Electron Transfer Ability from NADH to Menaquinone and from NADPH to Oxygen of Type II NADH Dehydrogenase of Corynebacterium glutamicum

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1 Biosci. Biotechnol. Biochem., 69 (1), , 2005 Electron Transfer Ability from NADH to Menaquinone and from NADPH to Oxygen of Type II NADH Dehydrogenase of Corynebacterium glutamicum Nawarat NANTAPONG, Asuka OTOFUJI, Catharina T. MIGITA, Osao ADACHI, Hirohide TOYAMA, and Kazunobu MATSUSHITA y Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi , Japan Received August 30, 2004; Accepted November 4, 2004 Type II NADH dehydrogenase of Corynebacterium glutamicum (NDH-2) was purified from an ndh overexpressing strain. Purification conferred 6-fold higher specific activity of NADH:ubiquinone-1 oxidoreductase with a 3.5-fold higher recovery than that previously reported (K. Matsushita et al., 2000). UV visible and fluorescence analyses of the purified enzyme showed that NDH-2 of C. glutamicum contained non-covalently bound FAD but not covalently bound FMN. This enzyme had an ability to catalyze electron transfer from NADH and NADPH to oxygen as well as various artificial quinone analogs at neutral and acidic phs respectively. The reduction of native quinone of C. glutamicum, menaquinone-2, with this enzyme was observed only with NADH, whereas electron transfer to oxygen was observed more intensively with NADPH. This study provides evidence that C. glutamicum NDH-2 is a source of the reactive oxygen species, superoxide and hydrogen peroxide, concomitant with NADH and NADPH oxidation, but especially with NADPH oxidation. Together with this unique character of NADPH oxidation, phylogenetic analysis of NDH-2 from various organisms suggests that NDH-2 of C. glutamicum is more closely related to yeast or fungal enzymes than to other prokaryotic enzymes. Key words: type II NADH dehydrogenase; NAD(P)H quinone oxidoreductase; superoxide; bacterial respiratory chain; Corynebacterium glutamicum NADH:quinone oxidoreductase, found in bacterial respiratory chains, can be divided into three different types: a proton-translocating type I NADH dehydrogenase (NDH-1), type II NADH dehydrogenase lacking an energy coupling site (NDH-2), and Na þ -translocating NADH:quinone oxidoreductase. NDH-1, which is homologous to mitochondrial complex I, is composed of different subunits and has FMN and several iron sulfur clusters as the prosthetic groups. This enzyme is able to pump protons from the cytosolic side to the periplasmic side. NDH-2 is a single subunit enzyme and bears flavin but no iron sulfur clusters. 1) Although the oxidation of NADH is extensively carried out by complex I in mammals, mitochondria from fungi contain an alternative NADH dehydrogenase, NDH-2, together with complex I in Neurospora crassa 2) or without complex I in Saccharomyces cerevisiae. 3) Similarly to fungi, the bacterial respiratory chain has NDH-1 and NDH-2, or either one of these. Escherichia coli has NDH-1 and NDH-2, 4) which are encoded by the nuo operon 5) and the ndh gene 6) respectively, but Paracoccus denitrificans has only NDH-1 7) and Bacillus subtilis has only NDH-2. 8) Although bacterial NDH-1 from E. coli 9,10) and P. denitrificans 11,12) has been well characterized, NDH-2 has not been well studied, except for E. coli NDH-2. 13,14) Prokaryotic NDH-2 has been isolated from B. subtilis, 15) Methylococcus capsulatus, 16) Acidianus ambivalens, 17) and Sulfolobus metallicus. 18) These enzymes lack iron sulfur clusters and contain a flavin, non-covalently bound FAD in most cases 13,15,16) but covalently bound FMN in other cases. 17,18) There is no evidence that NDH-2 from these microorganisms contains a metal binding motif. Only E. coli NDH-2 has been reported to contain a Cu(I) thiolate ligation domain. 