Characterization of the quinone reductase activity of the ferric reductase B protein from Paracoccus denitrificans

Archives of Biochemistry and Biophysics 483 (2009) 29–36

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Characterization of the quinone reductase activity of the ferric reductase B protein from Paracoccus denitrificans Vojteˇch Sedlácˇek a, Rob J.M. van Spanning b, Igor Kucˇera a,* a b

Department of Biochemistry, Faculty of Science, Masaryk University, Kotlárˇská 2, CZ-61137 Brno, Czech Republic Department of Molecular Cell Physiology, Faculty of Earth and Life Science, VU University Amsterdam, NL-1081 HV Amsterdam, The Netherlands

a r t i c l e

i n f o

Article history: Received 14 October 2008 and in revised form 12 December 2008 Available online 30 December 2008 Keywords: NADH Flavoprotein Quinone reduction Redox cycling

a b s t r a c t The ferric reductase B (FerB) protein of Paracoccus denitrificans exhibits activity of an NAD(P)H: Fe(III) chelate, chromate and quinone oxidoreductase. Sequence analysis places FerB in a family of soluble flavin-containing quinone reductases. The enzyme reduces a range of quinone substrates, including derivatives of 1,4-benzoquinone and 1,2- and 1,4-naphthoquinone, via a ping-pong kinetic mechanism. Dicoumarol and Cibacron Blue 3GA are competitive inhibitors of NADH oxidation. In the case of benzoquinones, FerB apparently acts through a two-electron transfer process, whereas in the case of naphthoquinones, one-electron reduction takes place resulting in the formation of semiquinone radicals. A ferB mutant strain exhibited an increased resistance to 1,4-naphthoquinone, attributable to the absence of the FerB-mediated redox cycling. The ferB promoter displayed a high basal activity throughout the growth of P. denitrificans, which could not be further enhanced by addition of different types of naphthoquinones. This indicates that the ferB gene is expressed constitutively. Ó 2008 Elsevier Inc. All rights reserved.

Ferric reductase B (FerB) was initially isolated from Paraccoccus denitrificans as a homodimeric, FAD-containing flavoprotein capable of reducing various Fe(III) complexes with either NADH or, less efficiently, NADPH as the electron donor [1]. Analysis of the first 27 N-terminal amino acid residues sequence revealed that FerB had a homology with the previously described chromate reductase (ChrR)1 from Pseudomonas putida [2]. Indeed it was subsequently shown that purified FerB was able to reduce chromate. Moreover, it turned out that it is also reduces certain benzo- and naphthoquinones, and hence could be termed a quinone reductase. Later, the same conclusion was drawn for the ChrR enzyme [3]. Among the currently known quinone reductases, one of the most intensively studied members is the NAD(P)H:quinone oxidoreductase 1 (NQO1; EC 1.6.99.2), formerly called DT-diaphorase. It is a cytosolic flavoenzyme that contains 2 FAD per homodimer of 55,000-Mr. Being widely distributed in animal tissues, NQO1 is generally regarded as a protective device because of its ability to reduce cytotoxic quinone xenobiotics directly to hydroquinones in a two-electron process, in this way preventing superoxide production resulting from one-electron reduction of quinones by other enzymes and the subsequent autooxidation of the semiquinones * Corresponding author. Fax: +420 54949 2690. E-mail address: [email protected] (I. Kucˇera). 1 Abbreviations used: ChrR, chromate reductase; E17 , one-electron redox potential at pH 7; FerB, ferric reductase B; IC50, half maximal inhibitory concentration; MdaB, modulator of drug activity B protein; NQO1, NAD(P)H:quinone oxidoreductase 1; NQR, NAD(P)H:quinone reductase; SOD, superoxide dismutase, WrbA, tryptophan repressor-binding protein; Tris, tris(hydroxymethyl)aminomethane. 0003-9861/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.12.016

formed [4]. In addition, NQO1 may act as an antioxidant enzyme by regenerating antioxidant forms of ubiquinone and tocopherolquinone [5]. A distinctive property of the enzyme is its sensitivity to inhibition by submicromolar concentrations of the anticoagulant dicoumarol [6]. Several reports now indicate that NQO1 found in animal tissues has functional counterparts in plants, fungi and bacteria [7]. The enzymes from tobacco leaves [8] and Arabidopsis thaliana [9] were found to resemble NQO1 with respect to the reaction mechanism and with respect to donor and acceptor specificity but to differ from it with regard to (i) the molecular structure (tetramers instead of dimers), (ii) type of flavin cofactor (FAD replaced by FMN), (iii) the hydride transfer stereospecificity and (iv) much lower sensitivity to dicoumarol. FMN-containing, dimeric quinone reductases have been described in basidiomycetes Phanerochaete chrysosporium [10] and Gloeophyllum trabeum [11], and in yeast Saccharomyces cerevisiae [12]. Escherichia coli expresses at least four soluble proteins able to shuttle electrons from reduced nicotinamide nucleotides to quinoid acceptors. The first one is NAD(P)H-dependent quinone oxidoreductase (QOR), which does not require any flavin cofactor, catalyzes the reduction of 1,4-benzoquinone derivatives and appears to be inactive towards naphthoquinones. Structurally it belongs to the medium-chain dehydrogenase/reductase superfamily, which also includes f-crystallin and the zinc-dependent enzymes alcohol dehydrogenase and glucose dehydrogenase [13,14]. The second on is the enzyme ‘modulator of drug activity B’ (MdaB), which was identified on the basis of the resistance that

