Báo cáo khoa học: Structure analysis of the flavoredoxin from Desulfovibrio vulgaris Miyazaki F reveals key residues that discriminate the functions and properties of the flavin reductase family pdf

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

Correspondence
M. Kitamura, Department of Applied
Chemistry and Bioengineering, Graduate
School of Engineering, Osaka City
University, 3-3-138 Sugimoto, Sumiyoshi-ku,
Osaka 558-8585, Japan
Fax: +81 666 05 2769
Tel: +81 666 05 3091
E-mail:
Y. Higuchi, Department of Life Science,
Graduate School of Life Science, University
of Hyogo, 3-2-1 Koto, Kamigori-cho,
Ako-gun, Hyogo 678-1297, Japan
Fax: +81 791 58 0177
Tel: +81 791 58 0179
E-mail:
Database
The coordinates and structure factor data
have been deposited in the PDB, under the
accession number 2D5M. The nucleotide
and amino acid sequence data may be
found in the DDBJ, EMBL and GenBank
sequence databases under the accession
numbers AB214904 and BAD99043,
respectively
(Received 30 March 2009, revised 28 May
2009, accepted 29 June 2009)
doi:10.1111/j.1742-4658.2009.07184.x
The crystal structure of flavoredoxin from Desulfovibrio vulgaris Miyazaki
F was determined at 1.05 A

˚
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

interest to understand the molecular bases for the dif-
ferent reactivities displayed by flavins in different pro-
tein contexts. Flavoredoxin is an electron-transfer
protein that has one FMN molecule per subunit or
monomer [1]. The only flavoredoxins characterized to
date are from the sulfate-reducing bacterium Desulfo-
vibrio gigas [1–3] and an Archaeon Methanosarcina
acetivorans [4]. Deletion and mutation analyses of this
bacterium have indicated that flavoredoxin is involved
in the thiosulfate reduction process [3]. It has been
proposed that flavoredoxin receives an electron, which
was originally generated by a hydrogenase, from
flavodoxin or ferredoxin, and transfers it to a sulfite
reductase, desulfoviridin [3].
The D. gigas flavoredoxin has an apparent sequence
identity of 22% with Archaeoglobus fulgidus ferric
reductase (FeR) [1]. FeR catalyzes the reduction of
Fe(III) chelates, such as Fe(III)–EDTA, in a NAD(P)H-
dependent manner [5,6]. The crystal structure of FeR
has been determined with and without NADP
+
[6].
These authors were unsuccessful in their attempts to
solve the structure in the Fe(III) ion-bound state. The
catalytic mechanism of ferric ion reduction by this
enzyme has been proposed based on biochemical [5] and
structural studies of FeR [6], although the key residues
for ferric ion binding need to be identified to elucidate
the complete reaction mechanism. It was surprising that,
as pointed out by Chiu et al. [6], FeR revealed a com-

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

(GAGG, nucleotides 737–740 in the PstI–KpnI frag-
ment) is present upstream of the initiation codon
(ATG), and there are potential promoter regions at
nucleotides 643–648 (TTGCCG) and 666–671 (CAA-
ACT) in the PstI–KpnI fragment. Nucleotides 1339–
1371 comprise the putative transcriptional terminator,
forming a stem-and-loop structure. The results of a
BLAST homology search indicate that the product of
this ORF is highly homologous to flavoredoxins from
other bacteria, especially that of D. vulgaris (Hilden-
borough), with an identity of 71%; therefore, we
confirmed this ORF to be the flavoredoxin gene.
Recombinant flavoredoxin purification
We used an Escherichia coli expression system to
express the flavoredoxin gene. Recombinant flavo-
redoxin was detected in transformed E. coli crude cell
extracts by SDS ⁄ PAGE. Through chromatographic
steps using DE52 and Superdex 75, a large amount of
flavoredoxin was purified to homogeneity by
SDS ⁄ PAGE (Fig. S1). The molecular mass of the
expressed flavoredoxin was estimated to be
 23 000 Da by SDS⁄ PAGE, which is different from
the value calculated based on the amino acid sequence
(20 800 Da). We also estimated the molecular mass in
the native state to be  37 000 Da using a Superdex
75 gel-filtration column. This value is about twice that
calculated from the amino acid sequence, indicating
that the native form of flavoredoxin is a dimer.
Amino acid sequence analysis of flavoredoxin
The N-terminal amino acid sequence of flavoredoxin

