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Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase Federico Fischer, Debora Raimondi, Alessandro Aliverti and Giuliana Zanetti Dipartimento di Fisiologia e Biochimica Generali, Universita ` degli Studi di Milano, Milano, Italy The gene fprA of Mycobacterium tuberculosis, encoding a putative protein with 40% identity to mammalian adreno- doxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50-kDa protein monomer contained one tightly bound FAD, whose fluor- escence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant (k cat /K m )forNADPHwastwo orders of magnitude larger than that of NADH. Enzyme full reduction, under anaerobiosis, could be achieved with a stoichiometric amount of either dithionite or NADH, but not with even large excess of NADPH. In enzyme titration with substoichiometric amounts of NADPH, only charge transfer species (FAD-NADPH and FADH 2 -NADP + ) were formed. At NADPH/FAD ratios higher than one, the neutral FAD semiquinone accumulated, implying that the semiquinone was stabilized by NADPH binding. Stabiliza- tion of the one-electron reduced form of the enzyme may be instrumental for the physiological role of this mycobacterial flavoprotein. By several approaches, FprA was shown to be able to interact productively with [2Fe)2S] iron-sulfur pro- teins, either adrenodoxin or plant ferredoxin. More inter- estingly, kinetic parameters of the cytochrome c reductase reaction catalyzed by FprA in the presence of a 7Fe ferre- doxin purified from M. smegmatis were determined. A K m value of 30 n M and a specificity constant of 110 l M )1 Æs )1 (10 times greater than that for the 2Fe ferredoxin) were determined for this ferredoxin. The systematic name for FprA is therefore NADPH-ferredoxin oxidoreductase. Keywords: flavoprotein; ferredoxin reductase; ferredoxin; Mycobacterium tuberculosis. Information available from the complete genome sequence of Mycobacterium tuberculosis [1] has promoted a wide investigation of new targets for drugs against tuberculosis [2]. The disease has regained ground in the developed world due to the increased appearance of resistant strains of the bacterium and the facile diffusion in the immunodepressed people. M. tuberculosis is strongly dependent on iron availability and on iron-containing cofactors for growth and survival [3]. It is well-known that iron availability in the host plays a very important role in promoting the infection by mycobacteria. Interestingly, it has been reported that Nramp1 (natural resistance-associated macrophage protein) protein of mouse macrophages confers resistance to myco- bacterial infection in mice [4]. Recently, a hyphothesis has been proposed based on the homology of Nramp1 to DCT1, a metal-ion transporter [5]. Thus, the action of Nramp1 in the phagosomal membrane may be to deplete Fe 2+ or other divalent cations from the phagosome, thus hampering the pathogen growth. Among possible strategies to effectively interfere with the pathogen metabolism, the blockage or limitation of Fe 2+ availability inside the mycobacterium seems a promising target to pursue. Redox systems called ferric reductases use intracellular redox cofactors to reduce the ferric Fe to the ferrous form for biosynthesis of iron-proteins. A NAD(P)H:ferrimycobactin oxidoreductase activity was measured in M. smegmatis cell extract [6]. In Escherichia coli, enzymes of the ferredoxin- NADP + reductase (FNR) protein family showing iron reductase activity, such as the flavin reductase, sulfite reductase and flavohemoglobin, have been implicated in such metabolism [7]. Searches of the M. tuberculosis genome for enzymes structurally related to the FNR family was unsuccessful but led to the identification of two genes, fprA and fprB, encoding putative adrenodoxin reductase- like proteins, expected to be functionally related to members of the FNR family [8], i.e. electron transferases that function as a switch between two-electron and one-electron flow systems. This class of enzymes is implicated in a variety of functions such as iron reduction, activation of ribonucleo- tide reductase, response to oxygen stress as well as reduction of P450 cytochromes [8]. Here, we report on production and biochemical charac- terization of the recombinant FprA. The homogeneous protein is shown to be a novel bacterial ferredoxin reductase Correspondence to G. Zanetti, Dipartimento di Fisiologia e Biochimica Generali, Via Celoria 26, 20133 Milano, Italy. Fax: + 39 02 50314895. Tel.: + 39 02 50314896, E-mail: gzanetti@mailserver.unimi.it Abbreviations: AdR, adrenodoxin reductase; Adx, adrenodoxin; FNR, ferredoxin-NADP + reductase; Fd I, ferredoxin I; DPIP, 2,6-dichlorophenol-indophenol; SQ, semiquinone; CT, charge-transfer complex. Proteins: Bos taurus adrenodoxin, SWISS-PROT entry ADX1_BOVIN; Spinacia oleracea ferredoxin I, SWISS-PROT entry FER1_SPIOL; Mycobacterium smegmatis ferredoxin, SWISS-PROT entry FER_MYCSM. Enzymes: adrenodoxin reductase (EC 1.18.1.2), ferredoxin-NADP+ reductase (EC 1.18.1.2) Note: a website is available at http://users.unimi.it/phybioch/ Index_htm (Received 1 February 2002, revised 11 April 2002, accepted 2 May 2002) Eur. J. Biochem. 