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it confers to the antibiotics DMP 840, adriamycin and etoposide [15]. It proved to be identical with a previously described diaphorase-like enzyme inducible in E. coli by 2-alkyl-1,4-quinones [16,17]. The crystal structure of MdaB has recently been determined both in its apo form and in complex with FAD, revealing a close structural relationship to established members of the diaphorase family [18]. The third protein, designated YieF, exists as a homodimer with FMN as cofactor. Although it is able to carry out a four-electron reduction of chromate plus molecular oxygen to Cr(III) plus superoxide, its physiological role is more likely be the two-electron reduction of quinones to quinols [19]. The fourth E. coli protein is WrbA, which was originally proposed to play a role in regulation of the tryptophan operon, but recent results implicate that it is involved in quinone reduction too [20]. Little is known about the role of microbial quinone reductases in cell protection in general and prevention of superoxide formation in particular. The observations are scarce and far from conclusive. For example, analyses of P. putida strains expressing different levels of ChrB suggest that this protein enables the cells to defend themselves against the oxidative stress generated by chromate [19] or H2O2 [3], but not by one-electron oxidizers Cu2+ or paraquat [3]. Deletion of the gene for the quinone reductase Lot6p from the yeast genome resulted in a considerably increased toxicity of 1,4naphthoquinone to the resulting mutant strain [12]. On the contrary, deletion of the mdaB gene in E. coli did not affect the growth in the presence of menadione [18]. We felt it therefore important to acquire further data on the interaction of FerB with quinones which might disclose similarities or dissimilarities in relation to other known quinone reductases and shed new light on various aspects of its involvement in bacterial metabolism.

30 °C during continual shaking. The turbidities of the cultures were recorded every 20 min at 600 nm. The test samples were performed in triplicate, and five replicates were used for each control. The specific growth rate was calculated from a semilogarithmic plot of the growth curve. Construction of a ferB promoter-lacZ (PrferB::lacZ) fusion strain A 500 bp DNA fragment upstream the coding region of the ferB gene was PCR amplified using Taq DNA polymerase with the primers 1 (50 -AAAGAATTCGCCGGCTCACGGCCAGAAGCTG-30 ) and 2 (50 AAAGGATCCCAGCCGGCCCTCGGCCAGTTTC-30 ) containing restriction sites for EcoRI and BamHI (in italic), respectively. The resulting fragment was ligated in the pGEM-T Easy vector (Promega), which was then transferred to E. coli TG1. The 500 bp EcoRI/BamHI fragment of the resulting pGEM derivative was then excised and inserted into the EcoRI/BamHI restricted vector pBK11 [24]. The resulting ferB promoter-lacZ reporter vector was transferred to P. denitrificans 1222 via triparental mating using E. coli HB101/ pRK2020 as a helper strain [25]. Clones of P. denitrificans that inserted the vector successfully in their chromosome via homologous recombination were selected on plates with rifampicin and streptomycin. Cells from the resulting colonies were checked for proper integration of the promoter lacZ fusion by site-specific PCR using the two primers described above together with the specific upstream primer 3 (50 -TGGGTCAGCGATTCCGCCCGGGTCTC-30 ) and downstream primer 2 bordering the integrated DNA with the PferB-lacZ fusion. One of the strains obtained in this manner was used for further studies and was designated Pd1222::pPr021. Isolation of FerB

