Báo cáo khoa học: Synechocystis DrgA protein functioning as nitroreductase and ferric reductase is capable of catalyzing the Fenton reaction potx

The DrgA protein exhibited potent quinone reductase activity and, furthermore, we newly found that it contained FMN and highly catalyzed nitroreductase, flavin reductase and ferric reduct

and ferric reductase is capable of catalyzing the Fenton reaction

Kouji Takeda1, Mayumi Iizuka1, Toshihiro Watanabe2, Junichi Nakagawa1, Shinji Kawasaki1 and Youichi Niimura1

1 Department of Bioscience, Tokyo University of Agriculture, Japan

2 Department of Food science and Technology, Tokyo University of Agriculture, Japan

Oxygen is a double-edged sword in that it is essential

for any aerobic organisms, but a part of it is

conver-ted to reactive oxygen species (ROS), which could kill

the cells Among such ROS, the hydroxyl radical is

the most cytotoxic agent, being generated via the

Fen-ton reaction from hydrogen peroxide The FenFen-ton

reaction is a collective designation for the reaction in

which hydrogen peroxide is reduced univalently

through the transfer of an electron in the presence of

Fe2+ to produce an hydroxyl radical It is thought

that most of intracellular iron exists as Fe3+ in order

not to trigger the Fenton reaction Therefore, when

the Fenton reaction occurs, the Fe3+must be reduced

to Fe2+ In some in vitro Fenton systems, superoxide was shown to be capable of reducing free iron [1–3] However, it is not likely that intracellular concentra-tion of superoxide is high enough to contribute in that way [4,5] Other candidate reductants, such as thiols, a-ketoacids, and NAD(P)H, are all abundant inside cells, and each of these can reduce Fe3+ in vitro[6–8] However it is still impossible to conclude that these candidates would function as predominant reductants

in vivo Under exceptional pressure to the cells, Wood-mansee and Imlay [9] demonstrated that in Escheri-chia coli, the Fenton reaction takes place through reduction of Fe3+by the reduced free flavin generated

Keywords

DrgA; Fenton reaction; flavin reductase;

iron(III) reductase; nitroreductase

Correspondence

K Takeda, The Department of Bioscience,

Tokyo University of Agriculture, 1-1-1

Sakuragaoka, Setagaya-ku, Tokyo 156–8502,

Japan

Fax ⁄ Tel: +81 3 54772764

E-mail: k2takeda@nodai.ac.jp

(Received 19 November 2006, revised 31

December 2006, accepted 8 January 2007)

doi:10.1111/j.1742-4658.2007.05680.x

In order to identify an enzyme capable of Fenton reaction in Synechocystis,

we purified an enzyme catalyzing one-electron reduction of t-butyl hydro-peroxide in the presence of FAD and Fe(III)-EDTA The enzyme was a

26 kDa protein, and its N-terminal amino acid sequencing revealed it to be DrgA protein previously reported as quinone reductase [Matsuo M, Endo

T and Asada K (1998) Plant Cell Physiol 39, 751–755] The DrgA protein exhibited potent quinone reductase activity and, furthermore, we newly found that it contained FMN and highly catalyzed nitroreductase, flavin reductase and ferric reductase activities This is the first demonstration of nitroreductase activity of DrgA protein previously identified by a drgA mutant phenotype DrgA protein strongly catalyzed the Fenton reaction in the presence of synthetic chelate compounds, but did so poorly in the pres-ence of natural chelate compounds Its ferric reductase activity was observed with both natural and synthetic chelate compounds with a better efficiency with the latter In addition to small molecular-weight chemical chelators, an iron transporter protein, transferrin, and an iron storage pro-tein, ferritin, turned out to be substrates of the DrgA propro-tein, suggesting it might play a role in iron metabolism under physiological conditions and possibly catalyze the Fenton reaction under hyper-reductive conditions in this microorganism

Abbreviations

ROS, reactive oxygen species.