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

452 nm, which are characteristic of proteins that bind
to flavin derivatives. The recombinant flavoredoxin
was subjected to reverse-phase HPLC on a C
8
column,
and the retention time of the obtained prosthetic group
was compared with those of flavin derivatives. The
retention time of the prosthetic group bound to recom-
binant flavoredoxin was identical to that of FMN
(Fig. S3). The A
448
: A
268
ratio of the holoprotein was
0.267, suggesting that the flavoredoxin expressed in
E. coli as a holoprotein binds to FMN as a prosthetic
group at a molar ratio of 1.
Overall structure of DvMF flavoredoxin
DvMF flavoredoxin was crystallized in the P3
1
21 space
group with one molecule in the asymmetric unit. The
structure was refined to a crystallographic R factor of
0.135 and R
free
of 0.162 at 1.05 A
˚
resolution (Table 1).
Residues 128–130 and 187–190 (four C-terminal resi-
dues) were excluded from the structural model because

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,

the structures of the FMN-binding protein and
flavodoxin were determined by X-ray crystallography;
the structure of DvMF flavoredoxin resembles
the former (rmsd = 2.5 A
˚
) rather than the latter
(rmsd = 3.4 A
˚
).
As deduced by gel-filtration chromatography,
DvMF flavoredoxin forms a dimer, as evidenced by
the crystallographic two-fold axis in the crystal. When
the dimeric structure of DvMF flavoredoxin was com-
pared with those of FeR and FMN-binding protein,
the flavoredoxin dimer was superimposed on the
former (Fig. 2B) but not on the latter (Fig. 2C). In
flavoredoxin, the twofold axis associated with the
dimer passes through the vicinity of the side chains of
Pro13, Ile126, Gln133 and Ile163. The corresponding
Table 1. Summary of x-ray data collection, phasing and refinement
statistics.
Native
Methylmercuric
chloride
derivative
Data collection
Beamline BL41XU, SPring-8 BL44XU, SPring-8
Space group P3
1
21 P3

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

Resolution range (A
˚
) 10–1.05 (1.09–1.05) -
R
work
0.135 (0.208) -
R
free
0.162 -
R.m.s. deviations from ideal values
Bond lengths (A
˚
) ⁄
angle distances (°)
0.016 ⁄ 0.031 -
Ramachandran plot
Most-favored 136 -
Additionally allowed 16 -
Generously allowed 0 -
Disallowed 1 (Val167) -
Structure of flavoredoxin N. Shibata et al.
4842 FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS
A
B
C
Fig. 2. Structures of flavoredoxin and other
related proteins. (A) Overall structure of the
flavoredoxin dimer. Each subunit is shown
in green–cyan and violet–red models. FMN
molecules are depicted as ball-and-stick

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-

mer, forming an anti-parallel intermolecular b sheet
(Fig. 2A). The subsequent residues of the loop (Ala7–
Pro13) turn into the interior of the dimer interface,
and Leu10 and Tyr12 form a hydrophobic core with
Ile70, Met116, Val141, Pro154, Ile156 and Pro161 of
the other monomer. Similarly, Gly175–Ala181 forms
an anti-parallel intermolecular b sheet with the b12 of
the other monomer, and towards the C-terminus, the
subsequent residues, Phe182–Lys186, pass through the
a2 vicinity of the other monomer. For the FeR dimer,
the N-terminal loop is replaced by an a helix, and side
chain-to-side chain interactions play a major role in
dimer interactions through this region. By contrast, a
similar intermolecular b sheet through the C-terminal
loop is conserved.
Structure of the FMN-binding region
The hydrogen bonds and salt bridge that encompass
the ribitol moiety and the phosphate group of
FeR and M. acetivorans flavoredoxin are moderately
and completely conserved in DvMF flavoredoxin
(Fig. 3A,B and Fig. S4A), respectively. In the case of
FeR, Ser84 replaces Asn29 of DvMF flavoredoxin,
which forms a hydrogen bond to the O3P atom of
FMN (Fig. 3A,B). DvMF flavoredoxin forms three
additional hydrogen bonds between the NH
2
moiety
of Arg51 and three atoms of FMN (N1, O2 and O3’).
In both FeR and M. acetivorans flavoredoxin, the cor-
responding residue is asparagine (Asn47 and Asn52,