Materials and methods Strains, media, and growth conditions The Paracoccus denitrificans strains employed included CCM982 (Czech Collection of Microorganism, Brno, Czech Republic), Pd1222 (a restriction-deficient derivative of the wild-type strain, [21]), Pd20021 (a mutant strain with the ferB gene interrupted by a kanamycin-resistance marker; Sedlácˇek, V., Van Spanning, R.J.M., Kucˇera, I., unpublished results) and Pd1222::pPr021. E. coli [22] served as a recipient strain for gene cloning and a donor for conjugative transfer into Paracoccus cells, with participation of the E. coli HB101 helper strain harboring pRK2020 [23]. P. denitrificans was grown at 30 °C in brain/heart infusion (BHI) broth (Oxoid) or in a chemically defined medium composed of: 17 mM Na2HPO412H2O, 33 mM KH2PO4, 50 mM NH4Cl, 1 mM MgSO47H2O, 30 lM ferric citrate, and 50 mM succinate. E. coli was grown at 37 °C in yeasttryptone (YT) broth (Oxoid). When necessary, antibiotics were added to final concentrations of 80 lg of rifampicin, 25 lg of kanamycin, 25 lg of streptomycin, and 12.5 lg of tetracycline per ml. Growth routinely proceeded on a rotary shaker (200 rpm) in 100 ml Erlenmeyer flasks with a working volume of 15 ml. To obtain a large mass of P. denitrificans CCM982 cells for FerB purification, we used a Biostat B-DCU fermentor (Sartorius AG) with a 10-l vessel, filled with 10 l of the synthetic succinate medium. During cultivation, the temperature (30 °C), pH (7.3), and rotation speed (300 rpm) were controlled online (MFCS/win 2.1; Sartorius AG) and sterile air was continuously supplied at a rate of 3.5 l/min. The growth inhibition assays were conducted using a Bioscreen C automated turbidometer (Thermo Labsystems, Finland). 10 ll of quinone sample solution and 3 ll of the diluted P. denitrificans culture were mixed with 287 ll of mineral aerobic medium with 50 mM succinate and 30 lM ferric citrate in each microtitre plate well. The plates were then sealed and incubated for five days at

In the light of the newly recognized ability of FerB to bind to Cibacron Blue, a triazine dye and nucleotide analog, our original purification protocol [1] was modified to include Cibacron Blue affinity chromatography at normal pressure as a central step. Typically, a 50–100 ml sample of cell-free extract was loaded onto a 12- by 65-cm column packed with Affi-Gel Blue (Fluka), pre-equilibrated with 25 mM Tris/HCl of pH 7.4, washed with the same buffer (about 300 ml), and eluted with a solution consisting of 25 mM Tris/HCl, pH 7.4, 2 M NaCl and 20% (v/v) ethylene glycol. The fractions with ferric reductase activity were pooled, concentrated by ultrafiltration using an Amicon concentrator with an YM-10 membrane (Millipore) and dialyzed against fresh Tris/HCl buffer. Two subsequent purification steps, anion-exchange chromatography on MonoQ HP 10/10 and chromatofocusing on MonoP HR 5/20, were carried out on a Pharmacia FPLC apparatus essentially as described [1]. The final gel permeation chromatography step employed previously could now be omitted without any significant impact on the purity of the product, as checked by SDS–polyacrylamide gel electrophoresis. Enzyme assays NADH:quinone reductase activity was assayed spectrophotometrically (Ultrospec 2000, Pharmacia Biotech, Sweden) at a constant temperature of 30 °C by following the oxidation of NADH at 340 nm using an absorption coefficient of 6.22 mM1 cm1. The reaction was started by adding FerB to a 1-cm pathlength cuvette that contained 1 ml of 25 mM Tris/HCl buffer of pH 7.4 with various concentrations of NADH and of a given quinone. If necessary, corrections were made for non-enzymatic reaction and for difference in absorption between quinone and quinol forms at the measuring wavelength. Ferric reductase activity was estimated from the increase rate of the absorbance at 562 nm, due to the formation of an Fe(II)-ferrozine complex [1]. Standard procedures were used

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to determine the enzymatic activity of superoxide dismutase [26] and b-galactosidase [27]. Analytical methods Oxygen consumption and evolution was monitored at 30 °C with a Clark-type electrode fitted to a 3-ml closed reaction chamber equipped with a magnetic stirring bar. Hydrogen peroxide production was determined colorimetrically in 200-ll samples, withdrawn from the reaction mixture, by means of a horseradish peroxidase/4-aminoantipyrine/phenol system [28]. Cytochrome c reduction was monitored by the absorbance increase at 550 nm [29]. Changes in the absorption spectrum of the protein-bound flavin chromophore were recorded with an UV S-2100 Scinco diode array spectrophotometer, and the resulting data processed using LabPro Plus software (Scinco, South Korea). Lowry method [30] with bovine serum albumin (Sigma) as a standard was used for a protein determination.