by flavin reductase in a hyper-reductive environment

when respiration is blocked in the bacteria In this

process, electrons are transferred from the enlarged

NADH pools to FAD, from FADH2 to iron, and

finally from iron to H2O2

In photosynthetic organisms, excess light energy

over the utilizing capacity leads to generation of ROS

Especially under high-intensity light and other stresses,

intercellular ROS accumulation tends to occur, but an

antioxidant protection system usually exists to

counter-act it [10–15] Whether the Fenton recounter-action is involved

in the production of ROS during photosynthesis as

demonstrated in E coli is an open question

In this study we investigated the Fenton reaction in

Synechocystis sp PCC6803, a prokaryote capable of

photosynthesis and categorized as an oxygenic

photo-synthetic bacterium From cell-free extracts, we

puri-fied an enzyme catalyzing one-electron reduction of

t-butyl hydroperoxide in the presence of FAD and

Fe(III)-EDTA The enzyme turned out to be DrgA

protein and its catalytic activities for ferric reductase,

nitroreductase and flavin reductase were demonstrated

Enzyme characterization and its possible involvement

in the Fenton reaction will be presented

Results

Cell free NAD(P)H oxidoreductase activity driving

the Fenton reaction

We examined the Fenton reaction by measuring t-butyl

hydroperoxide reducing activity using cell-fee extracts

after dialysis, NADH or NADPH (as electron donor),

FAD or FMN (as free flavin), and FeCl3 or

Fe(III)-EDTA (as iron compounds) In Fenton reactions using

the cell-free extracts prepared after dialysis

supplemen-ted with NADH and NADPH, we detecsupplemen-ted lower

activity with FeCl3 than with Fe(III)-EDTA in the

presence or absence of free flavin We detected the

highest Fenton activity with NADPH and

Fe(III)-EDTA, while a marked potentiation by flavin was

observed when using NADH and Fe(III)-EDTA

(Table 1)

Although the enzyme system in E coli proposed by

Woodmansee and Imlay [9] required free flavin for

activation, in Synechocystis, there are flavin-dependent

and flavin-independent systems In the Fenton reaction

with NADH using Synechocystis cell-free extracts

pre-pared prior to dialysis, we detected high activity in the

presence of Fe(III)-EDTA, but this was not further

po-tentiated by addition of free flavin (supplementary

Table S1) We attributed this to free flavin contained

in the cell-free extracts

Purification of the NAD(P)H oxidoreductase responsible for the Fenton reaction associated with free flavin

In an attempt to identify the presumed enzyme in the presence of the Fenton reaction, we purified an enzyme catalyzing flavin-dependent peroxide-reducing activity using t-butyl hydroperoxide The purification proce-dure was described in Experimental proceproce-dures The purified protein showed a single protein band of

26 kDa on a SDS⁄ PAGE gel (supplementary Fig S1) The N-terminal amino acid sequence was determined

to be MDTFDAIYQRRSVKHFDPDH, and it turned out to be identical to that of DrgA protein [16] The purification procedure, as described in the Experimental procedures section, gave a yield of 51%

in the terms of t-butyl hydroperoxide reducing activity (Table 2)

Characterization of DrgA protein Identification of FMN contained in DrgA protein The amino acid sequence predicted from the DNA sequence of the drgA gene displayed sequence homolo-gies to several bacterial flavoproteins [17–22] Several highly FMN-binding regions (at positions 10–14 and 148–151) have been identified in the amino acid sequences of DrgA Both endogenous and recombinant DrgA protein exhibited an absorption spectrum typical

of a flavoprotein (Fig 1 at 459 nm, arrow) Further-more, by HPLC analysis, the flavin coenzyme released from native DrgA protein by hot methanol treatment [23] was identified as FMN (data not shown) The

Table 1 NAD(P)H oxidoreductase activities responsible for Fenton reaction in the dialyzed cell-free extracts The activity was deter-mined following absorbance of NAD(P)H oxidation at 340 nm in a

50 m M sodium phosphate buffer (pH 7.0) at 30 C The reaction mixture contained 100 l M Fe(III)-EDTA, 15 l M flavin and 1 m M t-bu-tyl hydroperoxide Specific activity is expressed as enzyme activity per mg of total protein ND, not detected.

NAD(P)H oxidoreductase activities responsible for Fenton reaction (mU ⁄ mg protein)

NADH

Fe(III)-EDTA 67.9 ± 13.9 59.1 ± 14.3 10.7 ± 3.0 NADPH

Fe(III)-EDTA 119.2 ± 18.4 87.2 ± 0.7 76.2 ± 13.7

recombinant DrgA protein preparation also showed a

similar absorption maximum, confirming the

associ-ation of FMN with DrgA protein The absorption

maximum at 459 nm disappeared upon addition of

0.3 mm NADH under anaerobic conditions indicating

reduction of protein-bound FMN (Fig 1) The ratio

of absorbance at 280 and 459 nm was 4.32 : 1 for

native DrgA protein, and 4.36 : 1 for recombinant

DrgA protein

Substrate specificity

As summarized in Table 3, the Synechocystis DrgA

protein showed significant substrate preference to

qui-nones as previously reported by Matsuo et al [24] We measured quinone reductase activity using ubiquinone

