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: kitamura@bioa.eng.osaka-cu.ac.jp
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: hig@sci.u-hyogo.ac.jp
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 ª2009 The Authors Journal compilation ª2009 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 ª2009 The Authors Journal compilation ª2009 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 Rfactor 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 ahelices (a1–4), two 3
10
heli-
ces (3
10
1–2) and 12 bstrands (b1–12) as secondary
structural elements; it also has a Greek key motif with
seven anti-parallel bstrands (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 ª2009 The Authors Journal compilation ª2009 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 ª2009 The Authors Journal compilation ª2009 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 bsheet
(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 bsheet 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 ahelix, and side
chain-to-side chain interactions play a major role in
dimer interactions through this region. By contrast, a
similar intermolecular bsheet 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 ahelix [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 ª2009 The Authors Journal compilation ª2009 FEBS