Structure analysis of the flavoredoxin from
Desulfovibrio vulgaris Miyazaki F reveals key residues
that discriminate the functions and properties of the
flavin reductase family
Naoki Shibata
1
, Yasufumi Ueda
1
, Daisuke Takeuchi
2
, Yoshihiro Haruyama
2
, Shuichi Kojima
3
,
Junichi Sato
4
, Youichi Niimura
4
, Masaya Kitamura
2
and Yoshiki Higuchi
1
1 Department of Life Science, University of Hyogo, Japan
2 Department of Applied Chemistry and Bioengineering, Osaka City University, Japan
3 Institute for Biomolecular Science, Gakushuin University, Tokyo, Japan
4 Department of Bioscience, Tokyo University of Agriculture, Japan
Keywords
crystal structure; electron transfer; flavin
mononucleotide; flavin reductase family;
sulfate-reducing bacterium

˚
resolution and its ferric reductase activity was
examined. The aim was to elucidate whether flavoredoxin has structural
similarity to ferric reductase and ferric reductase activity, based on the
sequence similarity to ferric reductase from Archaeoglobus fulgidus.As
expected, flavoredoxin shared a common overall structure with A. fulgidus
ferric reductase and displayed weak ferric reductase and flavin reductase
activities; however, flavoredoxin contains two FMN molecules per dimer,
unlike A. fulgidus ferric reductase, which has only one FMN molecule per
dimer. Compared with A. fulgidus ferric reductase, flavoredoxin forms three
additional hydrogen bonds and has a significantly smaller solvent-accessible
surface area. These observations explain the higher affinity of flavoredoxin
for FMN. Unexpectedly, an electron-density map indicated the presence of
a Mes molecule on the re-side of the isoalloxazine ring of FMN, and that
two zinc ions are bound to the two cysteine residues, Cys39 and Cys40,
adjacent to FMN. These two cysteine residues are close to one of the puta-
tive ferric ion binding sites of ferric reductase. Based on their structural
similarities, we conclude that the corresponding site of ferric reductase is
the most plausible site for ferric ion binding. Comparing the structures
with related flavin proteins revealed key structural features regarding
the discrimination of function (ferric ion or flavin reduction) and a unique
electron transport system.
Abbreviations
DvMF, Desulfovibrio vulgaris Miyazaki F; FeR, ferric reductase; Fre, flavin reductase; PDB, Protein Data Bank.
4840 FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS
Introduction
Flavins play major roles as cofactors for a wide variety
of redox proteins and enzymes; these reactions depend
on the redox ability of the flavin species. Although the
basic redox reactions are identical or similar, it is of

mon overall fold with the FMN-binding protein from
Desulfovibrio vulgaris Miyazaki F (DvMF), whose crys-
tal structure was determined by our group [7]. However,
FeR and the FNM-binding protein from have relatively
low sequence identity (12%). FeR also has structural
similarity to the flavin reductase (Fre, NADH : flavin
oxidoreductase) family, which includes the Fre compo-
nent of the two-component flavin-diffusible monooxy-
genase [5,6]. The Fre component reduces a flavin with
NADH or NADPH to provide a reduced flavin, which
is used to activate molecular oxygen for the oxygenase
reaction [8]. In this family, neither component of the
enzyme binds flavin tightly as a cofactor, but rather
utilizes it as another substrate [8].
Considering the amino acid sequence similarities
between flavoredoxin and related flavin proteins, the
question arises as to whether flavoredoxin possesses
ferric ion or flavin reductase activity. Which structures
determine the unique functions of these flavin proteins?
In this study, we present the crystal structure of
DvMF flavoredoxin and discuss the key residues for
ligand binding and metal ion binding, based on the
crystal structures.
Results
Cloning and sequencing of the flavoredoxin gene
We determined the nucleotide sequence of the entire
flavoredoxin gene (accession number AB214904 in the
DDBJ, EMBL and GenBank nucleotide databases).
The ORF that encodes flavoredoxin comprises 190
amino acid residues. A potential ribosome-binding site