0 as substrate In the presence of NADH, the specific activities of endogenous Synechocystis DrgA protein and recombinant DrgA protein were 7.03 UÆmg)1 pro-tein and 7.67 UÆmg)1protein Those in the presence of NADPH were 11.33 UÆmg)1 protein and 11.98 UÆmg)1 protein, respectively, indicating endogenous and recombinant DrgA protein were equally potent as qui-none reductase

Moreover, the recombinant DrgA protein showed substrate specificity similar to that of endogenous Syn-echocystis DrgA protein (data not shown) Therefore,

we used recombinant DrgA protein in subsequent experiments

DrgA protein showed nitroreductase activity for nitrobenzene, dinoseb and nitrofurazone, with the highest activity for nitrofurazone

The flavin reductase activities of DrgA protein for FAD and FMN were 7.41 and 6.95 UÆmg)1 protein, respectively, in the presence of NADPH

Ferric reductase activities and peroxide reducing activities responsible for Fenton reaction Reduction of iron is known to require reduced flavins provided by flavin reductase In the presence of free FAD, we found ferric reductase activity of recombin-ant DrgA protein using various iron compounds (Table 4) The specific activity of ferric reductase of DrgA protein for natural chelators varied between 0.1 and 2.0 UÆmg)1 protein, and that for synthetic chela-tors varied between 1.7 and 5.2 UÆmg)1protein Surprisingly, as well as being active with small molecular weight chemicals, DrgA protein was also active with the iron transport protein transferrin (1.06 UÆmg)1 protein), and the iron storage protein ferritin (1.74 UÆmg)1protein)

For measurement of the Fenton reaction we used peroxide as substrate, and the specific activities for natural chelators were 0.5–3.5 UÆmg)1 protein and those for synthetic chelators were 12.3–39.0 UÆmg)1 protein Thus, the activity for the Fenton reaction was about 10 times higher for synthetic chelators than for natural chelators

Chemical stoichiometry of the Fenton reaction The chemical stoichiometry of hydrogen peroxide reducing activity of DrgA protein in the presence of NADH and Fe(III)-EDTA was investigated From a mass balance, we estimated that in this enzymatic reac-tion, 148 lm of hydrogen peroxide were reduced by

Table 2 Purification of NADH-dependent t-butyl

hydroperoxide-reducing activity responsible for the Fenton reaction The activity

was determined following absorbance of NAD(P)H oxidation at

340 nm in a 50 m M sodium phosphate buffer (pH 7.0) at 30 C.

The reaction mixture contained cell-free extracts, 150 l M NADH,

100 l M Fe(III)-EDTA, 15 l M FAD and 1 m M t-butyl hydroperoxide.

Specific activity is expressed as enzyme activity per mg of total

protein The cell-free extracts were prepared starting from 10 g

wet cells

Total

protein

(mg)

Total activity (U)

Specific activity (U ⁄ mg protein)

Purification index

Yield (%)

0.15

0.10

0.05

0.00

Wavelength (nm)

Fig 1 Absorption spectra of native, recombinant DrgA protein and

recombinant DrgA protein reduced by NADH Absorption spectra of

the purified native (16.8 l M ; ——), recombinant DrgA protein

(23.6 l M ; - - - -) and recombinant DrgA protein after anaerobic

reduc-tion with 0.3 m M NADH (– – – –) in a 50 m M sodium phosphate

buf-fer, pH 7.0, at 25 C.

consuming 84 lm of NADH, generating 148 lm of hydroxyl radical as final product

Collectively, the chemical stoichiometry of the reac-tion can be formulated as follows (a one-electron reduction):

2H2O2þ NADH þ Hþ! 2OHþ NADþþ 2H2O

In addition, when DNA degradation was measured using pBR322 plasmid as substrate (in the absence of FAD), the reaction resulted in complete degradation

of the DNA (no band was detected in Fig 2, lane V)

A partial DNA degradation was observed in the absence of iron compound (Fig 2, lane III)

Kinetic parameters for substrates DrgA protein catalyzed activity for nitroreductase, fla-vin reductase and ferric reductase In the presence of saturated concentration of the substrates for these activities, namely, 50 lm nitrofurazone (nitroreduc-tase), 30 lm FAD or FMN (flavin reduc(nitroreduc-tase), and

50 lm Fe(III)-EDTA in the presence and absence of

30 lm FAD (ferric reductase), we measured the Km

values of NADH and NADPH (supplementary Table S2) As the Km values for NADPH were much lower using any substrates than those for NADH, we meas-ured the Km and kcat values of these reactions with a

Table 3 Substrate specificity of purified DrgA protein in the

pres-ence of either NADH or NADPH Experimental details are described

in the Experimental procedures section Oxidation of 150 l M NADH

or NADPH was measured in the presence of an electron acceptor.