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(IWQMPGWWMGAPWTVKKYIDDVF; from the sequence in gi:15803569). The primary structure of FerB also contains two sequence motifs (GSLRKDSLN and SPGVIGAA) that share significant homology to FMN- and NADPH-binding sites, previously identified in the crystal structure of the NADPH-dependent FMN reductase from Pseudomonas aeruginosa PAO1 [32]. Pfam analyses grouped FerB to the NADPH-dependent FMN reductase family (PF03358) along with the proteins ChrR, YieF, NQR and Lot6p. In contrast, NQO1 and MdaB matched the flavodoxin 2 family (PF02525) and WrbA the flavodoxin 1 family (PF00258). All the three families are members of the flavoprotein superfamily (CL0042). Kinetic parameters of FerB for quinone substrates

Nonlinear regression analysis was performed with the kinetic software package EZ-FIT developed by Perrella [31]. The parameter values were evaluated using the kinetic model that gave the best fit to the data according to the following criteria: (i) the minimized Akaike information criterion (AIC) test, (ii) the t-statistics of the fitted kinetic parameters at the 0.05 level of significance, (iii) the number of outlying data points, and (iv) the ‘‘Runs” test of residuals at the 0.05 level of significance.

Steady state kinetic parameters Km, kcat and kcat/Km were determined for 7 benzoquinones and 6 naphthoquinones as substrates of FerB. In these assays, reaction rates were monitored by continuously recording the decrease in absorbance of NADH at 340 nm. The initial concentration of NADH was kept constant at 0.15 mM, i.e. well above the Km value (see below), while the initial quinone concentration was varied systematically. As is evident from the values of the kinetic specificity constant (kcat/Km) in Table 1, benzoquinones are somewhat better substrates than napthoquinones in accordance with that they have more positive redox potentials. However, the relation between kcat/Km and E17 is not so straightforward. For example, tetrachloro- and tetramethyl derivative, the best and worst electron acceptor among the benzoquinones tested, are both only very poor substrates of the enzyme.

Chemicals

Effects of NQO1 affecting inhibitors on FerB

All the quinones used were reagent grade, and further purified by vacuum sublimation. Their stock solutions (10 mM) were prepared in 20% (v/v) acetone. Reduction of 1,4-naphthoquinone to the corresponding hydroquinone was achieved by adding a few grains of solid NaBH4 to the acetone solution. After eliminating the unreacted NaBH4 by acidifying the sample with HCl and subsequent centrifugation, the supernatant was kept under nitrogen at 20 °C to prevent re-oxidation. Dicoumarol was dissolved in dimethyl sulfoxide.

The apparent functional similarity between FerB and NQO1 prompted an examination of the sensitivity of the former enzyme to the typical inhibitors of NQO1, dicoumarol and Cibacron Blue 3GA. The initial velocity studies were performed with NADH as the variable substrate in the presence of 0.2 mM 1,4-naphthoquinone and several fixed concentrations of the inhibitor. Plots of reciprocals of initial velocities against reciprocal concentrations of NADH gave a series of straight lines which intersected at one point on the vertical axis, typical for a competitive inhibition (data not shown). The Ki value of dicoumarol was found to be 60 ± 9 lM which value is higher than the 0.5 nM value reported earlier for human NQO1 [33]. A more potent competitive inhibition was observed with Cibacron Blue 3GA (Ki = 6 ± 1 lM), providing the rationale for the selection of this compound as a ligand for affinity chromatography of FerB (see the Material and methods section).

Analysis of kinetic data

Results Comparison of amino acid sequences The gene encoding FerB was identified by searching the publicly accessible P. denitrificans 1222 genome database at http://genome.ornl.gov/microbial/pden/, using the DNA sequence encoding the N-terminal sequence of the mature protein [1]. The 182 longamino acids sequence encoded by the ferB gene matched well with the relative molecular mass of 20 196 of purified FerB, experimentally determined by mass spectrometry [1]. A BLAST search revealed that FerB was most similar to P. putida ChrR (48% identity, 64% similarity), followed by E. coli YieF (36% identity, 54% similarity), NQR from A. thaliana (31% identity, 49% similarity) and Lot6p protein from Saccharomyces cerevisiae (27% identity, 46% similarity). A multiple alignment of these proteins is shown in Fig. 1. The longest conserved region common to all these five sequences (indicated by a box) is the LFVTPEYNXXXXXXLKNAIDXXS sequence, which is a protein signature mandatory for the attribution to the previously defined NADH_dh2 protein family [19]. Only 27% identity was observed between this segment of FerB and the corresponding amino acid sequences of NQO1 (IFQFPLQWFGVPAILKGWFERVF; from the sequence in gi:4505415) and MdaB