269, 3005–3013 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02989.x and to possess some properties similar to those of the bovine adrenodoxin reductase [9]. MATERIALS AND METHODS Materials All chemicals and pyridine nucleotides were purchased from Sigma–Aldrich Chemical Co. Cytochrome c (Sigma C2506) was further purified by ion-exchange chromatography on SP-Sepharose (Pharmacia Biotech.). Restriction endonuc- leases, DNA polymerase and DNA modifying enzymes were supplied by Amersham Pharmacia Biotech. M. tuber- culosis cosmid MTCY164 was kindly provided by S. T. Cole, Institut Pasteur, France. pGEM-T and pET11a were from Promega and Novagen, respectively. Bovine Adx 1 was a generous gift from F. Bonomi, University of Milano, Italy. Recombinant spinach ferredoxin I (Fd I) was purified as described previously [10]. M. smegmatis ferredoxin has been purified by a modification of the procedure described by Imai et al. [11]. DEAE-cellulose and Sepharose 4B steps were replaced by chromatoraphy on HiLoad Q-Sepharose High-Performance and HiLoad phenyl-Sepharose High- Performance columns (Pharmacia Biotech). Ferredoxin was eluted at about 0.7 M NaCl from the first column using a 0–1 M NaCl gradient in 50 m M Tris/HCl, pH 7.4. The pooled fractions were brought to 2 M ammonium sulphate and loaded on the second column. Elution was performed with a 2–0 M ammonium sulphate gradient in the same buffer as above. Ferredoxin was desalted by dialysis against 50 m M Tris/HCl, pH 7.4. PCR amplification and molecular cloning The open reading frame of the M. tuberculosis gene Rv3106, named fprA, was amplified from the cosmid MTCY164 (GenBank accession no. Z95150) by PCR using the nucleotides 5¢-GC CATATGATGCGTCCCTATTACA-3¢ and 5¢-GT CATATGTCAGCCGAGCCCAAT-3¢,which contained the NdeI restriction site (underlined). The result- ing DNA fragment was cloned into pGEM-T vector and sequenced. The NdeI DNA fragment from the recombinant plasmid containing fprA was recloned in the NdeIsiteofthe expression vector pET-11a, yielding pETfprA. Overexpression of fprA E.coliBL21(DE3) cells transformed with pETfprA were grown in flasks under vigorous shaking at various temper- atures in 2 · YT medium supplemented with 100 mgÆL )1 ampicillin. For enzyme purification, E.colicells were grown in a New Brunswick 12 L fermentor at 25 °C to midlog phase (D 600 ¼ 1.2–1.5). The culture, after cooling to 15 °C, was induced with 0.1 m M isopropyl thio-b- D -galactoside. Cells were harvested after 15–17 h. Purification of FprA All purification steps were performed at 4 °Cexceptfor FPLC, which was carried out at room temperature. E.coli cell paste were resuspended in 2 mLÆg )1 of buffer A (50 m M Na-phosphate, pH 7.0, containing 1 m M EDTA and 1 m M 2-mercaptoethanol) supplemented with 1 m M phenylmethanesulfonyl fluoride and disrupted by sonica- tion. After removal of cell debris by centrifugation at 43 000 g for 1 h, the protein concentration of the crude extract was adjusted to 25 mgÆmL )1 . The solution was then brought to 40% saturation of ammonium sulphate (1.64 M ), the precipitate discarded and the soluble fraction loaded on Sepharose 4B column (Pharmacia Biotech) pre- equilibrated with 1.64 M ammonium sulphate in buffer A. FprA was eluted with the same solution as a single peak well separated from the material eluting in the void volume. To the pooled FprA-containing fractions glycerol was added to 10% final concentration and the enzyme was precipitated with 85% saturation ammonium sulphate. The pellet was resuspended and dialysed against 25 m M imidazole-HCl, pH 7.0, containing 10% glycerol and 1m M 2-mercaptoethanol. The enzyme was loaded on a HiLoad Q-Sepharose High-Performance column (Pharma- cia Biotech) and eluted with a linear gradient from 150 to 250 m M NaCl. The purified FprA was desalted by gel- filtration on PD10 column (Pharmacia Biotech) using 50 m M Hepes/KOH, pH 7.0, containing 10% glycerol and 1m M DTT. The enzyme stored at )80 °Cretaineditsfull activity for more than 1 year. Molecular characterization methods SDS/PAGE was carried out on 10% polyacrylamide gels. Microsequencing was performed on an Applied Biosystems 477/A protein sequencer equipped with an on-line HPLC system. Analytical gel-filtration analyses were performed on a HPLC apparatus (Waters) equipped with either Superdex 75 or Superose 12 columns (Pharmacia Biotech) in 50 m M Hepes/KOH, pH 7.0, containing 0.15 M ammonium acetate and 2 m M 2-mercaptoethanol. FprA and ferredoxin (10 and 40 l M , respectively) were cross-linked by treatment with 5m M N-ethyl-3-(3-dimethylaminopropyl)carbodiimide in 25 m M Na-phosphate, pH 7.0 [12]. Spectral analyses Absorption spectra were recorded with a Hewlett-Packard 8453 diode-array spectrophotometer. The extinction coeffi- cient of the protein-bound flavin was determined spectro- photometrically quantitating the FAD released from the apoprotein following SDS treatment [13]. Fluorescence measurements were performed on a Jasco FP-777 spectro- fluorometer at 15 °C. The identity of the enzyme bound flavin was assessed fluorimetrically by treating with phos- phodiesterase