196 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) Ó 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,
1 lg 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.
Ó FEBS 2004 P. denitrificans Fe(III) reductases (Eur. J. Biochem. 271) 557

spectrum where the enzyme excited at 446 nm shows a
fluorescence emission with a maximum at 506 nm.
Identification of the flavin moiety of FerB
Exposure of FerB to heated methanol released a yellow
soluble compound from the holoenzyme. Thin-layer chro-
matography of the methanol extract gave a single fluores-
cent spot with a R
f
of 0.075. Under identical conditions,
riboflavin, FMN and FAD exhibited R
f
values of 0.40, 0.19
and 0.075, respectively. Thus, we concluded that FerB
contains a noncovalently bound FAD.
Substrate specificity
In order to investigate the catalytic abilities of the isolated
enzymes, a specificity study was undertaken with Fe(III)ni-
trilotriacetic acid and NADH as reference substrates. The
rate of reduction of different Fe(III) compounds, under the
conditions specified, is shown in Table 2. FerA has an
absolute requirement for the flavin and cannot effectively use
NADPH as an electron donor in place of NADH. A substi-
tution of riboflavin for FMN led to a decrease by 73% in the
reaction rate (not shown in Table 2). Unlike FerA, FerB
does not require exogenous flavin and utilizes both NADH
and NADPH. Probably the most striking difference between
FerA and FerB lies in the inability of the latter enzyme to
reduce Fe(III) complexed by vicinal dihydroxy groups, as
present in maltol, pyrocatechol and 2,3-dihydroxybenzoic
acid. In line with this, we also observed that FerA, but not

FerB, exhibited a reducing activity towards the complex
of Fe(III) with a crude isolate of parabactin, the natural
catecholic siderophore of P. denitrificans (data not shown).
By monitoring the absorbance at 340 nm, we observed
that the addition of FMN to FerA resulted in a significant
rate of NADH oxidation also in the absence of Fe(III),
which suggested the presence of an NADH : FMN oxido-
reductase activity. Its value typically amounted to
330 nmol NADHÆs
)1
Æmg protein
)1
. For this reason we
tested whether the reduction of Fe(III) could be due to the
FMNH
2
formed by FerA. This possibility gained support
from the stopped-flow experiments in which we compared
the rates of a direct nonenzymatic reduction of Fe(III)
complexes by the chemically prepared FMNH
2
.Therelative
rate values presented in the last column of Table 2 match
more closely those of FerA (r ¼ 0.92) rather than those for
FerB (r ¼ 0.77), demonstrating that FerA indeed may act
primarily as a flavin reductase, and, per se, not mediate the
subsequent redox reactions of Fe(III).
The identification of a sequence homology between
FerB and chromate reductases (Fig. 2B) prompted an
investigation of whether or not the Paracoccus enzyme

can reduce chromate. The positive answer is conveyed
by the results in Fig. 4, showing a significant speed-up in
Table 2. Substrate specificity of Fe(III) reductases from P. denitrificans, compared with the chemical reactivity of FMNH
2
. Experimental details are
described in the Material and methods section. The Fe(III) complexes were used at a final concentration of 0.2 m
M
. The relative reaction velocity for
NADPH was related to that of NADH as 100%. The relative rates for the different Fe(III) complexes were related to that of Fe(III)nitrilotriacetic
acid as 100%. The relative rate zero means a relative reaction velocity of > 0.1%.
Substrate
Relative rate of Fe(II)(ferrozine)
3
complex formation
FerA FerB No enzyme
+50 l
M
FMN –FMN +50 l
M
FMN FMNH
2
NADH 100
a
100
b
100 –
NADPH 0 82.3 69.0 –
Fe(III)nitrilotriacetic acid 100
a
100

b
100 100
c
Fe(III)EGTA 125.5 54.2 54.9 106.4
Fe(III)EDTA 3.8 2.4 1.1 5.5
Fe(III)(maltol)
3
91.4 3.9 3.1 83.1
FeCl
3
29.0 0.7 0.7 –
Fe(III)citrate 65.8 3.7 8.4 36.4
Fe(III)(pyrocat)
3
37.3 0 0 13.0
Fe(III)(DHB)
3
76.8 0 0 28.0
a
100% ¼ 2.04 · 10
)7
molÆs
)1
Æmg protein
)1
;
b
100% ¼ 8.43 · 10
)9
molÆs