Mechanistic aspects of quinone reduction by FerB Two of the best quinone substrates of FerB, ubiquinone-0 and 1,4-naphthoquinone, were chosen for use in more detailed kinetic studies. Double reciprocal plots obtained by varying the NADH concentration at several different fixed concentrations of ubiquinone-0 (0–250 lM) or 1,4-naphthoquinone (0–100 lM) showed essentially parallel lines (data not shown), which indicated a ping-pong bi-bi mechanism. The Km’s for ubiquinone-0 and 1,4naphthoquinone derived from these data were identical to those presented in Table 1, the Km for NADH was determined to be 6 ± 1 and 7 ± 2 lM with ubiquinone-0 and 1,4-naphthoquinone as the second substrate, respectively. The next set of experiments was carried out to determine the stoichiometry of quinone reduction. The spectrophotometric measurement of NADH consumption upon addition of known quantities of ubiquinone-0 (Fig. 2) revealed that one mol of NADH was consumed per mol of ubiquinone-0 added. The 1:1 stoichiometry

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Fig. 1. Multiple sequence alignment of FerB with homologous proteins identified by Blast search. The GenBank(http://www.ncbi.nlm.nih.gov/Genbank/) identifiers for the sequences are as follows: Paracoccus denitrificans (PADEN) FerB, gi:69933457; Pseudomonas putida (PSEPU) ChrR, gi:14209680; Arabidopsis thaliana (ARATH) NQR, gi:75273736; Escherichia coli (ECOLI) YieF, gi:84028019; Saccharomyces cerevisiae (YEAST) Lot6p, gi:74583672; Pseudomonas aeruginosa PA01 (PSEAE) FMNR, gi:81541544. The alignment was generated using ClustalW 1.84 and processed with BoxShade 3.31. Sequence identity (black) or similarity (gray) between at least 80% of the sequence members are shaded. The ‘LFVTPEYNXXXXXXLKNAIDXXS’ region, the hallmark of the NADH_dh2 protein family, is indicated by a box. The sequence motifs GSLRSGSYN and SAGRFGTA in the NADPH-dependent FMN reductase (FMNR, bottom line), implicated as binding sites for FMN and NADPH, respectively, are underlined.

Table 1 Apparent kinetic parameters of FerB for quinones as substrates. Initial velocity measurements were performed at various concentrations of quinones in the presence of a fixed concentration of NADH (150 lM). One-electron reduction potentials E17 are taken from [39]. Kinetic parameters are means ± SD, as calculated by nonlinear regression analysis. ND, not detectable activity. Electron acceptor

E17 (V)

kcat (s1)

Km (lM)

kcat/Km (s1 lM1)

1,4-Benzoquinones 2,3-Dimethoxy-5-methyl-1,4-benzoquinone 2-Methyl-1,4-benzoquinone 1,4-Benzoquinone 2-Chloro-1,4-benzoquinone 2,6-Dimethyl-1,4-benzoquinone 2,3,5,6-Tetrachloro-1,4-benzoquinone 2,3,5,6-Tetramethyl-1,4-benzoquinone

0.110 0.023 0.078 0.254 0.080 0.650 0.260

139 ± 23 88 ± 4 174 ± 26 89 ± 15 96 ± 13 33 ± 3 14 ± 1

24 ± 12 17 ± 3 51 ± 18 69 ± 31 76 ± 27 48 ± 13 286 ± 60

5.8 5.3 3.4 1.3 1.3 0.7 0.1

1,4-Naphthoquinones 1,4-Naphthoquinone 2-Methyl-1,4-naphthoquinone 5-Hydroxy-1,4-naphthoquione 2-Hydroxy-1,4-naphthoquinone

0.140 0.203 0.093 0.310

64 ± 3 69 ± 10 69 ± 5 ND

38 ± 7 72 ± 30 83 ± 21 ND

1.7 1.0 0.8 ND

1,2-Naphthoquinones 1,2-Naphthoquinone 1,2-Naphthoquinone-4-sulphonate

0.089 0.215

75 ± 6 43 ± 4

20 ± 4 61 ± 17

3.8 0.7

remained essentially unchanged for several subsequent additions of ubiquinone-0 either in the absence or presence of oxygen. 1,4naphthoquinone behaved strikingly differently. We observed that the molar ratio NADH consumed to 1,4-naphthoquinone added increased with successive additions of the latter compound, but only when oxygen was present. To investigate whether oxygen can act as a second electron sink at a later stage of the NADH oxidation process, we employed a Clark-type oxygen electrode to monitor oxygen disappearance from the reaction mixture in a closed sys-

tem (Fig. 3). In the presence of ubiquinone-0 at the beginning, the injection of NADH did not lead to any significant response. Replacing ubiquinone-0 with 1,4-naphthoquinone, however, caused oxygen to be consumed with a stoichiometry of 1:1 for O2/NADH, indicating a reduction of O2 to H2O2. The identity of the reaction product was confirmed in two ways. First, as illustrated in Fig. 3, it was found that addition of catalase released about a half of the oxygen previously consumed, in accordance with the known stoichiometry of the catalase reaction. Second, for-