from DvMF was determined to be Met–Lys–Lys–Ser–
Leu–Gly–Ala, and the Met was formylated. When the
flavoredoxin amino acid sequences of DvMF and other
N. Shibata et al. Structure of flavoredoxin
FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4841
organisms were compared, they were found to be
highly conserved. The three characteristic co-ordina-
tion motifs (
36
TSKP–
62
FGVSVL–
124
GTHTL) of the
FeR from A. fulgidus, which is linked to FMN or
NAD binding [5], were also found in DvMF flavore-
doxin (
40
CSQP–
66
FTISIP–
128
GLHTQ). These co-ordi-
nation motifs are not homologous to those of
flavodoxin or FMN-binding protein.
Identification of the prosthetic group
To identify the prosthetic group bound to the recombi-
nant flavoredoxin, UV-visible spectra of the purified
holoprotein were recorded (Fig. S2). In the visible
region, absorption maxima were observed at 381 and

of poor electron densities in these regions. DvMF fla-
voredoxin contains four a helices (a1–4), two 3
10
heli-
ces (3
10
1–2) and 12 b strands (b1–12) as secondary
structural elements; it also has a Greek key motif with
seven anti-parallel b strands (Figs 1 and 2A), which is
also found in DvMF FMN-binding protein [7] and
A. fulgidus FeR [6]. Flavoredoxin contains two FMN
molecules per dimer, unlike FeR, which has only one
FMN molecule per dimer (Fig. 2B). The FMN mole-
cule is located in the hollow, encompassed mainly by
a1, a2 and b3.
A structural homology search was carried out using
the DALI server [9]. Among the proteins of known
function, the M. acetivorans flavoredoxin exhibited
the lowest rmsd (1.5 A
˚
) and the highest Z score
(25.5), as expected from the highest sequence identity
(30%) of the known structures. A. fulgidus FeR
showed the second lowest rmsd (1.9 A
˚
) and the
second highest Z score (18.8), although the sequence
identity between DvMF flavoredoxin and A. fulgidus
FeR is low (17%). Among the flavin-containing
electron-transfer proteins of Desulfovibrio species,

1
21
Unit-cell
parameters (A
˚
)
a = b = 53.35,
c = 116.22
a = b = 53.5,
c = 116.2
Wavelength (A
˚
) 0.7100 1.0000
Resolution range (A
˚
) 50–1.05 (1.09–1.05) 50–1.71 (1.77–1.71)
Measured reflections 950,955 205,263
Unique reflections 89,930 21,327
Completeness (%) 99.6 (97.0) 99.6 (97.6)
R
merge
0.088 (0.573) 0.087 (0.158)
Multiplicity 10.6 (8.7) 9.6 (7.2)
I ⁄ r(I) 42.1 (3.7) 59.8 (14.0)
SAD phasing for methylmercuric chloride derivative
Figure of merit,
centric ⁄ acentric
- 0.165 ⁄ 0.609
Phasing power - 5.126
Refinement

models. (B) Superimposed Ca-traces of
flavoredoxin (green and violet) and FeR (light
gray). (C) Superimposed Ca-traces of
flavoredoxin (green and violet) and
FMN-binding protein (light gray).
Fig. 1. Amino acid sequence alignment of
DvMF flavoredoxin, Methanosarcina acetivo-
rans flavoredoxin, ferric reductase and HpaC
component of Escherichia coli 4-hydroxyph-
enylacetate 3-monooxygenase. Secondary
structure elements of flavoredoxin are
shown on the lines of residue numbers.
Residues involved in binding of FMN are
shown in red. Residues shown in bold are
aligned based on crystal structures. Resi-
dues shown in regular characters indicate
that structural information is unavailable or
that structurally equivalent residues are not
present. Alignment for HpaC was performed
with
CLUSTAL W [48].
N. Shibata et al. Structure of flavoredoxin
FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4843
residues in the FMN-binding protein are exposed to
the solvent.
In terms of dimer interactions, both N- and C-termi-
nal loops (Met1–Pro13 and Val174–Lys186, respec-
tively) appear to play important roles. The six
N-terminal residues (Met1–Gly6) are extended in the
opposite direction along the b10 of the other mono-