)1
Æmg protein
)1
;
c
100% ¼ 3.73 · 10
)5
M
Æs
)1
.
Fig. 4. Chromate reductase activity of FerB. Reaction mixture
(0.5 mL) containing 0.045 m
M
chromate, 0.15 m
M
NADH and 25 m
M
Tris/HCl buffer, pH 7.4, with (d) or without (s)5lg of FerB were
incubated at 30 °C. Aliquots were withdrawn at the indicated time
intervals and analyzed for the concentration of chromate.
558 J. Mazoch et al. (Eur. J. Biochem. 271) Ó FEBS 2004
the reduction of chromate by NADH, caused by the
presence of FerB. FerB also functioned as an efficient
quinone reductase. For a series of four quinones, the relative
activities were found as follows: 2,3-dimethoxy-5-methyl-
1,4-benzoquinone (ubiquinone-0), 100%; p-benzoquinone,
77%; menadione, 6%; duroquinone, 0.01% (100% ¼
2.42 lmol NADHÆs
)1

Æmg protein
)1
). The enzyme did not
catalyze the oxidation of NADH by FMN at an appreci-
able rate (specific activity less than 1 nmol NADHÆs
)1
Æ
mg protein
)1
).
Kinetic studies of reactions catalyzed by FerA and FerB
When studying the Fe(III) reductase reaction of FerA, there
are effectively three compounds to be considered in a kinetic
analysis: NADH, FMN and complexed Fe(III). Initial
velocity studies were carried out by varying the concentra-
tions of NADH in the presence of a series of concentrations
of Fe(III)nitrilotriacetic acid while maintaining FMN at a
constant concentration. As illustrated in Fig. 5(A), recipro-
cal plots are patterns of lines intersecting to the left of the
vertical axis. The FMN reductase reaction of FerA was
analyzed with NADH and FMN in the absence of Fe(III).
Under these conditions, kinetic patterns similar to those
found above were obtained (Fig. 5B). This behavior indi-
cates a sequential mechanism with NADH and FMN as
substrates.
The results obtained on studying the Fe(III) reductase
reaction of FerB are in Fig. 6. It can be seen that the
reciprocal plot for NADH as variable substrate at a series of
concentrations of Fe(III)nitrilotriacetic acid consists of a
series of parallel lines. Kinetic patterns of this type occur in

cases of ping-pong mechanisms. At the highest concentra-
tion of NADH the initial velocity of the enzyme turned out
to be slower than that predicted by the hyperbolic law.
Similar substrate inhibition was also observed in the 1/v vs.
1/[Fe(III)nitrilotriacetic acid] plot; this issue was, however,
not pursued further at this stage.
Table 3 lists values of the kinetic parameters for individ-
ual substrates, as estimated by computer fitting of velocity
data of Figs 5 and 6 to the appropriate kinetic equations.
Besides the data for Fe(III)nitrilotriacetic acid in Table 3,
we also determined the kinetic parameters for some other
Fe(III) complexes reducible by FerB. In the presence of a
saturation concentration (0.15 m
M
)ofNADH,thevalues
of K
m
and k
cat
are: (0.4 ± 0.2) m
M
and (0.91 ± 0.02) s
)1
for Fe(III)EGTA (1.6 ± 0.8) m
M
and (0.16 ± 0.03) s
)1
for
Fe(III)EDTA (0.4 ± 0.2) m
M

and (0.08 ± 0.01) s
)1
for
Fe(III)citrate, and (0.4 ± 0.2) m
M
and (0.015 ± 0.003) s
)1
for Fe(III) chloride. The kinetics of the reduction of Fe(III)
ligated with maltol could not be measured reliably due to
the difficulty caused by an intensive coloration of the
complex. The fact that all the obtained values K
m
are higher
than 0.2 m
M
confirms that the measurements reported in
Table 2 were performed under conditions of first-order
Fig. 5. Initial-velocity kinetics of the reactions catalyzed by FerA.
Measurements were performed with 0.2 lgFerAproteinÆmL
)1
in
25 m
M
Tris/HCl buffer, pH 7.4 at 30 °C. (A) Fe(III) reductase reac-
tion velocity as a function of NADH concentration in the presence of
eight (s), 12 (d), 20 (h), 40 (j)and67(n) lM Fe(III)nitrilotriacetic
acid at constant concentrations of FMN (50 l
M
) and ferrozine
(0.8 m