Specific activity is expressed as enzyme activity per milligram of

purified native or recombinant DrgA protein ND, not detected.

Electron acceptor

Enzyme activity (UÆmg protein)1)

Quinone reductase

Flavin reductase

Other related enzyme activity

Nitroreductase

Table 4 Effect of different iron compounds on the ferric reductase

activities and NAD(P)H oxidoreductase activities responsible for the

Fenton reaction Experimental details are described in the

Experi-mental procedures section Oxidation of 150 l M NADPH was

meas-ured at 340 nm in a reaction mixture containing Fe(III) complexes,

15 l M FAD and recombinant DrgA protein for ferric reductase

activ-ity, and the same reaction mixture was used the addition of 200 l M

H 2 O 2 for the Fenton reaction The final concentration of the Fe(III)

complexes was 10 l M except for Ferritin, where the reaction

mix-ture contained 382.5 lg ferritin Specific activity is expressed as

enzyme activity per milligram of purified recombinant DrgA protein

Iron compounds

Enzyme activity (UÆmg protein)1)

Ferric reductase activity

Fenton reaction Natural chelate iron compounds

Fe(III) ammonium citrate 1.98 ± 0.06 2.82 ± 0.18

Fe(III)-deoxymugineic acid 0.13 ± 0.05 0.53 ± 0.16

Fe(III)-nicotianamine 0.58 ± 0.25 3.46 ± 0.11

Synthetic chelate iron compounds

Fe(III)-nitrilotriacetic acid 1.74 ± 0.3 12.33 ± 0.12

Natural iron transporter protein

Natural iron storage protein

Ferritin from horse spleen 1.74 ± 0.03 0.95 ± 0.06

a

Diethylenetriamine-N,N,N ¢,N ¢¢,N ¢¢-pentaacetic acid.

Fig 2 DNA degradation Experimental details are described in the Experimental procedures section The reaction mixture contained 3.2 lg pBR322 (lane I), 3.2 lg pBR322 plus 300 l M H 2 O 2 (lane II), 3.2 lg pBR322 plus recombinant DrgA protein (lane III), 3.2 lg pBR322 plus Fe(III)-EDTA plus 300 l M H2O2 (lane IV), 3.2 lg pBR322 plus Fe(III)-EDTA plus 300 l M H 2 O 2 plus recombinant DrgA protein (lane V).

saturated concentration of NADPH (150 lm) Table 5

summarizes the values of Km, kcat and kcat⁄ Km for

these reactions

The kcat⁄ Km value of DrgA protein for

nitrofura-zone was 8.57 ± 0.67· 105m)1Æs)1, a value similar to

those reported for nitrofurazone of E coli

nitro-reductase NfsA and NfsB (6.5· 106m)1Æs)1 and

8.3· 104m)1Æs)1, respectively) [25,26] The kcat⁄ Km

value for FMN reductase activity of DrgA protein was

2.65 ± 0.14· 105m)1Æs)1 It is relatively low

com-pared with the corresponding value of Vibrio harveyi

NADPH-flavin oxidoreductase, which is 5.5·

106m)1Æs)1[27–29]

Many flavin reductases display ferric reductase

activ-ity [30–34] In parallel, iron compounds, such as FeCl3

or Fe(III)-EDTA have been used as model substrates

to study ferric reductase, and such effort has yielded in

identification of several ferric reductases from various

organisms [35–39] The kcat⁄ Km value for

Fe(III)-EDTA of DrgA protein was 10.9 ± 0.21· 106m)1Æs)1

in the presence of free FAD In the absence of free

FAD, it was 3.67 ± 0.05· 104m)1Æs)1, indicating that

this activity is markedly stimulated by addition of free

flavin

Discussion

The Fenton reaction generates compounds that are

toxic to cells and presumably plays a role in

restrain-ing bacterial growth under severe environmental

pressure In E coli, the Fenton reaction takes place

when the respiratory chain is blocked, as shown by

Woodmansee and Imlay [9] The photosynthetic

bacterium Synechocystis would be under stress when

exposed to strong light due to overproduction of ROS,

and the enzyme responsible for the Fenton reaction is

identified here as DrgA protein DrgA protein was first

purified by Matsuo et al [24], who showed that its

reductase activity worked best towards quinone, among other substrates tested; weak activity for nitro-benzene was also demonstrated However, upon homology search for DrgA protein using BLAST, the amino acid sequence of the DrgA protein deduced from its DNA sequence was found to be similar to that of several nitroreductase-like proteins [17–22] The highest sequence homology (67% identity) was assigned to a nitroreductase from Trichodesmium ery-thraeumIMS101