the flavin released after thermal denaturation at 100 °C of the holoenzyme [13]. Activity assays Enzyme catalyzed reactions were monitored continuously on a Hewlett-Packard 8453 diode-array spectrophotometer. Ferric reductase activity was assayed in both aerobic and anaerobic conditions in 50 m M Tris/HCl, pH 7.5 at 25 °C as described previously [14]. Diaphorase activity was measured in 0.1 M Tris/HCl, pH 8.2 at 25 °C with either K 3 Fe(CN) 6 or DPIP as electron acceptor and NADPH or NADH as reductants. Cytochrome c reductase activity was assayed in the same buffer as above with either 5 l M spinach Fd I, bovine Adx or M. smegmatis ferredoxin, 3006 F. Fischer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 using 50 l M cytochrome c as the terminal electron acceptor. Unless otherwise stated, the NADPH concentration was kept constant by regeneration with 2.5 m M glucose 6-phosphate and 2 lgÆmL )1 glucose 6-phosphate dehydro- genase. Steady-state kinetic parameters for the diaphorase activities and for the cytochrome c reductase activity with mycobacterial ferredoxin were determined by varying the concentrations of the substrates. Double-reciprocal plots of the data yielded parallel lines. Initial rate data (v)werefitted by nonlinear regression using GRAFIT 4.0 (Erythacus Software Ltd, Staines, UK) to a ping-pong Bi-Bi mechan- ism equation (Eqn 1): v ¼ V  A  B=ðK a  B þ K b  A þ A  BÞð1Þ where A and B,andK a and K b are the molar concentrations and the Michaelis constants for the two substrates, respect- ively. Enzyme titrations and photoreductions Titrations of oxidized FprA with NADP + ,NAD + ,or spinach Fd I were performed spectrophotometrically at 15 °C. The enzyme was diluted to a final concentration of 12–15 l M in 10 m M Tris/HCl, pH 7.7. NADP + titrations were carried out at different ionic strength by varying the NaCl concentration between 0 and 150 m M .Difference spectra were computed by subtracting from each spectrum that obtained in the absence of ligand, after correction for dilution. K d values were obtained by fitting data sets by nonlinear regression to the theoretical Eqn (2) for a 1 : 1 binding, using the software GRAFIT 4.0 (Erythacus Software Ltd, Staines, UK). DA ¼ De  L þ P þ K d À ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi L þ P þ K d ðÞ 2 À 4  L  P q 2 ð2Þ DA is the value of the difference spectrum at a selected wavelength; De is the difference extinction coefficient at that wavelength of the protein-ligand complex; L is the total molar concentration of added ligand; P is the total molar concentration of FprA. All reduction experiments were carried out in anaerobic cuvettes at 15 °C. Solutions were made anaerobic by successive cycles of equilibration with O 2 -free nitrogen and evacuation. Reductive titrations with Na-dithionite, NADPH, or NADH were carried out using 15–50 l M FprA solutions in 10 m M Tris/HCl, at pH 7.4. Photoreductions of FprA using EDTA/light [15] were performed in 10 m M Hepes/KOH, at pH 7.0, containing 15 m M EDTA and 1.8 l M 5-deazariboflavin. NADP + titration of reduced enzyme was carried out by additions of an anaerobic solution of NADP + to FprA previously photoreduced as described above. The amount of FAD SQ was calculated by subtraction of the contribution of the CT species [16,17] from the absorbance at 625 nm according to Eqn (3): A sq ¼ A 625 Àð2:79  A 750 Þð3Þ The contribution of CT species to A 625 can be estimated taking into account that SQ does not absorb at 750 nm [18] and that a A 625 /A 750 value of 2.79 for CT species could be determined from experiments in which no SQ was formed. RESULTS Identification of fprA and fprB The search of M. tuberculosis genome [1] for enzymes potentially involved in iron metabolism led to the identifi- cation of two genes, fprA and fprB, whose predicted protein products are related to each other. They share a domain with significant similarity (% 40% identity) with mamma- lian AdR (Table 1). FprB contains a C-terminal domain homologous to FprA (42% identity) plus an N-terminal moiety comprising an iron-sulfur binding region signature typical of bacterial 7Fe ferredoxins. It is remarkable that AdR homologs are present in very few bacteria, whereas two such proteins are found in mycobacteria (Table 1). To our knowledge, the fusion protein does not have a counter- part in other organisms, except for other mycobacteria. Production of FprA We tried to heterologously express both cloned genes, yet we were only successful in obtaining FprA in a soluble active form. In a preliminary series of experiments, E.coli BL21(DE3) strain harboring pETfprA was grown at 37 °C. Upon induction, a novel protein band of 50-kDa was clearly visible in SDS/PAGE, but most of the protein was present in insoluble form. Growth and induction conditions were varied to optimize the production of the recombinant protein in a soluble form (not shown). The amount of the soluble recombinant protein increased greatly by lowering the growth temperature. Concomitantly, the NADPH-ferricyanide reductase specific activity of the soluble cell extracts also increased, being highest in cells grown at 15 °C and harvested