respectively) to which only the O2 atom of FMN
forms a hydrogen bond (Figs 3A,B and Fig. S4A).
The isoalloxazine ring of FMN is surrounded by
hydrophobic residues, Leu16, Trp35, Ile84, Phe164,
Tyr171 and Phe182, the first five residues of which
correspond to Leu13, Thr31, Phe81, Tyr147 and
Tyr150 in FeR (Fig. 3A,B), and Val18, Trp36, Leu85,
Leu162, Tyr169 and Leu180 in M. acetivorans flavo-
redoxin (Fig. S4A).
It should be noted that a positively charged residue,
Lys92, is involved in the binding of the phosphate
group(s) of FMN or FAD. Lys92 forms a salt bridge
with the O3P of FMN. A salt bridge that involves fla-
vin species has not been reported in the structures of
the other electron-transfer flavoproteins; however, a
salt bridge between the lysine ⁄ arginine residue and
FMN ⁄ FAD is found frequently in flavin-dependent
enzymes. To date, from the 130 FMN protein PDB
entries 22 have at least one FMN–lysine and 62 have
at least one FMN–arginine interaction. For FAD
proteins, from the 210 entries 9 have at least one
FAD–lysine and 55 have at least one FAD–arginine
interaction. One of these proteins, FeR, has a Lys89
residue that interacts with the phosphate group of
FMN. As pointed out by Chiu et al. [6], the structure
of FeR resembles the flavin-binding domain of ferre-
doxin : NADP
+
reductase [10]. The lysine residue is
not conserved; instead, an arginine residue interacts

revealed that the nicotinamide moiety of NADP
+
faces the re-side of the isoalloxazine ring, and that the
2¢-P-AMP moiety is held in the groove between the 3
10
helix and the third a helix [6]. Unexpectedly, in fla-
voredoxin, this site is occupied by Mes, which was
added to crystallization buffer solution. The Mes mole-
cule is held in place through a salt bridge with Arg169,
hydrogen bonds with Thr9 and Val167, and hydropho-
bic interactions with Trp35 and FMN (Fig. 3C).
Structure of the metal ion binding site
The electron-density map displayed three isolated
spheres with significantly greater density than normal
water oxygen atoms. Two of these are close to the
FMN binding site, and the other is on the opposite
surface of the protein. An anomalous-difference map
calculated from the native dataset showed significant
Structure of flavoredoxin N. Shibata et al.
4844 FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS
peaks at each of these sites. These densities were
assigned as zinc ions derived from the crystallization
solution as an additive. The zinc ion closest to FMN,
Zn201, is coordinated by the Sc atom of one of the
two conformers of Cys40 and two water molecules
(Fig. 3D). Zn203, which is 5.1 A
˚
from Zn201, is also
A
B

totally different in both FeR and M. acetivorans fla-
voredoxin. These cysteine residues are replaced by
threonine and leucine in FeR and asparagine and
valine in M. acetivorans flavoredoxin (Figs 1 and 3D;
Fig. S4C). In the case of FeR, however, Cys45 is adja-
cent to this site instead (Fig. 3D).
Redox potential of recombinant flavoredoxin
Figure 4 shows the results of linear regression analysis
of the logarithms for the redox ratio of the mediator
versus that of recombinant flavoredoxin. The redox
potential of oxidized flavoredoxin ⁄ reduced flavoredox-
in (E
flr
) was calculated as )343 mV at pH 7.0, deter-
mined using Neutral Red (E
m,7
= )325 mV, n =2)
[11] or benzyl viologen (E
m,7
= )359 mV, n=1) [12].
An n value of 2 was used in these experiments, which
fit the experimental data closely. Although recombi-
nant flavoredoxin was fully reduced by sodium dithio-
nite, no semiquinone intermediate was found.
Flavoredoxin reduction by NAD(P)H
Reduction experiments were performed under anaero-
bic conditions substituted by oxygen-free argon, and
NADH or NADPH was used as the reductant. The
observed increase in absorption around 340 nm is
caused by the addition of NADH or NADPH. Based

and 3.22 units ÆmgÆprotein
)1
under
aerobic conditions, respectively [13]. Even though both
activities of flavoredoxin could be detected, FeR and
Fre activities of flavoredoxin were 33.6-fold and 1200-
fold lower than those of DrgA, respectively. However,
FMN-binding protein [14] showed neither FeR nor
Fre activity.
Discussion
Based on the high structural similarity between DvMF
flavoredoxin and A. fulgidus FeR, we expected that the
flavoredoxin would have FeR and Fre activities, both
of which confer reduction of a flavin by receiving elec-
trons from NADH or NADPH; however, these activi-
ties of flavoredoxin were much lower than those of
A. fulgidus FeR. The FeR activity of A. fulgidus, which
is also a sulfate reducer, was reported to be 3503
unitsÆmgÆprotein
)1
at 85 °C using NADPH and FMN
[5]. This value is  1.36 · 10
6
-fold higher than that of
DvMF flavoredoxin. DvMF FMN-binding protein,
which also shares structural similarity with A. fulgidus
FeR, did not exhibit any detectable FeR activity. It
should be noted that the FeR activity of the A. fulgidus
enzyme is at least 1000-fold higher than all other
bacterial enzymes [5]. In addition, the FeR activity