Furthermore, by examining DrgA mutant strains, Elanskaya and co-workers demonstrated that the pro-tein could be involved not only in quinone reduction [40,41], but also in the reduction of nitroaromatic com-pounds [40,42]

In the present study, nitroreductase activity of purified DrgA protein was first demonstrated by using nitrobenzene, dinoseb and nitrofrazone as sub-strate, with the highest activity for nitrofurazone The kcat⁄ Km value of DrgA protein for nitrofurazone was 8.57 ± 0.67· 105m)1Æs)1 Together, our data indicate that DrgA protein functions as nitroreduc-tase in vitro

The two crystallized nitroreductases of E coli and Enterobacter cloacae which are homologous to DrgA protein were reported to contain FMN [43–47] and their highly conserved FMN binding sites (NCBI database, Conserved domains cd02149.2) are also found in the DrgA protein sequence at positions 10–

14 and 148–151 Indeed our DrgA protein was also shown to contain FMN, although this was not so in the previous report by Matsuo et al [24] As pro-tein-bound FMN is known to be readily released by dialysis and gel filtration, we kept these procedures

at a minimum Therefore, it is likely that the differ-ence in the FMN content in the two DrgA protein preparations is due to the difference in the purifica-tion scheme

Table 5 Kinetic parameters of DrgA protein (recombinant DrgA protein was used) Experimental details are described in the Experimental procedures section Oxidation of 150 l M NADPH (saturated concentration) was measured in the presence of an electron acceptor.

Substrate

K m value for substrate (l M ) kcat(s)1) kcat⁄ K m ( M )1Æs)1)

Nitroreductase

Flavin reductase

Ferric reductase

An amino acid sequence homology search of DrgA

protein picked up flavin reductase as the second

high-est score after nitroreductase Indeed in this study it

was demonstrated that DrgA protein has a reductase

activity to flavin as well as to nitroaromatic

com-pounds Flavin reductases are known to be capable of

ferric reduction [30–34] and, recently, Woodmansee

and Imlay [9] proposed that this reaction can be

involved in the Fenton reaction both in vivo and

in vitro Our DrgA protein also catalyzed the Fenton

reaction as well as iron(III) reduction in vitro

There are two types of ferric reductase reactions:

namely, a reaction using flavin, and a reaction

inde-pendent of flavin While ferric reductase observed in

E colirequired flavin, it was not essential for iron(III)

reduction by DrgA protein, though addition of flavin

stimulated the reaction The kcat⁄ Km values of DrgA

protein for Fe(III)-EDTA were 10.9 ± 0.21·

106m)1Æs)1 in the presence of FAD and

3.67 ± 0.05· 104 m)1Æs)1 in its absence, much higher

than the reported kcat⁄ Km value of a ferric reductase,

FerB, of Paracoccus denitrificans, which is only

1· 102m)1Æs)1 [39] Although variation caused by

technical differences in the measurement of the two

experiments should be considered, these results support

the idea that DrgA protein probably functions as a

fer-ric reductase using free flavin rather than functioning

simply as a flavin reductase

We have showed here that DrgA protein utilizes both

a synthetic iron chelator, such as EDTA, and natural

chelators such as citric acid In addition to these small

molecular weight chemical chelators (natural chelate

compounds and synthetic chelate compounds),

transfer-rin and ferritin could be substrates of the ferric

reduc-tase activity of DrgA protein These observations

indicate that DrgA protein might function in iron

meta-bolism under physiological conditions

Collectively, DrgA protein is an oxidoreductase

util-izing NADH or NADPH as electron donors, and

qui-none, nitroaromatic compounds, flavin and iron

chelated compounds as electron acceptors Enzyme

kinetic studies indicate that DrgA protein exerts an

efficient reductase reaction to iron in the presence of

flavin

The driving force of the Fenton reaction is a

diva-lent iron generated from the ferric reductase reaction

In a hyper-reductive environment, possibly caused by

exposure to strong light, this enzyme system might

trigger the Fenton reaction It would now be

interest-ing to compare wild-type strains and drgA gene

dele-tion mutant strains for growth rate and the reguladele-tion

of DrgA protein expression under environmental

stresses such as iron depletion

Experimental procedures

Cell culture and preparation of cell-free extracts Synechocystis sp PCC6803 cell culture and preparation of cell-free extracts were carried out as described previously [48]