about 16 h after induction. The purification of FprA was achieved by a three-step procedure as described in Materials and methods. An ammonium sulphate fractionation coupled to a salt-pro- moted adsorption chromatography on Sepharose 4B, and followed by an anion-exchange chromatography on Table 1. Sequence comparison matrix stating percentage of identical residues of bacterial and selected eukaryotic adrenodoxin reductase-like proteins. Proteins with sequence identity lower than 20% were omitted. Individual proteins are: A, FprA (M. tuberculosis); B, FprA (M. lep- rae); C, FprB C-terminal domain (M. tuberculosis); D, FprB C-term- inal domain (M. leprae); E, probable ferredoxin reductase (Deinococcus radiodurans); F, putative ferredoxin reductase (Strepto- myces coelicolor); G, Arh1p (Saccharomyces cerevisiae); H, bovine adrenodoxin reductase. ABCDEFGH A 100 82 42 41 48 41 30 41 B 100 42 41 46 38 30 40 C 100 76 41 36 28 40 D 100 42 36 28 36 E 100 44 30 42 F 100 28 34 G 100 33 H 100 Ó FEBS 2002 M. tuberculosis NADPH-ferredoxin reductase (Eur. J. Biochem. 269) 3007 Q-Sepharose, yielded about 2 mg of FprA per gram of cells, with an overall yield of 25% and a purification factor of 18. SDS/PAGE of the various fractions of the purification is showninFig.1. FprA is a flavoprotein The visible absorption spectrum of the purified protein is presented in Fig. 2. The absorbance in the visible region is that typical of a flavoprotein with bands centered at 381 and 452 nm and shoulders at 422 and 473 nm. Maximal absorbance in the ultraviolet region was at 272 nm. A value of 7.0 for the A 272 /A 452 ratio was calculated from the spectrum. Flavin fluorescence was almost completely quenched. The non covalently bound flavin in FprA was shown to be FAD. The flavin fluorescence of the released cofactor increased about 10-fold after phosphodiesterase treatment, as expected for the conversion from FAD to FMN. The extinction coefficient of the enzyme at 452 nm was calculated to be 10 600 M )1 Æcm )1 from the amount of FAD released after protein denaturation by SDS. A stoichiometry of 0.98 mol FAD per mol of 50 kDa monomer was established. The flavin was reducible by dithionite (Fig. 2) and an anaerobic titration of FprA with this reductant showed that 0.92 molÆmol FAD )1 or about two electrons per flavin were required for full reduction (see inset of Fig. 2). This excludes the presence of additional redox cofactors in the enzyme. No changes in absorbance beyond 550 nm were observed, indicating that the flavin semiquinone (SQ) did not accumulate [18]. Thus, only two forms of the FAD prosthetic group were present during titration, the oxidized form and the fully reduced one, as can be deduced from the presence of an isosbestic point at 340 nm. The same pattern of reduction was obtained by photoreduction [15]. FprA was rapidly reduced by succes- sive periods of irradiation in the presence of 5-deazaribo- flavin and EDTA, yielding the hydroquinone spectrum after 8 min of light exposure (data not shown). Reoxidation of fully reduced enzyme by molecular oxygen occurred without any detectable SQ formation. Thus, the one-electron reduced form of FAD is not stabilized in the enzyme. Molecular properties FprA showed a M r of about 50 000 in denaturing PAGE (Fig. 1). This value is in good agreement with that of 49 341 calculated from the sequence. The identity of the overpro- duced protein was assessed by N-terminal analysis. The first 21 amino-acid residues of the purified protein were identical to those deduced from the gene sequence: MRPYYIAIVG SGPSAFFAAAS. The M r of the recombinant FprA in solution was determined in several conditions. Gel filtration experiments in FPLC, either on Superose 12 or Superdex 75, allowed the determination of a value of 53 ± 5 kDa, when the protein was maintained in 10% glycerol and 1 m M dithiothreitol, indicating that under these conditions the protein is a monomer. The addition of glycerol and 2-mercaptoethanol were required to avoid formation of aggregates. Catalytic properties The ferric reductase activity of the purified protein was investigated by using Fe 3+ -EDTA in the presence of the Fe 2+ -chelator ferrozine [14]. The activity was very low both in the presence and absence of oxygen and/or FAD: 0.5–1 (mol NADPH)Æmin )1 Æ(mol FAD) )1 .Furthermore, addition of 1 l M 7Fe ferredoxin from M. smegmatis (see Fig. 1. Purification of recombinant FprA as analysed by SDS/PAGE. Lanes 1 and 5, molecular mass markers (values in kDa are indicated); lane 2, crude extract; lane 3, after Sepharose 4B; lane 4, after Q-Sepharose. Fig. 2. Electronic absorption spectrum of purified FprA and dithionite titration. Theenzymewas26l M in 10 m M Tris/HCl, pH 7.4, con- taining 10% glycerol and 1 m M dithiothreitol. FprA was stepwise reduced with dithionite under anaerobiosis. The spectra recorded at 0, 0.2, 0.4, 0.5, 0.7, 0.9, and 1 reductant/FAD molar ratios are reported. The inset shows the plot of the fractional absorbance change at 452 nm as a function of dithionite/FAD molar ratio. A i and A f are the initial and final values of absorbance at 452 nm, respectively. 