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Fig. 2. Stoichiometry of the FerB-catalyzed reaction of NADH with ubiquinone-0 or 1,4-naphthoquinone. The indicated amounts of NADH and quinone were added to 1 ml of 25 mM Tris/HCl- (pH 7.4) containing 35 nM FerB and the decline in NADH concentration was monitored by recording the absorbance at 340 nm. The assays were conducted either anaerobically, in stoppered cuvettes flushed with nitrogen, or aerobically in open cuvettes.

mation of H2O2 was quantified directly by a peroxidase assay. Analysis of the reaction mixture at the end of reaction showed that the oxidation of 300 lM NADH indeed resulted in the formation of 276 ± 26 lM H2O2 (mean ± SEM, n = 3) as expected. An explanation for the observed reactivity differences between ubiquinone-0 and 1,4-naphthoquinone (Figs. 2 and 3) may relate to a participation of free naphthosemiquinone radicals (Fig. 4). If they were produced by FerB (reaction 1), they would either disproportionate to quinone and hydroquinone (reaction 2) or reduce O2 to O 2 , the superoxide anion radical (reaction 3). Superoxide then would either react with hydroquinone yielding semiquinone and H2O2 (reaction 4) or disproportionate to O2 and H2O2 (reaction 5). Thus it can be predicted that addition of superoxide dismutase should cause superoxide disproportionation to prevail over radical chain propagation with the consequence of diminishing the amount of oxygen converted into H2O2. The result presented by a dashed line in Fig. 3 verifies this expectation. With SOD added, the oxygen level decreased only transiently and increased again after a few minutes most likely as a result of superoxide disproportionation. An alternative approach to testing for the production of radicals during the reaction catalyzed by FerB was to measure the rate of one-electron reduction of ferricytochrome c to ferrocytochrome c. The results contained in Fig. 5 show that only 1,4-naphthoquinone,

but not ubiquinone-0, mediated electron transfer from NADH to ferricytochrome c in the presence of FerB (compare traces 2 and 3). Superoxide dismutase slowed down the reaction rate (trace 3 vs. trace 4), but the relative decrease never exceeded 10%, even at the highest concentrations of this enzyme. The chemically prepared naphthohydroquinone did not induce an obvious increase in the oxygen consumption rate when added at the same concentrations as naphthoquinone. Furthermore, 1,4-naphthoquinone functioned as redox mediator also when the experiment was repeated anaerobically under nitrogen atmosphere (results not shown). All together, these results support the idea that a semiquinone is the true product of the FerB-catalyzed reduction of 1,4naphthoquinone, superoxide is formed secondarily and both radical species can independently donate an electron to cytochrome c. Fig. 6 depicts the spectroscopic changes accompanying the FerB-catalyzed reduction of quinones. The purified FerB displayed an absorption maximum at around 450 nm (trace 1), typical of the oxidized flavin cofactor, previously shown to be FAD [1]. This signal disappeared upon reduction of the enzyme by NADH (trace 2). Ubiquinone-0 addition led to a rapid partial reoxidation of the flavin (trace 4). By contrast, mixing of the reduced enzyme with 1,4-naphthoquinone produced a broad absorption band above 500 nm, with maxima situated at 540 and 580 nm (trace 3) which, in analogy with the behavior of other flavoprotein enzymes, can be

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Fig. 3. Oxygen uptake during the FerB-catalyzed reaction of NADH with ubiquinone-0 or 1,4-naphtoquinone. The reaction mixture of total volume 3 ml consisted of 25 mM Tris/HCl (pH 7.4), 0.3 mM of the respective quinone substrate and 23 nM of FerB. Following NADH addition, O2 uptake was monitored with a Clark-type electrode. The dashed line illustrates the effect of superoxide dismutase, when present at 70 U/ml from the beginning of incubation. Catalase (580 U/ml) was added as indicated to degrade the H2O2 generated.

Fig. 4. Schematic representation of the reactions involved in quinone redox cycling. 1, enzyme-catalyzed univalent reduction of quinone (Q) to semiquinone (QH); 2, spontaneous disproportionation of semiquinone to quinone and hydroquinone (QH2); 3, generation of superoxide anion (O 2 ) in the reaction of semiquinone with molecular dioxygen (O2); 4, oxidation of hydroquinone by superoxide to produce semiquinone and hydrogen peroxide (H2O2); 5, spontaneous or enzyme-catalyzed dismutation of superoxide to dioxygen and hydrogen peroxide. Acceleration of reaction 5 by the addition of superoxide dismutase should slow down the reaction 4 and cause hydroquinone to be a final reaction product. Reduction of the added cytochrome c is indicative of the presence of radical species (QH, O 2 ).