19) The Gram-positive coryne-form bacterium Corynebacterium glutamicum is an amino acid producing strain used industrially for the production of L-lysine and L- glutamate. The respiratory chain of this bacterium consists of several different primary dehydrogenases, 20,21) such as NADH dehydrogenase, succinate dehydrogenase, L-lactate dehydrogenase, and malate: quinone oxidoreductase, and at least three terminal oxidases, CN-sensitive cytochrome aa 3, 22,23) CN-resistant bypass oxidase, 20) and cytochrome bd. 24) The NADH y To whom correspondence should be addressed. Tel: ; Fax: ; kazunobu@yamaguchi-u.ac.jp Abbreviations: DEPMPO, 5-diethoxyphosphonyl-5-methyl-1-pyrroline N-oxide; EGTA, O,O 0 -Bis<2-aminoethyl>ethyleneglycol-N,N,N 0 -N 0 -tetra acetic acid; FR, ferricyanide; KPB, potassium phosphate buffer; MD, menadione; MQ 2, menaquinone-2; NDH-1, type I NADH dehydrogenae; NDH- 2, type II NADH dehydrogenase; SDS PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Q 1 or Q 2, ubiquinone-1 or -2

2 150 N. NANTAPONG et al. dehydrogenase of C. glutamicum is NDH-2, which is able to work partly as an NADPH oxidase. 25) In spite of the fact that there is only NDH-2 and no NDH-1 in C. glutamicum, the disruption of NDH-2 does not lead to severe growth defects. 26) In this organism, NADdependent malate and/or lactate dehydrogenases are active and are coupled with malate:quinone oxidoreductase and/or L-lactate dehydrogenase as part of an NADH oxidizing system. 26,27) Thus NDH-2 of C. glutamicum has unique character compared with NDH-2 from other bacterial species. In this study, NDH-2 was purified from the overexpressing strain of C. glutamicum and subsequently characterized. The results show that it contains noncovalently bound FAD, which has an ability to catalyze electron transfer from NADH or NADPH to quinone and oxygen, the latter reaction being a source of superoxide as well as hydrogen peroxide. Materials and Methods Materials. Ubiquinone-1 or -2 (Q 1 or Q 2 ) and menaquinone-2 (MQ 2 ) was kindly supplied by Eizai Co., Japan, and by Professor N. Sone and Dr. J. Sakamoto of the Kyushu Institute of Technology, respectively. FAD, FMN, deamino-nad, and horseradish peroxidase were from Sigma. 5-Diethoxyphosphonyl-5-methyl-1-pyrroline N-oxide (DEPMPO) was from Oxis (Portland, OR, U.S.A.). All other materials were of reagent grade and were obtained from commercial sources. (EGTA), and 4 mm MgCl 2 at a final concentration of 10 mg protein per ml. The suspension was subsequently used for purification. Purification of NDH-2. All steps were performed at 0 4 C. The membrane suspension was incubated on ice for 30 min in the presence of 2% Triton X-100 and then ultracentrifuged at 40,000 rpm for 30 min. The supernatant was directly applied on a DEAE-Toyopearl column (1 ml of bed volume per 2 3 mg of protein applied) which had been equilibrated with 50 mm KPB, ph 6.5, containing 1 mm EGTA and 4 mm MgCl 2. The column was washed with 3 bed volumes of column buffer (50 mm KPB, ph 6.5, containing 1 mm EGTA, 4mM MgCl 2, 0.1% Triton X-100, and 20 mm FAD), followed by washing with 3 bed volumes of the column buffer containing 0.1 M KCl. Then the enzyme was eluted by a linear gradient consisting of 3 bed volumes each of the column buffer containing 0.1 M KCl and 0.3 M KCl. Enzyme activity appeared at 0.2 M KCl from the column. The pooled active fractions were dialyzed against 30-fold volumes of column buffer at 4 C for 12 h. The dialyzate was applied on a Heparin Sepharose column (1 ml bed volume per mg of protein) which had previously been washed with 50 mm KPB, ph 6.5, containing 1 mm EGTA and 4 mm MgCl 2. After the column was washed with 3 bed volumes of column buffer containing 0.15 M KCl, the enzyme was eluted with 3 bed volumes of column buffer containing 0.35 M KCl. The active fractions were then combined and used for further experiments. Bacterial strains and growth conditions. An NDH-2 over-expressing strain derived from C. glutamicum KY ) was used in this study. For cultivation, the strain was inoculated into Luria Bertani (LB) medium supplemented with 25 mg/ml kanamycin and incubated at 30 C under 200 rpm shaking overnight. The LBgrown cells were then transferred with the 1% inoculum into 1-liter of glucose minimum medium 20) containing 25 mg/ml kanamycin in a 3-liter Erlenmeyer flask and grown at 30 C until the late exponential phase. Membrane