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

with the FAD molecule of the enzyme. In the case of
FMN-binding protein, Lys53 is adjacent to FMN,
forming a hydrogen bond with the phosphate group of
FMN through its main-chain N atom, whereas the side
chain amino group is 7 A
˚
from the FMN molecule.
The surface charge models of these proteins indicate
that the level of positive charge at the phosphate
group binding site is considerably higher in both
flavoredoxin and FeR than in the FMN-binding
protein (Fig. S5A–C).
The accessible surface areas of FMN in flavoredoxin
and FeR have been calculated to be 57 and 95 A
˚
2
,
respectively. Three residues, Trp35, Arg51 and Phe182,
are responsible for this difference (Fig. 3A,B). Both
Trp35 and Arg51 of flavoredoxin have larger volumes
than the corresponding residues (Thr31 and Asn47) of
FeR. No residue in FeR corresponds to Phe182
(Fig. 3A,B). The surface model of flavoredoxin indi-
cates that the re-side of the isoalloxazine ring is par-
tially covered by these residues (Fig. S5A). By
contrast, the re-side of the isoalloxazine ring of FeR is
completely exposed to the solvent (Fig. S5B).
Resolution of the structure of NADP
+
-bound FeR

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

C
D
Fig. 3. FMN binding site. (A,B) Superim-
posed models of FMN binding sites of fla-
voredoxin and FeR, showing residues that
interact with the FMN molecule. Hydrogen
bonds are shown as green dotted lines. Fla-
voredoxin is displayed by atom color, with
the exception that the carbon atoms of
FMN are in yellow. FeR is shown as a trans-
parent model. The Mes molecule is omitted
for clarity. The view in (B) is rotated 180°
about the vertical axis. Residue labels are
shown as flavoredoxin ⁄ FeR. (C) Residues of
flavoredoxin involved in Mes binding.
NADP
+
derived from the superimposed
model of FeR is also shown as a transpar-
ent model. (D) Zinc ion-binding site. Zinc
ions are depicted as green spheres. The
remaining color codes are the same as in
(A) and (B).
N. Shibata et al. Structure of flavoredoxin
FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4845
coordinated by Cys39 and the other conformer of
Cys40. This zinc ion is bound to His131, which corre-
sponds to the histidine residue that is completely con-
served among the FeR homologs [5,6]. Interestingly,
the residues corresponding to metal ion binding are

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

on the amino acid comparison (Fig. 1) and the crystal
structure of flavoredoxin, we propose that flavoredoxin
has a NAD(P)H binding site similar to that of A. fulgi-
dus FeR, and therefore we expect that NAD(P)H is
bound to this site and reduces the FMN of flavo-
redoxin; however, no decrease in absorption in the
visible region because of flavoredoxin reduction was
observed, even when NADH or NADPH was added
(data not shown); therefore, we conclude that flavore-
doxin has no oxidase activity.
Ferric reductase and flavin reductase activities of
recombinant flavoredoxin and related proteins
Flavoredoxin uses both NADH and NADPH as elec-
tron donors to reduce Fe
3+
–EDTA and FMN; how-
ever, we found that both FeR and Fre activities using
NADH were lower than those using NADPH (data
not shown). FeR and Fre activities of flavoredoxin
were 2.58 · 10
)3
and 2.70 · 10
)3
unitsÆmgÆprotein
)1
,
respectively, whereas those of DrgA from Synechocystis
sp. PCC6803, which was used as a positive control,
were 8.66 · 10
)2

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

of A. fulgidus enzyme was  1070-fold higher than
DrgA, which we used as a positive control, even under
anaerobic conditions (3.28 unitsÆmgÆprotein
)1
) [13].
Fig. 4. Linear regression analysis of the logarithms of concentration
ratios for oxidized and reduced forms of a mediator versus that of
recombinant flavoredoxin. (°) Neutral Red used as the mediator; (D)
Benzyl viologen is used as the mediator.
Structure of flavoredoxin N. Shibata et al.
4846 FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS
Thus, it appears that only A. fulgidus FeR possesses
extremely high FeR activity, compared with other
FeRs. The FeR activity of flavoredoxin is 10
2
–10
4
times
lower than that of other bacterial FeR-active enzymes.
Taking this into account, we postulate that DvMF fla-
voredoxin reacts similarly to FeR, although its activity
is not comparable with that of A. fulgidus FeR.
The same may be true of flavin reductase activity.
Two explanations can be proposed for the large differ-
ences noted in ferric ion and flavin reductase activities
between DvMF flavoredoxin and A. fulgidas FeR. The
first possibility is that NAD(P)H binds to flavoredoxin,
but hardly transfers an electron to FMN. The fact that
DvMF flavoredoxin has a lower redox potential than
NADH ()343 versus )320 mV) supports this explana-