3008 F. Fischer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 below) to the assay did not increase the iron reduction rate. On the other hand, the protein was found able to catalyze electron transfer from NADPH as well as NADH to artificial electron acceptors like ferricyanide and DPIP. The steady-state kinetic parameters for the ferricyanide and DPIP activities were determined at pH 8.2 (Table 2). The double-reciprocal plots of initial velocities obtained by varying the reduced pyridine nucleotide at various fixed levels of the artificial dye showed a pattern of parallel lines. Data were fitted to Eqn (1). For the K 3 Fe(CN) 6 reductase activity, the experiments revealed that ferricyanide concen- trations above 1 m M were inhibitory. A 100- to 150-fold lower K m values for NADPH with respect to NADH were observed in the diaphorase reactions, whereas similar values of k cat were obtained with both coenzymes, thus the specificity constant ratio NADPH/NADH was 225 in the ferricyanide reaction and 116 in the DPIP one. The catalytic efficiencies of FprA with respect to the acceptors differed by 10-fold with preference for the one-electron reducible substrate, i.e. ferricyanide. To study the interaction with pyridine nucleotides in details, FprA was titrated with both NADP + and NAD + . In both cases, the visible spectrum of the enzyme was perturbed. The difference spectra elicited by the ligand binding are shown in Fig. 3A. The features of the difference spectra produced by NADP + or NAD + are very similar, but the intensity of the 500 nm peak was fourfold higher in the case of NADP + (Fig. 3A). Titrations with this coenzyme were performed at increasing ionic strength to obtain an accurate estimate of the K d by extrapolation of the linear part in the graph of log K d vs. ÖI. Thus, K d values of FprA for NADP + of 6 n M at I ¼ 0, and 0.4 l M at I ¼ 50 m M were calculated (data not shown). In contrast, the K d value for NAD + was in the millimolar range. Identification of a physiological electron acceptor The physiological activity of the mammalian homolog of FprA is to reduce the [2Fe)2S] iron–sulfur protein Adx [9,19,20]. Nevertheless, there are no genes coding for [2Fe)2S] ferredoxins in the M. tuberculosis genome [1]. At first, we studied the interaction of the recombinant enzyme with the bovine Adx and with another [2Fe)2S]protein,the spinach leaf Fd I. Cytochrome c was used as final electron acceptor in these reactions. Its reduction was observed only when either Adx or Fd I was added in the assay, indicating that FprA was able to interact productively with both these electron carrier proteins. FprA was 10-fold more active with the plant type Fd I than with Adx under the same conditions. In the mean time, we cloned M. tuberculosis genes coding for 7Fe and 3Fe ferredoxins, but failed in obtaining the overexpression in E.coli. Several years ago, a 7Fe ferredoxin was purified from M. smegmatis [11]. By using a similar procedure, we obtained a reasonable amount of the M. smegmatis 7Fe ferredoxin in homogeneous form as judged by several criteria (native and denaturing PAGE, protein determination/molarity determined by using the reported extinction coefficient at 406 nm). N-Terminal analysis of the purified protein confirmed its identity with Fig. 3. Spectral perturbations elicited by ligand binding to FprA. All measurements were performed in 10 m M Tris/HCl, pH 7.7 with 15 l M enzyme. Difference spectra were computed by subtracting from spectra recorded at titration end-points those of unbound FprA and ligand. (A) difference spectra of the complexes between FprA and NADP + (solid line) or NAD + (dashed line). (B) difference spectrum of the complex between FprA and Fd I. Table 2. Kinetic parameters for the ferricyanide and DPIP reductase reactions of FprA. Electron acceptor k cat (e – Æs )1 ) K NADðPÞH m (l M ) k cat /K m (e – Æs )1 Æl M )1 ) K acceptor m (lM) k cat /K m (e – Æs )1 Æl M )1 ) NADPH K 3 Fe(CN) 6 63.0 ± 1.3 0.45 ± 0.02 140 ± 0.1 22 ± 2 2.9 ± 0.1 DPIP 25.6 ± 0.8 0.89 ± 0.08 29 ± 0.1 58 ± 3.8 0.44 ± 0.07 NADH K 3 Fe(CN) 6 42 ± 0.9 68 ± 4 0.62 ± 0.06 14.6 ± 1 2.87 ± 0.07 DPIP 21 ± 0.6 83 ± 5 0.25 ± 0.07 56 ± 3 0.37 ± 0.06 Ó FEBS 2002 M. tuberculosis NADPH-ferredoxin reductase (Eur. J. Biochem. 269) 3009 the ferredoxin isolated by Imai et al. [11]. This ferredoxin has 88% identity with FdxC of M. tuberculosis [1]. The steady-state kinetic parameters for both the 2Fe and 7Fe ferredoxin reductase activities are reported in Table 3. The kinetic data obtained with the protein substrates yielded parallel lines in double-reciprocal plots and were fitted to Eqn (1). The k cat measured with the 7Fe ferredoxin was 30% of that with the spinach protein, whereas the K m for the homologous protein substrate was about 30-fold lower, suggesting a much higher affinity of FprA for the mycobacterial ferredoxin. Due to the ability to reduce iron-sulfur proteins using preferentially the pyridine nuc- leotide phosphate, the systematic name for FprA is thus NADPH-ferredoxin oxidoreductase or NFR. Although Fd I is not the physiological substrate of the bacterial reductase, a titration of FprA with Fd I was attempted to demonstrate