preparation. The cells were harvested by centrifugation and washed twice with 20 mm potassium phosphate buffer (KPB), ph 7.5. The washed cells were resuspended in the same buffer at 2.5 ml per g wet cells in the presence of 0.5 mg/ml of lysozyme and then incubated at 30 C under 80 rpm rotation for 1 h. Then the lysozyme-treated cells were disrupted by passing them twice through a French pressure cell press at 16,000 psi, and the cell debris was removed by centrifugation at 6,000 rpm for 20 min at 4 C. The supernatant was ultracentrifuged at 40,000 rpm for 90 min at 4 C to separate the soluble fraction from the precipitate. The membrane precipitated was resuspended with 50 mm KPB, ph 6.5, containing 1 mm O,O 0 -Bis<2- aminoethyl>ethyleneglycol-n,n,n 0 -N 0 -tetra acetic acid Enzyme assays. All enzyme assays were performed at 25 C. NAD(P)H oxidase activity was spectrophotometrically measured at 340 nm by following the decrease in NAD(P)H concentration. The reaction mixture (1 ml) contained an appropriate amount of enzyme, 0.2 mm NAD(P)H, and 50 mm KPB or Na acetate buffer. To see full enzyme activity, 20 mm FAD was also added to the reaction mixture when indicated. The amount of enzyme that oxidized 1 mmol of NAD(P)H per min was defined as 1 unit, where a millimolar extinction coefficient of 6.2 or 6.3 was used for the calculation of NADH or NADPH oxidase activity respectively. Q 1 or Q 2 reductase activity was spectrophotometrically measured by following the decrease in absorbance at 340 nm in a 1 ml reaction mixture consisting of an appropriate amount of enzyme, 0.2 mm NAD(P)H, 50 mm Q 1 or 30 mm Q 2, and 50 mm KPB or Na acetate buffer. A unit of activity was defined as 1 mmol of NAD(P)H oxidized per min, calculated using a millimolar extinction coefficient of Ferricyanide (K 3 Fe(CN) 6 ; FR) reductase activity was spectrophotometrically measured by following the decrease in absorbance at 420 nm. The reaction mixture consisted of an appropriate amount of enzyme, 0.2 mm NAD(P)H, 1 mm K 3 Fe(CN) 6, and 50 mm KPB. A unit of activity was defined as 2 mmol of K 3 Fe(CN) 6 reduced

3 per min (equivalent to 1 mmol of NAD(P)H oxidized), where a millimolar extinction coefficient of 1 was used for the calculation. MQ 2 - or menadione (MD)-dependent FR oxidoreductase activity was also spectrophotometrically measured by following the decrease in absorbance at 420 nm. The reaction mixture consisted of an appropriate amount of enzyme, 0.2 mm NADH, 1mM K 3 Fe(CN) 6,30mM MQ 2 or 50 mm MD, and 50 mm KPB or Na acetate buffer. A unit of activity was defined as 2 mmol of K 3 Fe(CN) 6 reduced per min, and FR reduction activity without MQ 2 or MD was subtracted to lead MQ 2 - or MD-dependent FR reductase activity. K m and V max values were determined from a Hofstee plot of several electron acceptors, oxygen, Q 1,Q 2, FR, MD, and MQ 2, for NDH-2 in the presence of NADH or NADPH as an electron donor. Detection of superoxide and hydrogen peroxide. Production of the superoxide radical was quantitatively determined by the reduction of cytochrome c. The reaction was performed in an appropriate buffer along with 0.2 mm NAD(P)H, 30 mm horse heart cytochrome c, 2mM EDTA, and purified NDH-2 with a total volume of 1 ml. Cytochrome c reduction was monitored at 550 nm at 25 C, where a millimolar extinction coefficient of 19.0 was used. The production of hydrogen peroxide by NDH-2 was measured using a horseradish peroxidasebased o-dianisidine assay. 28) The initial reaction was carried out with 60 mm NAD(P)H and purified NDH-2 in an appropriate buffer (total volume 1 ml) by incubation at 25 C until NAD(P)H was completely oxidized. After the reaction, 100 to 200 ml of the initial solution was transferred to a new 1 ml-cuvette (final volume 1 ml) containing 50 mm KPB, ph 7.0, 0.15 mm o-dianisidine, 7.5 U horseradish peroxidase, and mm EDTA. The absorbance change at 460 nm was followed, and the hydrogen peroxide concentration of the initial solution was determined from a standard curve. Spectroscopic analyses. To remove FAD and also exchange Triton X-100 with dodecylmaltoside in the