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

the case, flavoredoxin cannot bind a ferric ion at this
site unless Phe182 moves away from the site. Then,
because NAD(P)H was not the most suitable electron
donor for DvMF flavoredoxin, the FeR and flavin
reductase activities of DvMF flavoredoxin might have
been underestimated, and we cannot ignore that the
physiologic function of flavoredoxin is to transfer an
electron from reduced ferredoxin or flavodoxin to a
sulfite reductase, i.e. desulfoviridin in the thiosulfate
reduction process [3].
It has been reported that A. fulgidus FeR activity
decreases during purification because of the loss of a
flavin molecule, and is restored by adding FMN or
FAD, which suggests that the affinities for flavin
species are not sufficiently high. In D. gigas flavore-
doxin, however, FMN binds tightly with a dissociation
constant of 0.12 nm [1], i.e. 2500-fold lower than the
K
m
value (0.3 lm) for FMN of FeR [5]. This large dif-
ference can be explained in part by the difference in
the exposed surface areas of FMN, as described above.
Among the NADH : flavin oxidoreductase family, one
structure, the HpaC component of Sulfolobus tokodaii
4-hydroxyphenylacetate 3-monooxygenase, which is a
member of the two-component flavin-diffusible mono-
oxygenases, in the FMN-bound form has been
reported [15]. The K
m
of S. tokodaii HpaC for FMN 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

)1
with NADPH as the
electron donor and 1.3 lm and 30.6 s
)1
with NADH
[13]. For FMN, K
m
and k
cat
values of E. coli Fre were
calculated as 2.67 lm and 0.724 s
)1
with NADPH as
the electron donor and 0.67 lm and 1.84 s
)1
with
NADH (our unpublished data). These differences may
influence FeR and Fre activities.
Chiu et al. [6] claimed that His126 of A. fulgidus
FeR is one of the candidates for the ferric ion-binding
residue based on the structure of the mercury-bound
derivative, although a ferric ion was not found at the
site of either the native or NADP
+
-bound form;
instead this residue is bound to the nicotinamide moi-
ety of NADP
+
in the NADP
+

-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

are unlikely to have high affinity to ferric ions. It
should be noted that E. coli HpaC displayed FeR
activity [16]. Sequence alignment indicates that it has a
cysteine residue at the position that corresponds to
Cys45 (Fig. 1); therefore, flavin proteins, which have
similar folding and ferric reductase activity, have at
least one cysteine residue at or around the site corre-
sponding to the zinc-binding site of flavoredoxin.
These lines of evidence raise questions as to why a zinc
ion binds robustly to DvMF flavoredoxin and why no
metal ions were found in the A. fulgidus FeR structure
even in a ferric ion atmosphere. FeR may have low
affinity for ferric ions in the oxidized state. As Chiu
et al. [6] have suggested, the solvent-exposed dimethyl-
benzene moiety is one candidate for binding the ferric
ion. In this case, the ferric ion would bind only weakly
to FeR, because none of the residues that could act as
a ligand for the ferric ion are found at this position. A
zinc ion is preferentially trapped at the metal ion bind-
ing site of DvMF flavoredoxin. Indeed, the ferric
reductases of Azotobacter vinelandii [25] and
Legionella pneumophila [26] are inhibited in the pres-
ence of zinc ions. This is probably because a zinc ion
is bound tightly at the ferric ion binding site, although
the detailed inhibition mechanism remains unknown.
In the case of A. vinelandii ferric reductase, the enzyme
was purified as a mixture of two proteins or a hetero-
dimer with molecular masses of 44 600 and 69 000 Da
[25]. Although none of the amino acid sequences of
A. vinelandii is recorded as ferric reductase, an NCBI