tion. The second possibility is that the binding of
NAD(P)H is sterically hindered by the surrounding
residues. Regarding this second possibility, when the
flavoredoxin–Mes complex is superimposed on the
FeR–NADP
+
complex, steric hindrances occur
between flavoredoxin and NADP
+
at three different
sites (Fig. 3C). First, the adenine ring of NADP
+
has
severe steric contacts with Pro166, Val167 and Ser168,
which comprise the loop between b11 and b 12. Sec-
ond, the phosphate and ribose groups overlap with the
phenyl group of Phe182. Third, the nicotinamide moi-
ety is immediately adjacent to Trp35. At the first site,
the adenine ring has to move away from these overlap-
ping residues to bind to the corresponding site of fla-
voredoxin. At the second site, such steric repulsions
could be relieved by displacement of the residues
and ⁄ or a modified configuration of NADP
+
, because
most Phe182 is exposed to solvent and the side chain
rotates freely about its Ca–Cb and Cb–Cc bonds. The
steric contacts of Trp35 at the third site are not severe.
Chiu et al. [6] have suggested that the dimethylbenzene
moiety is a candidate for ferric ion binding. If this is

unknown, but that of E. coli HpaC is reported to be
2.1 lm [16], which is comparable with the values
obtained for other NADH : flavin oxidoreductases
[16–23]. The accessible surface area of FMN of
S. tokodaii HpaC was calculated to be 152 A
˚
2
.As
expected, this area is significantly larger than those of
flavoredoxin (57 A
˚
2
) and FeR (95 A
˚
2
). The large acces-
sible surface area for FMN of HpaC is quite reason-
able considering that HpaC releases the FMN
molecule immediately upon reduction, with which
the oxygenase component catalyzes the oxygenation
reaction.
A. fulgidus FeR is reported to be able to use FMN,
but not riboflavin, as the electron acceptor, although
most FeR molecules can use both. For example, the
K
m
and k
cat
values of E. coli Fre for riboflavin are
reported as 2.5 lm and 52.4 s

-bound form. These
authors also proposed that Cys45 of FeR, which corre-
sponds to Ser49 of DvMF flavoredoxin, is another
candidate [6]. The Oc atom of Ser49 does not bind to
a zinc ion but is only 5.68 and 5.50 A
˚
from Zn201
and Zn203, respectively (Fig. 3D). In FeR, Cys39 and
Cys40 are replaced by leucine and threonine, and
Cys45 rather than threonine would be the ligand for a
ferric ion, because the hydroxyl group of threonine has
a lower affinity for ferric ions than the thiol group of
cysteine. DrgA, which was used as a positive control
in activity measurements, has a cysteine (Cys147) and
three histidine (His15, His20 and His45) residues,
which could be candidates for ferric ion-binding
N. Shibata et al. Structure of flavoredoxin
FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4847
residues. However, DrgA seems to have a different
folding from flavoredoxin and FeR, as the secondary
structure prediction by PSIPRED [24] suggests that
DrgA has a helix-rich structure (data not shown),
unlike the b-rich structures of flavoredoxin and FeR.
Structural analysis needs to be carried out to elucidate
the ferric ion-binding site of DrgA. Suharti et al. [4]
have recently shown that M. acetivorans flavoredoxin
does not transfer an electron to ferric and chelated
ferric ion, and does not have a cystein residue at the
metal-binding site. The corresponding region is formed
by Val40, Asn41, Gly50 and Phe127 (Fig. S4C), which