tentatively assigned to the formation of the blue neutral FADH radical. The accumulation of this species during the reaction of FerB with 1,4-naphthoquinone can be viewed as further evidence of a one-electron process (see the Discussion). Susceptibility of the wild-type and ferB mutant strains to quinones To examine the physiological role of the quinone reductase activity of FerB, we constructed a mutant strain in which the ferB gene was inactivated by insertion of the kanamycin resistance gene and analyzed the resultant effects on its growth properties. Exponential phase cultures of wild-type and ferB mutant were used to inoculate fresh media that contained increasing concentrations of 1,4-naphthoquinone. Continuous turbidimetric recording of bacterial growth revealed that in the absence of 1,4-naphthoquinone,

Fig. 5. Reduction of ferricytochrome c related to the enzymatic activity of FerB. Assays were performed in in 25 mM Tris-HCl- (pH 7.4) containing 60 lM NADH and 12 lM ferricytochrome c. Cytochrome reduction was monitored by measuring the absorbance at 550 nm as a function of time. The following additions were made prior to the initiation of enzymic reaction by 35 nM FerB (at the arrow): trace 1, no addition (control); trace 2, 20 lM ubiquinone-0; trace 3, 20 lM 1,4-naphthoquinone; trace 4, 20 lM 1,4-naphthoquinone plus 110 U/ml of superoxide dismutase.

the mutant grew as rapidly as the wild type and had a cell yield comparable to that of the wild type. In the presence of micromolar concentrations of 1,4-naphthoquinone the growth of wild-type P. denitrificans was effectively inhibited with a 50% inhibitory concentration (IC50) of 6 ± 2 lM (mean ± SEM, n = 3) (Fig. 7). The IC50 of the FerB mutant strain was 3-fold higher (IC50 = 17 ± 5 lM, n = 3) indicating that this strain was more resistant to the toxic effect of 1,4-naphthoquinone. Parallel experiments with ubiquinone0 showed that both strains did not differ in their responses to this compound (IC50 = 0.75 ± 0.22 mM, n = 3).

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grown on a reciprocal shaker to a turbidity of 0.2 at 600 nm at which phase of growth the tested quinone was added and the shaking was continued for another 17 h. Samples were taken periodically and assayed for turbidity at 600 nm as a measure of the biomass concentration and for b-galactosidase activity (expressed in Miller units) as a measure for the ferB promoter strength. We have consistently observed a relatively high basal expression of b-galactosidase of about 1 200 Miller units which could not be further induced by 1,4-naphthoquinone (0–6 lM), menadione (0– 10 lM) or juglone (0–2 lM). Higher concentrations of these compounds proved toxic to the cells, as indicated by substantially reduced growth rates.

Discussion

Fig. 6. Absorption spectra changes during the reaction of FerB with NADH and quinones. FerB (25 lM) in 25 mM Tris/HCl buffer of pH 7.4 is represented by scan 1. Scan 2 was taken after the addition of 0.1 mM NADH; scans 3 and 4 were taken after the addition of 1,4-naphthoquinone and 0.1 mM ubiquinone-0, respectively, to the NADH-reduced enzyme. All spectra were recorded with a photodiode array yielding a complete spectrum in 3 s. Repetitive scanning over a period of 1 min revealed no qualitative changes in the spectra.

Fig. 7. Effect of 1,4-naphthoquinone on the specific growth rates of the wild-type (closed circles) and FerB-deficient (open circles) strain. Turbidimetric growth curves were generated and used to determine the specific growth rates as described in the Materials and methods. Each data point represents the mean of three individual cultures and standard deviations of the mean are plotted as vertical bars.

Activity of the ferB promoter The fact that soluble quinone reductases are often inducible by their own substrates [7] prompted us to investigate whether the same holds true for FerB. A plasmid was constructed with the reporter gene lacZ under control of a promoter region in front of the ferB gene and this vector was allowed to integrate into the genome of P. denitrificans via homologous recombination. Cultures of the strain with this ferB promoter-reporter construct were then