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 [

32
P]ATP[cP] (185 TBqÆm-
mol
)1
) was obtained from MP Biomedicals (Irvine, CA,
USA). All other chemicals were of analytic grade for bio-
chemical use. Genomic DNA isolated from DvMF was pre-
pared by the method of Saito & Miura [29]. To amplify the
flavoredoxin gene, we searched for the published amino
acid sequences of flavoredoxin from other bacteria, because
the DvMF flavoredoxin amino acid sequence was
unknown, but we could not find the PCR conditions; how-
ever, we noted that the ABC transporter gene, the amino
acid sequence of which is highly conserved across species,
was in the complementary strand upstream of the flavore-
doxin gene in sulfate-reducing bacteria. Assuming that gene
mapping is similar among sulfate-reducing bacteria, we
designed two primers according to the conserved regions of
the amino acid sequence of the ABC transporter gene
from D. vulgaris (Hildenborough), and amplified part
of the ABC transporter gene using PCR with DvMF geno-
mic DNA as a template. The PCR primer sequences were
as follows: ABC01, 5¢-TGGATAGCCGCCAAGATGGG
GTT-3¢ (23-mer corresponding to the amino acid sequence
58
W–I–A–A–K–M–G–F); and ABC02, 5¢-GCGAGCGG
GGCCGAATCGTAGAA-3¢ (23-mer complementary
sequence to the corresponding amino acid sequence
163
F–Y–D–S–A–P–L–A). The PCR products were separated

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

pUC18, and the resultant ligation mixture was transformed
into E. coli JM109. One such transformant, isolated by the
colony hybridization method, was found to harbor a
plasmid that carried the  4.3-kbp PstI–KpnI fragment of
DvMF DNA, and was designated pABCFLR. The nucleo-
tide sequence of the inserted fragment was determined by
sequencing the restriction fragment that was cloned into
pUC18 using the dideoxy chain termination method [30]
with a DNA sequencer.
Expression and purification of recombinant
flavoredoxin
For high-level expression in E. coli, we used pMK2 [31],
which is an expression vector that carries the tac promoter.
The coding region of the flavoredoxin gene was amplified
by PCR with KOD-Plus- DNA polymerase (Toyobo).
DvMF genomic DNA was used as the PCR template. The
PCR primer sequences were as follows: flr09, 5¢-
CGACCCGGGTCATGAAGAAATCCCTGG-3¢ (27-mer);
and flr10, 5¢-TTTGTCGACTGATCAGGAGCGCAGGC
C-3¢ (27-mer). PCR was carried out at 94 °C for 2 min, fol-
lowed by 35 cycles of 94 °C for 15 s, 55 °C for 30 s, and
68 °C for 1 min. The PCR products were digested with
SmaI and SalI and ligated into similarly digested pUC18.
The cloned fragment was confirmed by sequencing, digested
with SmaI and HindIII, and then ligated into pMK2, which
was initially digested with EcoRI, blunt-ended with the
Klenow fragment and then digested with HindIII to give
the expression vector pMKFLR9-10. E. coli was trans-
formed with pMKFLR9-10, and transformants were grown
in 1.7 mL LB medium containing 100 lgÆmL

)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 carried out using the Pharmacia FPLC system
(Uppsala, Sweden) in 200 mm NaCl, 10 mm Tris ⁄ HCl (pH
8.0) at a flow rate of 0.5 mLÆmin
)1
, and the purified recom-
binant flavoredoxin was eluted after 23 min. SDS ⁄ PAGE
(15%) was carried out according to the method of Laemmli
[32]. For amino acid sequence analysis, further purification
was performed. The purified recombinant flavoredoxin was
loaded onto a TSK-GEL TMS-250 (Tosoh, Tokyo, Japan).
The column was washed with 0.1% trifluoroacetic acid, and
then developed with a gradient of 0–80% acetonitrile in
0.1% trifluoroacetic acid. The flow rate was 0.8 mLÆ min
)1
.
Purified apo-flavoredoxin was separated by SDS ⁄ PAGE
and electroblotted. For deformylation, a sample was treated
with 0.6 m HCl overnight at room temperature. The total
protein concentration was determined using the BCA pro-
tein assay kit (Thermo Scientific Pierce, Waltham, MA,
USA) or calculated from the absorbance value at 448 nm.
Identification of the prosthetic group
To identify the prosthetic group, purified recombinant fla-
voredoxin and a mixture of riboflavin, FAD and FMN
were loaded onto an HPLC C
8
column (NUCLEOSIL 10
C
8
; Shinwa Chemical Industries Ltd., Kyoto, Japan). The