entry, ‘Oxidoreductase FAD ⁄ NAD(P)-binding: Oxido-
reductase FAD-binding region’ (ZP_00418100), might
be the amino acid sequence of the smaller component
of A. vinelandii ferric reductase. This entry contains
443 residues with a calculated molecular mass of
49 180 Da and has apparent identity with the FAD
and NADH domains of BenC, which is the reductase
component of benzoate dioxygenase reductase. The
crystal structure of BenC [27] shows that Cys307,
which corresponds to Cys412 of the entry, is only 6 A
˚
from the isoalloxazine ring of FAD, which is compara-
ble with flavoredoxin and FeR. If the entry corre-
sponds to the smaller component of the A. vinelandii
ferric reductase, Cys412 could be the ferric ion binding
site.
Experimental procedures
Cloning and sequencing of the flavoredoxin gene
E. coli JM109 (recA1, supE44, endA1, hsdR17, gryA96,
relA1, thi, D(lac-proAB), F’[traD36, proAB
+
, lacI
q
,
lacZDM15]) was used for cloning and expression of the fla-
voredoxin gene. DvMF was grown [28] and used for geno-
mic DNA preparation. Restriction and modification
enzymes were purchased from New England BioLabs (Pic-
kering, Ontario, Canada), Nippon Gene (Tokyo, Japan)
and Toyobo (Osaka, Japan). The [

by agarose gel electrophoresis and a fragment of  340 bp
was extracted using the MinElute Gel Extraction Kit (Qia-
gen, Venlo, Netherlands). The nucleotide sequence of this
fragment revealed a putative amino acid sequence that was
similar to the amino acid sequence of another ABC trans-
porter from sulfate-reducing bacteria. We then synthesized
five primers, which were used to determine the sequence
upstream of the ABC transporter gene using genomic DNA
as the template. The primer sequences were as follows:
ABC04, 5¢-CCAGCTTCACCTTGCCCTTC-3¢ (20-mer);
ABC05, 5¢-CTTGTCCACGTAGGCGAAGG-3¢ (20-mer);
Flr05, 5¢-TCTCGTGGGCACATACGACC-3¢ (20-mer); Flr06,
5¢-TCAACAAGG TGGATCCGGTG-3¢ (20-mer); and Flr07,
Structure of flavoredoxin N. Shibata et al.
4848 FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS
5¢-ACGTGAAGGTGGACGAATCC-3¢ (20-mer). We identi-
fied the flavoredoxin gene in the complementary strand
upstream of the ABC transporter, and then designed a
30-mer probe DNA (5¢-TCGGAGGTACCGCGCTGCAC
GCCCAGCTTC-3¢), which is a complementary sequence
corresponding the amino acid sequence
133
V–K–L–G–V–Q–
R–G–T–S–E. We carried out Southern hybridization with
this labeled oligonucleotide at 65 °C and detected a band
that hybridized to an  4.3-kb PstI–KpnI fragment using a
Bioimaging Analyzer (BAS1000; Fuji, Tokyo, Japan). Then,
we digested the genomic DNA with PstI and KpnI, and
fractionated the products on an agarose gel. The separated
fragments were ligated into the corresponding sites in

)1
ampicillin
for 9 h at 37 °C. Twelve flasks containing 167 mL of the
same medium were inoculated with 1.7 mL culture and
incubated overnight with shaking at 37 °C. Cells were
harvested by centrifugation at 4000 g for 10 min at 4 ° C.
The cell pellet was resuspended in 10 mm Tris ⁄ HCl buffer
(pH 8.0), sonicated using a Model 201M sonicator (Kubot-
a, Tokyo, Japan) at 9000 Hz and 200 W for 10 min, then
ultracentrifuged at 100 000 g for 2 h at 4 °C. The superna-
tant was then dialyzed against distilled water overnight at
4 °C.
For flavoredoxin purification, the dialysate was loaded
onto a DEAE-cellulose column (DE52, 2.2 · 15 cm) equili-
brated with 10 mm Tris ⁄ HCl (pH 8.0). The column was
washed with 150 mL of 100 mm NaCl and 10 mm Tris ⁄ HCl
(pH 8.0). Flavoredoxin was eluted with 200 mL of 300 mm
NaCl and 10 mm Tris ⁄ HCl (pH 8.0). The colored eluent
was dialyzed against distilled water overnight at 4 °C, and
then reloaded onto a DE52 column equilibrated with
10 mm Tris ⁄ HCl (pH 8.0). Flavoredoxin was eluted with a
linear gradient of 100–300 mm NaCl in 10 mm Tris ⁄ HCl
(pH 8.0) in a total volume of 300 mL. The flavoredoxin-
containing fractions were identified based on absorbance at
448 nm. The colored fractions were collected and dialyzed
against distilled water, and then lyophilized or concentrated
using Vivaspin (MW 5000 cut-off; Sartorius AG, Go
¨
ttin-
gen, Germany). Gel filtration on a Superdex 75 HR10 ⁄ 30