Enzyme purification All purification steps were carried out below 4C The cell-free extracts from 10 g wet cells were ultracentrifuged

at 100 000 g for 2 h (XL-100K centrifuge, Beckman, rotor type 45 Ti) and the supernatant (38 mL) was treated with streptomycin (final concentration 2%) to remove nucleic acids and stirred for 30 min on ice After centrifugation at

17 400 g for 20 min (Avanti HP-25 centrifuge, Beckman, rotor type JA 25.5), the supernatant (47 mL) was supplied with 1.14 m ammonium sulfate and the pH of the cell-free extracts was adjusted to 7.0 with 2.8% ammonium solu-tion, followed by stirring for 30 min After centrifugation

at 17 400 g for 15 min (Avanti HP-25 centrifuge, Beck-man, rotor type JA 25.5), the supernatant (49 mL) was subjected to a butyl toyopearl (Tosoh, Tokyo, Japan) col-umn (3.5· 22.0 cm) equilibrated with a 50 mm sodium phosphate buffer, pH 7.0, containing 1.14 m ammonium sulfate The column was washed with four column vol-umes of the same buffer, and the protein was eluted with

a linear gradient of 1.14 m ammonium sulfate to 0 m The pooled fraction (100 mL) was dialyzed twice against 5 L

of a 10 mm sodium phosphate buffer, pH 8.0 The dialy-sate was subjected to a DEAE Sepharose Fast Flow (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) column (3.3· 23.5 cm) equilibrated with a 10 mm sodium phos-phate buffer, pH 8.0 The column was washed with three column volumes of the same buffer, and the enzyme was eluted with a linear gradient of NaCl (0–250 mm) The active fractions (62 mL) were pooled, concentrated and dialyzed against 10 mm sodium phosphate buffer, pH 8.0,

by an Apollo membrane (cut-off size 10 kDa, Orbital Bio-science, Topsfield, MA, USA) Pooled fractions (6.2 mL) were put on a POROS HQ-H (Applied Biosystems, Tokyo, Japan) column (1.0· 10.0 cm) equilibrated with the same buffer The column was washed with five column volumes of the same buffer, and the enzyme was eluted with a linear gradient of NaCl (0–250 mm) The active fractions (120 mL) were pooled, concentrated and dialyzed against a 50 mm sodium phosphate buffer, pH 7.0, by an Apolo membrane (cut-off size 10 kDa, Orbital Bioscience) The purity and molecular mass of the enzyme were deter-mined by SDS⁄ PAGE by the method of Laemmli [49] The proteins were electro-transferred to a polyvinylidene difluoride membrane and the N-terminal sequence was determined by a protein sequencer (model 492, Applied Biosystems)