that an interaction between the two proteins was indeed occurring thus supporting the activity data. Figure 3B shows the difference spectrum obtained at saturating concentration of Fd I. Two positive peaks appeared centered around 450 and 380 nm, where FprA has absorption maxima. An approximate K d value of 2 l M was obtained by titration. The interaction between the two proteins was further investigated by using cross-linking agents. Following incubation of the two proteins with N-ethyl-3-(3-dimethylaminopropyl)carbodiimide, FprA was fully converted to protein adducts of about 66 kDa as determined by SDS/PAGE. This is the expected value for a 1 : 1 cross-linked complex between the flavopro- tein and Fd I [12]. The cross-linked species acquired the capacity to reduce directly cytochrome c as judged by measuring the cytochrome c reductase activity in the absence of added Fd I. The same type of experiments were repeated replacing the spinach protein with the 7Fe ferredoxin. A cross-linked protein of about 66 kDa was also obtained, although at a lower rate of formation with respect to the plant ferredoxin. Anaerobic reduction of FprA with NAD(P)H Bovine AdR shows peculiar behavior when anaerobically reduced by NADPH [21]. We therefore tried to verify whether FprA presented the same reduction pattern when treated with physiological reductants. Identification of reduced intermediates could help in elucidating the mech- anism of action of FprA. The titration of FprA with the less efficient substrate NADH practically superimposed to that with dithionite (Fig. 2). About 1 (mol NADH)Æ(mol FAD) )1 was sufficient to fully reduce the enzyme, again without significant changes at wavelengths longer than 550 nm (data not shown). The full reduction of the enzyme FAD by just an equimolar amount of NADH implies that the enzyme redox potential is far more positive than that of the pyridine nucleotide couple. A completely different pattern was observed when FprA was titrated with NADPH (Fig. 4A). Clearly, upon reduction with substoi- chiometric amounts of the reduced coenzyme, absorption in the 500–800 nm region built up with a broad peak at 550 nm. These spectral changes are usually ascribed to formation of charge-transfer (CT) species (FAD-NADPH and/or FADH 2 -NADP + ) [16,17]. Only two species are present during titration, as indicated by the presence of two isosbestic points (373 and 490 nm, respectively). After addition of more than 1 mol NADPH per mol FAD (Fig. 4B), the spectra in the long wavelength region changed. A peak at 580 nm with a shoulder at 625 nm developed. This type of spectrum (peaks in the 600 nm region with no absorption beyond 700 nm) can be attrib- uted to the flavin neutral SQ [18]. In an attempt to further characterize the various species formed during NADPH titration of FprA, a titration with NADP + of the fully Fig. 4. NADPH reduction of FprA. The titration was performed in 10 m M Tris/HCl, pH 7.4 under anaerobiosis. 47 l M FprA was titrated with NADPH. The spectra recorded at 0, 0.3, 0.45, 0.6, 0.7, 0.9, 1 (A) and at 1.3, 1.6, 1.9, 3, 6 (B) NADPH/FAD molar ratios are reported. The inset shows the plot of the absorbance at 625 nm due to SQ, obtained by subtracting the contribution of charge-transfer species as detailed in Materials and methods, as a function of NADPH/FAD molar ratio. Table 3. Kinetic parameters for the 2Fe and 7Fe ferredoxin reductase reactions of FprA. Electron acceptor k cat (e – Æs )1 ) K NADPH m (l M ) k cat /K m (e – Æs )1 Æl M )1 ) K acceptor m (l M ) k cat /K m (e – Æs )1 Æl M )1 ) S. oleracea Fd I 11 ± 0.20 2.6 ± 0.12 4.2 ± 0.27 0.86 ± 0.04 13 ± 0.63 M. smegmatis ferredoxin 3.4 ± 0.27 3.5 ± 0.72 0.97 ± 0.21 0.03 ± 0.004 110 ± 17 3010 F. Fischer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 reduced enzyme obtained by photoreduction was performed (Fig. 5A). The spectra resemble those already observed during the early steps of NADPH titration of oxidized enzyme (Fig. 4A). It can be noted that both the absorbance at 450 and 340 nm of the solution increased at each addition of NADP + up to 1 NADP + per FAD (see inset) and no SQ was formed. Thus, the spectrum of the CT formed in this experiment, which is superimposable to that formed in the titration of the oxidized enzyme with a molar amount of NADPH, is mostly due to FAD-NADPH charge transfer, as can be judged from the high absorbance at 340 and 450 nm, and low absorbance at 750 nm. The SQ amount present during NADPH titration could then be calculated by subtracting from the spectra the contribution of the CT species as obtained from the experiment shown in Fig. 5A. In the inset of Fig. 4B, the absorption changes due to SQ accumulation are plotted against the NADPH/flavin molar ratio. It can be observed that the SQ built up only after one NADPH/flavin was added, reached its maximum after addition of slightly more than two NADPH/flavin, and then remained at this level notwithstanding the high amount of NADPH added. Indeed, full reduction of the bound flavin to FAD dihydroquinone was not achieved even by prolonged incubation or by using NADPH in the presence of a NADPH regenerating system. This suggests