column was washed with 0.1% trifluoroacetic acid, and
then developed with a gradient of 0–20% acetonitrile in
0.1% trifluoroacetic acid at a flow rate of 0.8 mLÆmin
)1
.
Crystallization, data collection and processing
The details of crystallization and data collection have been
reported previously [33]. The purified protein solution was
concentrated to 25 mgÆmL
)1
by centrifugation using a Viva-
spin (M
r
5000 cut-off; Sartorius). Flavoredoxin was crystal-
lized using the sitting-drop vapor diffusion method. Protein
droplets were prepared by mixing 2 lL protein solution
with 2 lL reservoir solution and equilibrated against
100 lL reservoir solution containing 10% (w ⁄ v) poly(ethyl-
ene gycol)8000, 0.2 m zinc acetate and 100 mm Mes (pH
N. Shibata et al. Structure of flavoredoxin
FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4849
6.0), at 283 K. A methylmercuric chloride derivative crystal
was prepared by soaking the crystals for 24 h in a reservoir
solution that contained 1 mm methylmercuric chloride.
Before data collection, the crystals were passed quickly
through a cryoprotectant solution that contained 30%
(v ⁄ v) glycerol in addition to well solution components. The
crystals were flash-cooled to 100 K in a stream of nitrogen
gas. The native dataset extending to 1.05 A
˚

riding-on mode. Standard restraints were applied through-
out the refinement. Most parts of the electron- density
maps were well-defined, although residues 128–130 and
187–190 could not be built because of poor electron densi-
ties in these regions. The Ramachandran plot indicated that
all residues were in the most-favored and allowed regions,
except for Val167 (Table 1) located at the Mes-binding site,
which was well-defined by the electron- density map.
Structural comparison and analysis
Structures were compared using the DALI server [9]. Root
mean square deviation and rotation-translation matrices for
superimposing structures were calculated with ssm [41]. Sol-
vent-accessible surface areas were calculated with a 1.4 A
˚
probe using cns [39]. Electrostatic potentials were calcu-
lated with the delphi program [42], which uses finite differ-
ence methods to solve the linearized Poisson–Boltzmann
equation. Interior and exterior dielectric constants of 2 and
80, respectively, and a probe radius of 1.4 A
˚
were used.
The atomic charge parameters for the molecules were taken
from the default library. The model figures were generated
with molscript [43] ⁄ raster3d [43,44] for Figs 2 and 3 and
with chimera [45] for Fig. S4.
Determination of oxidation ⁄ reduction potential
The redox potential for oxidized ⁄ reduced flavoredoxin (E
flr
)
was determined by means of equilibrium reactions and

flr
, sodium dithionite solution
was added to the dye along with the flavoredoxin in the
oxidized state. Redox potentials were calculated by linear
regression analysis of the logarithms of the concentration
ratios for oxidized and reduced forms of the mediator
versus that of recombinant flavoredoxin.
Reduction titration of flavoredoxin by NAD(P)H
The reduction of flavoredoxin by NAD(P)H was observed
at 25 °C by spectral change in the visible region, using a
U-3000IR spectrophotometer (Hitachi, Tokyo, Japan). A
solution of flavoredoxin in 50 mm Tris ⁄ HCl (pH 7.5) in a
closed all-glass cuvette was made anaerobic by repeated
cycles of evacuation and flushing with oxygen-free argon.
For the observation of flavoredoxin reduction, 5 mm
NADH in 50 mm Tris ⁄ HCl (pH 7.5) was added to flavo-
redoxin in the oxidized state.
Measurements of ferric reductase and flavin
reductase activities
FeR and Fre activity were examined using flavoredoxin,
DrgA [13] and FMN-binding protein [14]. DrgA from Syn-
echocystis sp. PCC6803 and FMN-binding protein from
Structure of flavoredoxin N. Shibata et al.
4850 FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS
DvMF were prepared according to the methods of Takeda
et al. [13] and Kitamura et al. [14], respectively. On the
basis of structural homologies, both flavoredoxin and
FMN-binding protein are considered to have FeR motifs,
and DrgA was used as a positive control. Both activities
were measured under aerobic conditions, because purified

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Supporting information
The following supplementary material is available:
Fig. S1. SDS ⁄ PAGE of purified recombinant flavore-
doxin.
Fig. S2. Ultraviolet and visible-light spectra of purified