Enzyme assay

Fenton reaction activity

Enzyme activities were measured anaerobically Enzyme

solu-tions containing cell-free extracts (0.14–0.48 mg protein), or

1 lg purified enzyme in the presence or absence of flavin in a

50 mm sodium phosphate buffer (pH 7.0) were loaded into a

Tunberg tube After anaerobiosis was established by repeated

evacuation and equilibration with oxygen-free argon at

30C, the reaction was initiated by addition of enzyme

solu-tion to mixtures of iron(III) compounds and NADH solusolu-tion

The reaction was monitored at 340 nm in a

spectrophotome-ter (Hitachi U-3000) The iron(III) compounds and 150 lm

NADH solution in a 50 mm sodium phosphate buffer

(pH 7.0), in the presence or absence of 1 mm t-butyl

hydro-peroxide, were made anaerobic by bubbling with oxygen-free

argon at 30C Fenton reaction activity was determined by

measuring the difference of NAD(P)H consumption in the

presence and absence of t-butyl hydroperoxide

The absorbance coefficient of NADH and NADPH were

set to be 6.22 and 6.20 m m)1Æcm)1, respectively One unit

activity of the Fenton reaction is defined as the amount of

enzyme that oxidizes 1 lmole of NAD(P)H per minute

Ferric reductase activity

Ferric reductase activity was measured anaerobically in

the same reaction mixture as for the Fenton reaction, but

without t-butyl hydroperoxide, at 30C The activity was

determined by measuring the difference of NAD(P)H

consumption at 340 nm in the presence and absence of

iron(III) compounds

Flavin reductase activity

Flavin reductase activity was measured anaerobically using

the same reaction mixture as for ferric reductase, but

with-out iron(III) compounds, at 30C Flavin reductase activity

was determined by measuring the difference in NAD(P)H

consumption at 340 nm in the presence and absence of the

enzyme

Nitroreductase activity

The nitroreductase activity was measured aerobically at

30C The reaction mixture contained 50 mm sodium

phos-phate buffer (pH 7.0), 150 lm NAD(P)H, nitro compounds

and enzyme Nitroreductase activity was determined by

measuring NAD(P)H consumption at 340 nm in the presence

and absence of an enzyme

Substrate specificity for NAD(P)H oxidation

Substrate specificity was examined under aerobic conditions

because purified DrgA protein does not react with oxygen

NAD(P)H solution (final concentration 150 lm, in a 20 mm Tris⁄ HCl buffer, pH 7.5) was prewarmed to 30 C and placed in a micro black-cell and set into a spectrophoto-meter (Hitachi U-3000) NAD(P)H oxidation measurement was immediately started at 340 nm, and substrates were added to the mixture After baseline equilibrium was reached, DrgA protein was added to the mixture 2,3-dimethoxy-5-methyl-1, 4-benzoquinone (ubiquinone 0), duroquinone, ferricyanide, FAD, FMN, nitrobenzene, di-noseb and nitrofurazone were used as substrates at a final concentration of 100 lm each In the case of cytochrome C, the concentration was set to 50 lm and absorbance was measured at 550 nm The absorbance coefficient of NADH and NADPH was set as described above

Stoichiometry of the Fenton reaction Stoichiometry and confirmation of the product of the Fen-ton reaction were carried out under anaerobic conditions DrgA protein (140.8 lg), deoxyribose (final concentration 0.6 mm) and Fe(III)-EDTA (final concentration 5 lm) were mixed in a 15 mm sodium phosphate buffer, pH 7.0, in a Tunberg tube (final volume, 1.6 mL), then the air was sub-stituted with argon for 15 min NADH (final concentration

100 lm) and hydrogen peroxide (final concentration

300 lm) were added in a 15 mm sodium phosphate buffer,

pH 7.0, in another aerobic cuvette, and air was substituted with argon for 15 min The anaerobic cuvette and the tube were warmed at 30C for 5 min, and the initial concentra-tion of NADH was determined on site by measuring its absorption at 340 nm Then, the content of the Tunberg tube was transferred to an anaerobic cuvette using a syringe and the solution was mixed well The reaction was monit-ored by measuring the consumption of NADH at 340 nm

In parallel, the amount of hydrogen peroxide and hydroxyl radicals was measured before and after the reaction The quantitation of hydrogen peroxide and hydroxyl radicals was carried out as described previously [50,51]

DNA degradation in the Fenton reaction DNA degradation was measured under anaerobic condi-tions The recombinant DrgA protein (140.8 lg), Fe(III)-EDTA (final concentration 5 lm) and 3.2 lg pBR322 DNA were mixed in a 15 mm sodium phosphate buffer, pH 7.0,

in a Tunberg tube (final volume 1.6 mL), and the air was substituted with argon for 15 min In another aerobic cu-vette NADH (final concentration 100 lm) and hydrogen peroxide (final concentration 300 lm) were added in a

15 mm sodium phosphate buffer, pH 7.0, and the tubes were warmed at 30C for 5 min Following confirmation

of the initial concentration of NADH by measuring its absorption at 340 nm, the content of the Tunberg tube was transferred to an anaerobic cuvette using a syringe, mixed well and incubated for 5 min The reaction was monitored

by measuring the decrease of absorption of NADH at

340 nm Each 20 lL of reaction mixture was subjected to

agarose gel electrophoresis and DNA bands were visualized

on the gel by staining with ethidium bromide

Steady-state kinetics

The values of Km and kcat for Fe(III)-EDTA, FAD, FMN

and nitrofurazone was determined from Lineweaver)Burk

plots of the kinetic data obtained at 30C at various

sub-strate concentrations in a 50 mm sodium phosphate buffer,

pH 7.0, containing 150 lm NADPH The consumption of

NADPH was monitored with a spectrophotometer at

340 nm (Hitachi U-3000)