that the SQ is stabilized by complexation with NADPH. This is further confirmed by photoreduction experiments carried out in the presence of 1.5 (mol NADP + )Æ(mol FAD) )1 (Fig. 5B). Whereas photoreduction of uncomplexed FprA did not elicite accumulation of reduced intermediates, in the presence of NADP + the formation of a long wavelength band with a peak at 550 nm (ascribable to CT species) in the early steps of reduction was observed. With further irradiation, the spectral features typical of the SQ appeared. This indicates that the SQ accumulated only after NADPH was formed, thus suggesting that this intermediate is a complex between flavin SQ and NADPH. These data can be rationalized according to the scheme presented below: E ox þ NADPH $ CT CT þ NADPH $ E red -NADPH þ NADP þ CT þ E red -NADPH $ 2E sq -NADPH where CT indicate an equilibrium mixture of the two charge-transfer species FAD-NADPH and FADH 2 - NADP + . DISCUSSION The functional annotation of proteins identified in genome sequencing projects is based on protein sequence similarities to homologs in the databases. However, due to the possibility of divergent evolution, homologous enzymes may not catalyze the same reaction. Thus, a biochemical characterization of the gene product is required to establish the protein’s real function in that organism. This was particularly necessary in the case of the fprA gene product of M. tuberculosis, because of the absence in the bacterial genome of genes coding for [2Fe)2S] ferredoxins, the expected protein substrate for an adrenodoxin reductase- like enzyme. To our knowledge, this is the first adrenodoxin reductase-like protein from a bacterium to be characterized. The recombinant enzyme was shown to be a flavoprotein containing noncovalently bound FAD, whose fluorescence was nearly fully quenched. This is a remarkable difference from the mammalian enzyme, the flavin of which is fluorescent [22,23]. The fprA gene product did not show significant activity as ferric reductase as was at first hypothesized. Instead, it possesses the activities typical of the mammalian AdR [9,24], including the capacity to reduce the mammalian Adx. However, FprA was more efficient with plant Fd I and more interestingly, with a 7Fe ferredoxin of M. smegmatis. This ferredoxin is a homolog of M. tuberculosis FdxC (88% identity between the sequences). The higher affinity of the reductase for the 7Fe ferredoxin is in keeping with the absence of 2Fe ferredoxins in mycobacteria. The elucidation of the three-dimensional structure of the enzyme will provide more information on the structural basis for the specificity in protein–protein recognition. The enzyme can use both NADPH and NADH as a reductant; however, the specificity constant (k cat /K m )of NADPH is two orders of magnitude larger than that of NADH. Furthermore, binding of NADP + to FprA is extremely tight with K d values in the nanomolar region. The affinity of FprA for NADP + is at least 10 times higher than Fig. 5. Effect of NADP + addition after or before FprA photoreduction. Photoreduction of FprA was performed in 10 m M Hepes-KOH, pH 7.0, in the presence of 15 m M EDTA and 1.8 l M 5-deazaribofla- vin. (A) NADP + titration of 26.5 l M photoreduced FprA. The spectra recorded at 0, 0.1, 0.3, 0.4, 0.5, 0.7, 0.8, 1 NADP + /FAD molar ratios are reported. The inset shows the absorbance changes at 452 (s), 550 (d)and750nm(h) as a function of NADP + /FAD molar ratio. The absorbance change at 750 nm has been multiplied by four for clarity. (B) photoreduction of 20 l M FprA in the presence of 30 l M NADP + . The spectra recorded before and after 1.5, 2.5, 3.5 min irradiation (dashed line) and after 6, 10, 13, 17 min irradiation (solid lines) are shown. The inset shows an enlargement of the spectral data in the 500– 750 nm region. Ó FEBS 2002 M. tuberculosis NADPH-ferredoxin reductase (Eur. J. Biochem. 269) 3011 that of bovine AdR [9]. The tight binding of NADP(H) may have physiological implications. Anaerobic titrations with NADPH of FprA revealed a completely different pattern from that obtained with dithionite, NADH or photoreduc- tion. In the latter cases, only two forms of the enzyme, the oxidized and the fully reduced ones, were observed. With NADPH or NADP + present during reduction of FprA, two additional forms were identified: CT species (FAD- NADPH and FADH 2 -NADP + ) and FAD semiquinone. Unlike bovine AdR, FprA highly favored the CT species FAD-NADPH, as judged by comparison of the spectra [21]. By analysis of the conditions in which the SQ accumulated, it can be inferred that this intermediate results from NADPH binding to the flavin SQ, as observed in the case of bovine AdR [21]. This complex is assumed to be a compulsory intermediate in the catalytic cycle of these enzymes, whose functional role is to mediate electron transfer between two-electron donors (NADPH) and one- electron acceptors (iron-sulfur protein substrates) [9]. This enzyme must be of relevance to mycobacteria because a homolog is present in M. leprae, whose genome is greatly downsized and degraded [25]. On the basis of the high similarity of FprA with mammalian AdR (Table 1), its