M
). (B) Flavin reductase reaction velocity as a function of FMN
concentration in the presence of 5 (s), 10 (d), 20 (h)and30(j) l
M
NADH. In this case both ferrozine and Fe(III)nitrilotriacetic acid were
absent. Data are presented as double-reciprocal plots.
Fig. 6. Initial-velocity kinetics of the Fe(III) reductase reaction cata-
lyzedbyFerB.Measurements were performed at 30 °Cwith
3 lgFerBproteinÆmL
)1
in 25 m
M
Tris/HCl buffer, pH 7.4, contain-
ing 0.8 m
M
ferrozine. Reciprocal initial velocity is plotted against the
reciprocals of NADH concentration at a series of fixed concentrations
of Fe(III)nitrilotriacetic acid equal to 40 (s), 66.7 (d), 100 (n)and200
(m) lM.
Ó FEBS 2004 P. denitrificans Fe(III) reductases (Eur. J. Biochem. 271) 559
kinetics and hence the relative rates listed here, being
proportional to the k
cat
/K
m
ratios, correctly express the
substrate specificity of FerB.
Discussion
In this study, we have demonstrated a coexistence of two
types of soluble Fe(III) reductases, FerA and FerB, in the

soil bacterium Paracoccus denitrificans. Both enzymes are
flavin-dependent, but in different ways: a flavin serves as a
dissociable cosubstrate for FerA (a flavin reductase-type
enzyme) and as a tightly bound redox-active cofactor of
FerB (a flavoprotein-type enzyme).
Flavin reductases are present in a variety of organisms
[12]. Extensive studies have been conducted, for instance,
with enzymes from E. coli [7,32] or luminous bacteria
Vibrio harveyi [33–40] and Vibrio fischeri [41–44]. Although
these enzymes catalyze similar reactions, they differ in
respect to substrate specificities and reaction mechanisms.
Accordingly, they can be classified into D, P and G types
based on whether NADH, NADPH or both NADH and
NADPH are utilized. Obviously, FerA falls into the
D
-type.
Another classification criterion distinguishes between flavin
reductases lacking a detectable prosthetic group and those
having a noncovalently bound flavin cofactor. Considering
the negative result of the spectroscopic search for a bound
flavin group and the sequential kinetic pattern obtained
(Fig. 5), we conclude that the first possibility applies,
namely that FerA accelerates a direct redox reaction
between NADH and a flavin. In this respect, it seems to
be similar to the
D
-type flavin reductase of V. harveyi [35].
The observed substrate specificity of FerA (Table 2) adds to
the previous appreciation of the biological role of a free
reduced flavin as a reducing agent for Fe(III) [2,12,13,32]

and broadens it to account for the reduction of the catechol-
type Fe(III) complexes. The high effectiveness of the redox
process mediated by free flavin is reflected by a low value of
the apparent Michaelis constant for the Fe(III) complex
with nitrilotriacetic acid (Table 3).
In the case of FerB, the distinctive spectral features
(Fig. 3) and the ping-pong kinetic patterns (Fig. 6) make it
likely that the enzyme shuttles between the forms containing
oxidized and reduced bound flavin during the catalytic
cycle. The fact that FerB appears rather inefficient in
reducing some substrates which are otherwise prone to
uncatalyzed reactions with free FMNH
2
(Table 2) suggests
that the protein moiety contributes significantly to the
observed substrate specificity. Although more direct infor-
mation on the three-dimensional structure of FerB is
obviously required before any firm conclusion can be drawn
as to how this specificity is achieved, we can tentatively
imagine two situations. In the first, the reduced flavin binds
more tightly than the oxidized one, so that the cofactor
becomes poorer electron donor and reactions of electron
acceptors with low standard redox potentials are hampered.
Our voltammetric measurements (unpublished results) have
revealed that the Fe(III)/Fe(II) pairs complexed with maltol,
pyrocatechol and dihydroxybenzoate, which do not accept
electrons from FerB, have indeed the lowest standard redox
potentials among the iron compounds listed in Table 2.
Alternatively, FerB may carry a buried substrate binding
cavity whose size allows for interaction with smaller electron