Cloning, expression, and purification of DrgA

from Synechocystis sp PCC6803

We cloned the gene of drgA from Synechocystis sp

PCC6803 A Synechocystis DNA fragment containing the

open reading frame, slr 1719, was amplified by the PCR using

the forward primer, 5’-acg aat tcc acc acc acc acc acc aca tgg

aca cct ttg acg cta tt-3’ and the reverse primer, 5’-tag ctc gag

tta ggc aaa gga gtt ttc cca-3’ The forward primer was

designed to introduce six His Tags following an EcoR I site,

and the reverse primer contained a Xho I site as underlined

Amplified DNA fragments were subcloned into the

pTrc99A vector for transformation of E coli strain JM109

IPTG-induced recombinant protein was purified

All steps of the purification procedure of recombinant

Synechocystis DrgA were carried out at 4C and

monit-ored by SDS⁄ PAGE Cells (23 g wet weight) were

suspen-ded in 92 mL of 50 mm sodium phosphate buffer, pH 7.0

The suspension was stirred at 4C for 20 min Cells were

thawed and passed through a French pressure cell (Thermo

IEC, Needham, Heights, MA, USA) twice at 88.99 kgÆcm)2

and then sonicated for 3 min Phenylmethylsulfonyl fluoride

(final concentration, 2 mm) was added to the suspension

three times, i.e immediately before and after the passage

through the French pressure cell, and after sonication The

resultant suspension was centrifuged at 64 000 g for 20 min

(Avanti HP-25 centrifuge, Beckman, JA 25.5 rotor) to

remove unbroken cells The supernatant was treated with

streptomycin to remove nucleic acids and was stirred at

4C for 30 min After centrifugation at 64 000 g for

20 min (Avanti HP-25 centrifuge, Beckman, JA 25.5 rotor),

the supernatant (112 mL) was dialyzed twice against 5 L of

the 50 mm sodium phosphate buffer, pH 7.0, containing

300 mm NaCl The dialysate (120 mL) was subjected to a

Talon (Takara, Tokyo, Japan) column (2.2· 5.3 cm)

equili-brated with 50 mm sodium phosphate buffer, pH 7.0,

con-taining 300 mm NaCl The column was washed with five

volumes of the same buffer The enzyme was eluted

step-wise with 50, 100 and 150 mm imidazole from the column

The pooled fraction from 50 to 100 mm imidazole elution

was dialyzed three times against 5 L of a 10 mm sodium phosphate buffer, pH 8.0 The dialysate (35.5 mL) was sub-jected to a DEAE Sepharose Fast Flow (GE Healthcare Bio-Sciences) column (3.3· 23.5 cm) equilibrated with a

10 mm sodium phosphate buffer, pH 8.0 The column was washed with five column volumes of the same buffer, and the enzyme was eluted with a linear gradient of NaCl (0–

250 mm) Active fractions were pooled, concentrated and dialyzed against 45 mL of 50 mm sodium phosphate buffer,

pH 7.0, by an Apollo membrane (cut-off size 10 kDa, Orbi-tal Bioscience) The measurement of maximum absorption wavelength and extinction coefficient of DrgA protein pre-paration was carried out as described previously [52] The extinction coefficient for the bound FMN at 459 nm was estimated to be 11.9 mm)1Æcm)1

Acknowledgements

We are grateful to Professor S Mori of The University

of Tokyo for providing deoxymugineic acid and nico-tianamine We thank A Sekine and M Fujiya for their technical assistance

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Supplementary material

The following supplementary material is available online:

Fig S1 SDS⁄ PAGE of the purified t-butyl hydro-peroxide reducing enzyme SDS⁄ PAGE was carried out as described in Experimental procedures using 15% polyacrylamide gels

Table S1 NAD(P)H oxidoreductase activities responsible for the Fenton reaction in the pre-dialysis cell-free extracts The activity was determined follow-ing absorbance of NAD(P)H oxidation at 340 nm in a

50 mm sodium phosphate buffer (pH 7.0) at 30 C The reaction mixture contained 100 lm Fe(III)-EDTA,

15 lm flavin and 1 mm t-butyl hydroperoxide Specific activity is expressed as enzyme activity per milligram

of total protein

Table S2 Km for NADH or NADPH Experimen-tal details are described in the ExperimenExperimen-tal proce-dures section Oxidation of 150 lm NADH or NADPH was measured in the presence of an electron acceptor

This material is available as part of the online article from http://www.blackwell-synergy.com

Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corres-ponding author for the article