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
˚

resolution and
the methylmercuric chloride derivative extending to 1.7 A
˚
resolution were collected at 100 K on an ADSC Q315
charge-coupled device detector at the BL41XU beamline
and on a DIP2040 image-plate detector at the BL44XU
beamline (SPring-8, Hyogo, Japan). Data processing, scal-
ing, and reduction were performed using the HKL-2000
system [34].
Structure determination and refinement
The structure was solved by single-wavelength anomalous
diffraction using the methylmercuric chloride derivative.
Two mercury sites, located by the program snb [35], were
used for phasing with sharp [36]. The initial model was
built automatically with arp ⁄ warp [37], followed by man-
ual building with xfit [38] for the unbuilt regions. The ini-
tial crystallographic refinement was performed using cns
[39]. The initial model was first subjected to cycles of
energy minimization and individual B-factor refinement.
After the refinement step, a SIGMAA-weighted 2F
o
–F
c
map was calculated and used for manual model rebuilding
of the molecule. Next, water molecules were added using
the waterpick protocol of cns. After several cycles of refine-
ment, the structure was further refined using shelxl [40].
In the later stages, anisotropic B-factor refinement was per-
formed and several double conformations were applied. In
the final stage of refinement, protons were included in the

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

spectrophotometric measurements [46] with mixtures of
flavoredoxin and a mediator, which was Neutral Red or
benzyl viologen. The redox potential, E
h
, for the system at
equilibrium was calculated with the Nernst equation,
E
h
¼ E
m;7
ðdyeÞþ
RT
nF
ln
oxidized dye½
reduced dye½
where R denotes the gas constant, T is the absolute temper-
ature, F is the Faraday constant and n is the number of
electrochemical equivalents. A solution of flavoredoxin and
a dye in 100 mm potassium phosphate buffer (pH 7) in a
closed all-glass cuvette was made anaerobic by repeated
cycles of evacuation and flushing with oxygen-free argon.
When Neutral Red was used as the mediator, absorbances
at 452 and 540 nm were monitored. Neutral Red in the
reduced state and flavoredoxin do not absorb at 540 nm,
and flavoredoxin in the fully reduced state does not absorb
at 452 nm. When benzyl viologen was used as the mediator,
absorbances at 452 and 400 nm were monitored. Benzyl
viologen in the oxidized state does not absorb at either 452
or 400 nm. To determine E

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

flavoredoxin and FMN-binding protein do not react with
oxygen. The assay was performed using NADPH as a sub-
strate. To measure FeR activity, the reaction was initiated
by the addition of 100 lm Fe
3+
–EDTA solution to 41 lm
flavoredoxin or 97 lm FMN-binding protein or 0.44 lm
DrgA and 150 lm of NADPH in 50 mm sodium phosphate
buffer (pH 7.0) in the presence or absence of 15 l m FMN
in quartz cuvettes. The total volume was 1 mL at 30 °C.
The reaction was monitored at 340 nm in a spectrophotom-
eter [47]. Activity was determined by measuring the differ-
ence in NADPH consumption at 340 nm in the presence
and absence of Fe
3+
–EDTA. The absorbance coefficient
used for NADPH was 6.20 mm
)1
Æcm
)1
. Flavin reductase
activity was measured using the same reaction mixture as
for FeR, but using a final concentration of 100 mm FMN
solution and omitting Fe
3+
–EDTA. One unit of activity is
defined as 1 lmol NADPH oxidized per minute.
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Structure of flavoredoxin N. Shibata et al.
4852 FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS
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

flavoredoxin expressed in Escherichia coli transformed
with pMKFLR9-10.
Fig. S3. Identification of the prosthetic group by
HPLC.
Fig. S4. Superimposed models of the vicinity of FMN
binding sites of DvMF and Methanosarcina acetivorans
flavoredoxins as in Fig. 3.
Fig. S5. Surface charge models viewed from the same
perspective as in Fig. 2.
This supplementary material can be found in the
online article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
N. Shibata et al. Structure of flavoredoxin
FEBS Journal 276 (2009) 4840–4853 ª 2009 The Authors Journal compilation ª 2009 FEBS 4853