acceptors like 1 : 1 Fe(III) complexes, chromate or qui-
nones, but not with the rather bulky 1 : 3 Fe(III) complexes
or exogenous free flavins.
A survey of the literature indicates that FerB shares some
biochemical properties with other enzymes. Its relatedness
to the earlier described chromate reductase of Pseudomonas
putida [27] is apparent from a match in the N-terminal
sequence (Fig. 2B) and the ability to reduce chromate at
a comparably high rate (Fig. 4). The subunit and native
masses of FerB found here agree favorably with the values
of 20 and 50 kDa reported before for Pseudomonas enzyme
[27]. However, there is a marked difference in isoelectric
points. While FerB is an acidic protein (pI 5.5 by
chromatofocusing), pI of chromate reductase was claimed
to be higher than 7.0 from chromatographic behavior; our
own calculation based on the known amino acid sequence
(gi:14209680) and exploiting the pI/Mw tool [45] on http://
www.expasy.org gave the value of 8.55. Unfortunately, the
Pseudomonas enzyme was not tested for the presence of
flavin nor the activity towards electron acceptors other than
chromate, and hence a more detailed comparison with FerB
cannot be presented at the moment. It may also be of
interest to mention here the existence of the FAD-contain-
ing DT-diaphorase [46] and the FMN-containing flavin-
Table 3. Michaelis constants (± SE) for the substrates of FerA and FerB. Values were calculated from the data in Figs 5 and 6. The best fitting
kinetic models, selected according to the Akaike’s information criterion, correspond to a sequential bi–bi mechanism for FerA and to a ping-pong
mechanism for FerB. Non-linear regression values of k
cat
are (14 ± 1) s
)1

and (28 ± 6) s
)1
for the Fe(III) reductase and FMN reductase activity of
FerA, respectively, and (3.1 ± 0.5) s
)1
for FerB.
Substrate
(variable concentration)
Second substrate
(fixed concentrations)
Third substrate
(constant concentration)
K
m
(l
M
)
FerA
NADH Fe(III)nitrilotriacetic acid FMN (50 l
M
) 5.5 ± 0.7
Fe(III)nitrilotriacetic acid NADH FMN (50 l
M
)17±2
NADH
a
FMN – 11 ± 5
FMN
a
NADH – 20 ± 8

FerB
NADH Fe(III)nitrilotriacetic acid – 2.6 ± 0.7
Fe(III)nitrilotriacetic acid NADH – 800 ± 200
a
Fe(II)(ferrozine)
3
production was used to quantify reaction progress except in these cases where NADH oxidation by flavin was measured.
560 J. Mazoch et al. (Eur. J. Biochem. 271) Ó FEBS 2004
reductase [43,44] and nitroreductase [47] that resemble FerB
in being homodimers of comparable molecular size, utilizing
both NADH and NADPH as electron donors, exhibiting
ping-pong kinetics and having a number of activities
including that of quinone reductase. Indications exist that
a quite small number of amino acid residues determine
electron acceptor specificity of these enzymes. For example,
a single point mutation was enough to convert NfsB, one of
the oxygen-insensitive nitroreductases of Escherichia coli,to
a highly active flavin reductase [48]. Constrains of similar
type as in NfsB may exist in FerB and preclude in this way
its reaction with free flavin. We hope that future compar-
ative studies will clarify relationships (if any) between FerB
and the other mentioned proteins on a molecular level. A
part of this effort is currently aimed at the cloning of the
gene for FerB for further characterization.
Acknowledgements
We thank Dr Z. Vobu˚ rka (Institute of Organic Chemistry and
Biochemistry, AS CR, Prague) for performing N-terminal amino acid
sequencing, Dr Z. Zdra
´
hal (Laboratory of Mass Spectrometry of

Biomolecules, Faculty of Science, MU, Brno) for MALDI-MS analyses
and Dr Z. Prokop (National Centre for Biomolecular Research,
Faculty of Science, MU, Brno) for assisting us with the stopped-flow
measurements. This work was supported by a grant from the Grant
Agency of the Czech Republic to I. K. (203/01/1589) and partly by a
contract from Ministry of Agriculture of the Czech Republic to J.T.
(M-3-99-01).
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