enzymatic function may be inferred. In mitochondria, AdR, with a [2Fe)2S] ferredoxin, is part of an electron chain which delivers electrons from NADPH to cytochrome P450 enzymes, mainly involved in hydroxylation reactions [9,19,20]. The M. tuberculosis genome is rich in genes encoding P450 cytochromes (22 genes, see [1]), whereas it lacks genes coding for Adx-type ferredoxins and it contains only genes encoding 7Fe and 3Fe ferredoxins [1]. In bacteria, different systems for P450 cytochrome reduction are employed. Well known is the system comprising putidaredoxin reductase, a NADH-dependent flavoprotein, and putidaredoxin (2Fe ferredoxin), which transfers elec- trons to P450 cam [26]. This system is similar to the mammalian AdR-Adx. A microsomal-type P450 reductase instead is present in Bacillus megaterium [27]. Apparently, purification of the reductase from other bacteria was unsuccessful due to protein instability and low expression level. A microbial cytochrome P450 reduction system was purified from Streptomyces griseus grown in a soybean flour-enriched medium [28]. The ferredoxin reductase was a NADH-dependent flavoprotein of 60 kDa with a N-terminal sequence comprising a FAD binding consensus sequence (GXGXXG), which is typical of the glutathione reductase large family [29], to which AdR also belongs. They showed that this enzyme can couple electron transfer from NADH to cytochrome P450 soy in the presence of S. griseus 7Fe ferredoxin. The activity value measured in the cytochrome c assay is in agreement to that obtained with FprA and M. smegmatis ferredoxin. The low K m value of FprA for this iron-sulfur protein strengthens the hypothesis that a 7Fe ferredoxin could be the physiological partner of the enzyme. Nevertheless, in herbicide-induced S. griseolus cells [30], two small 3Fe ferredoxins were found highly expressed, which could reconstitute an in vitro electron chain to P450 cytochromes using spinach FNR. Recently, a cytochrome P450 and a 3Fe ferredoxin were purified from Mycobacterium sp. strain HE5, grown on morpholine [31]. In both these cases, it was hypothesized that the reductase is constitutively formed and it has a broad specificity with respect to the ferredoxin substrate. Further roles for AdR have been discovered. The AdR-Adx system of the lower eukaryote Saccharomyces cerevisiae was shown to be essential for yeast viability by gene knockout [32–34] and to be involved in the biosynthe- sis of the cell iron-sulfur clusters [35–37]. Furthermore, mammalian AdR has been recently identified to play a role in the p53-dependent apoptosis, due to its potential to produce reactive oxygen species (ROS) [38]. Accordingly, it can be assumed that the mycobacterial FprA may have similar functions in iron-sulfur cluster synthesis or oxidative stress response. It is likely that FprA is primarily involved in the reduction of P450 enzymes as is the case of the other bacerial reductases cited above. Recently, the P450 14a-demethylase of M. tuberculosis has been characterized and suggested to be involved in the cholesterol biosynthetic pathway [39]. Cholesterol has been shown to be essential to M. tuberculosis infection [40]. Furthermore, some of the cytochrome P450 enzymes could be involved in the synthesis of the complex cell wall components. Thus, if FprA provides electrons to several pathways through the inter- action with several ferredoxins, it represents a potential target for antimycobacterial drugs. Crystals of FprA have been obtained and the three-dimensional structure is being currently determined. ACKNOWLEDGEMENTS This work was carried out with funds from the Ministero dell’Univer- sita ` e della Ricerca Scientifica e Tecnologica (Prin 1999) and European Union (EU Cluster QLK2-2000–01761). We thank Dr G. Riccardi (University of Genova), Dr R. Cantoni and Dr M. Branzoni (University of Pavia) for help in cloning and DNA sequencing, Dr A. Negri and Dr G. Tedeschi for protein microsequencing, and Dr M. A. Vanoni and Dr B. Curti for helpful discussions. REFERENCES 1. Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry III, C.E. et al. (1998) Deciphering the biology of Mycobacterium tuber- culosis from the complete genome sequence. Nature 393, 537–544. 2. McKinney, J.D. (2000) In vivo veritas: the search for TB drug targets goes live. Nat. Med. 6, 1330–1333. 3. De Voss, J.J., Rutter, K., Schroeder, B.G. & Barry, C.E. III (1999) Iron acquisition and metabolism by mycobacteria. J. Bacteriol. 181, 4443–4451. 4. Vidal, S.M., Malo, D., Vogan, K., Skamene, E. & Gros, P. (1993) Natural resistance to infection with intracellular parasites: isola- tion of a candidate for Bcg. Cell 73, 469–485. 5. 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Ó FEBS 2002 M. tuberculosis NADPH-ferredoxin reductase (Eur. J. Biochem. 269) 3013 . Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase Federico Fischer, Debora Raimondi, Alessandro Aliverti and Giuliana Zanetti Dipartimento. Purification of recombinant FprA as analysed by SDS/PAGE. Lanes 1 and 5, molecular mass markers (values in kDa are indicated); lane 2, crude extract; lane

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