FerB was originally detected in cytoplasmic fractions of P. denitrificans on the basis of its ability to reduce certain ferric-organic complexes [1]. Enzyme-catalyzed reduction of Fe(III) to Fe(II) is generally considered as an essential step in the assimilation of ferric iron from the environment. Two lines of evidence, however, argue against a role of FerB in iron acquisition. First, FerB does not reduce Fe(III) ligated to catechol, which is the functional moiety of the natural siderophore parabactin produced by P. denitrificans [1]. Second, disruption of the ferB gene in this bacterium does not alter its capacity to utilize low concentrations of iron (Sedlácˇek, V. unpublished results). Sequence analysis in Fig. 1 now identifies FerB as a member of a protein family, which includes soluble flavin-containing quinone reductases from both prokaryotic and eukaryotic sources. This finding together with our results of enzymological studies indicate that the FerB enzyme functions physiologically as a quinone reductase rather than as a ferric iron reductase. It may be pertinent at this point to note that NQO1, the FerB counterpart in animal cells, has also the ability to reduce Fe(III) in situ [34], although the physiological relevance of this phenomenon is far from clear. The P. denitrificans FerB enzyme shares the characteristics of other soluble quinone reductases, as exemplified by the mammalian NQO1 enzyme. These include (i) a homodimeric nature of the enzymes, (ii) the presence of a flavin moiety as non-covalent cofactor, (iii) a considerable sensitivity to inhibition by dicoumarol, and (iv) that the enzymes display a ping-pong kinetic mechanism. Some differences, however, became also noticeable in our work. The most important one is that the observed stoichiometry of electron transfer from the enzyme-bound FADH2 depends on whether a benzoquinone or a naphthoquinone is being used as an electron acceptor. While in the former case, a hydroquinone is the main product, in the latter case significant amounts of semiquinone are formed. Evidence in favor of one-electron reduction of 1,4naphthoquinone comes largely from experiments measuring the stoichiometric ratio of reactants (NADH:quinone) or the semiquinone-dependent redox reactions of oxygen and cytochrome c. The same basic methodology was successfully employed earlier in demonstrating one-electron reaction mechanism for f-crystallins from guinea pig lens [35] and A. thaliana P1 [29]. Based on the increase in the absorbance in the 580 nm region (Fig. 7), a radical flavin species accumulates upon the addition of 1,4-naphthoquinone to the NADH-reduced FerB. Thermodynamic stabilization of the blue neutral radical form of the flavin cofactor is a general property inherent to flavoproteins involved physiologically in single-electron transfers, e.g., flavodoxins, ferredoxin-NADP reductase, and NADPH-cytochrome P-450 reductase [36]. Unlike the situation with FerB, no transient formation of flavin semiquinone could be detected in rat liver NQO1, not even with the obligatory one-electron acceptor hexacyanoferrate (III) [37]. The authors explained this negative result by considering that the rate constant

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for electron transfer from flavin radical exceeds that for electron transfer from the fully reduced flavin by a factor of at least 80. Obviously, this value must be substantially lower for FerB to account for the high steady-state level of the flavin radical during the reaction with naphthoquinones. A bi-substrate kinetic analysis, carried out by the same authors, of the overall reaction catalyzed by NQO1 gave infinite maximum velocity, indicating negligible contribution of enzyme-NADH and enzyme-acceptor complexes. Our data do not support a similar conclusion for FerB, since the maximum velocities calculated from nonlinear regression were clearly finite. Formation of enzyme-quinone complexes has recently been demonstrated for the Lot6p quinone reductase, homologous to FerB (Fig. 1), by means of spectrophotometric titrations [12]. The reported dissociation constants were in the order of 105 M, which compares with our findings presented here (Table 1). There is still considerable uncertainty regarding the actual contribution of quinone reductases in protection against oxidative stress associated with redox cycling in bacteria. By comparing individual derivatives in Table 1, it can be seen that decreases in redox potential (i.e., electron accepting potency) in the series 1,4-benzoquinone, 2-methyl-1,4-benzoquinone and 2,3-dimethoxy-5methyl-1,4-benzoquinone (ubiquinone-0) are in fact paralleled by increases in the kcat/Km specificity ratio. This means that structural factors, rather than the mere energetics of electron transfer, play a role in determining reaction rates. We now hypothesize, based on these findings, that FerB specifically recognizes benzoquinone ring of ubiquinone, an electron carrier naturally present at high amounts in P. denitrificans [38]. The enzyme thus may well exert a protective role in this bacterium by balancing the proton/electron state of reactive ubiquinone derivatives similarly as found for its close homologue ChrR in P. putida [3]. Nevertheless, it is also obvious that FerB not only does not overcome the toxic effect of the exogenously added 1,4-naphtoquinone but even potentiates it, probably due to increased semiquinone formation. Since, in addition, the FerB promoter activity fails to respond to quinone concentration, FerB does not meet the criterion of a defensive enzyme with respect to this particular class of xenobiotics. In conclusion, we have shown FerB to be a quinone reductase with atypical behavior toward naphthoquinones. We hope that the structure determination of FerB in the near future will provide an interpretative framework for understanding variations in efficiency of sequential electron transfer among individual quinonereducing enzymes. Acknowledgments This research was supported by grants from the Czech Science Foundation (525/07/1069) and the Ministry of Education, Youth and Sports (MSM0021622413).

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