purified enzyme, the purified enzyme (about 1.5 mg protein) was dialyzed against 50 mm KPB, ph 6.5, and then applied on a DEAE-Toyopearl column (1 ml bed volume) equilibrated with the same buffer. The column was washed with 10 ml of the same buffer containing 0.1% dodecylmaltoside, and the enzyme was eluted with the same buffer containing 0.1% dodecylmaltoside and 0.35 M KCl. UV visible spectra of the purified enzyme (4 mm) were recorded over a wavelength range of 250 to 600 nm on a double wavelength spectrophotometer (Hitachi 557) at 25 C. Fluoresence spectra of purified NDH-2 were recorded at 25 C with a Hitachi S fluorescence spectrophotometer. EPR spectra were recorded on a Bruker ELEXSYS E 500 spectrometer operating at 9.85 GHz at room temperature. The EPR conditions were typically a microwave power of 10 milliwatts and a modulation amplitude of Type II NADH Dehydrogenase of Corynebacterium glutamicum Gauss. A flat quartz cell (JEOL LC11, inner volume 60 ml) was used for the EPR measurement. The EPR sample contained 1.2 mm purified NDH-2, 0.2 mm FAD, 1mM NAD(P)H, and 45 mm DEPMPO in 50 mm KPB, ph 6.5, or 50 mm Na acetate buffer, ph 5.5. The sample was incubated for 15 min at room temperature. Hydroxyl radical ( OH) and superoxide radical (O 2 ) can be trapped and stabilized by DEPMPO, and the adducts show different characteristics of the EPR spectrum. 29) SDS PAGE analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was performed according to the method of Laemmli 30) followed by staining with Coomassie brilliant blue R250. The protein standard marker used consisted of phosphorylase b (94 kda), albumin (67 kda), ovalbumin (43 kda), carbonic anhydrase (31 kda), trypsin inhibitor (21.1 kda), and lysozyme (14.4 kda). Protein determination. The concentration of protein was determined by a modified Lowry method, 31) and bovine serum albumin was used as a standard. Results Purification of NDH-2 Although NDH-2 has previously been purified from C. glutamicum KY9714, 25) the enzyme was unstable and recovery during purification was low, suggesting that FAD might have been lost. In this study, we attempted to purify the enzyme from an NDH-2 overexpressing strain derived from KY9714 and tried to improve its recovery by adding FAD throughout the purification steps. Purification was performed by two-step column chromatography using DEAE-Toyopearl and Heparin Sepharose, as described in Materials and Methods. As summarized in Table 1, NADH:Q 1 reductase activity was enriched 6.2-fold with a 15.6% yield. Enzyme purity was confirmed on SDS PAGE, where a single polypeptide of 55-kDa corresponding to NDH-2 was observed (Fig. 1). Spectral analysis of the purified enzyme To avoid interference in spectral analysis, excess FAD supplemented during the purification was removed by dialysis and Triron X-100 was exchanged with dodecylmaltoside, as described in Materials and Methods. The purified enzyme thus prepared exhibited an Purification steps Table 1. Summary of NDH-2 Purification Total protein (mg) Specific activity (U/mg) Total activity (U) Yield (%) Membrane , Triton supernatant , DEAE-Toyopearl , Heparin Sepharose ,

4 152 N. NANTAPONG et al. Fig. 1. SDS PAGE of Purified NDH-2. Lane 1, Protein standard marker; Lane 2, Membrane fraction (10 mg of protein); Lane 3, Triton supernatant (10 mg of protein); Lane 4, DEAE-Toyopearl (10 mg of protein); Lane 5, Heparin Sepharose (10 mg of protein). Fig. 3. Determination of the Flavin Moiety from Purified NDH-2. Panels A and B are fluorescence emission (excited at 452 nm) and excitation (detected at 530 nm) spectra respectively. Panel C is the intensity of the fluorescence emission peak (excited at 452 nm) of the purified enzyme in arbitrary units at various phs. Fig. 2. UV Visible Spectrum of Purified NDH-2 (4 mm) of C. glutamicum in the Presence of 0.1% Dodecyl Maltoside. The purified enzyme was treated as described in Materials and Methods before this measurement. The oxidized (curve 1) and dithionite-reduced (curve 2) spectra are shown. absorption spectrum with a maximum at 273 nm and a broad shoulder between nm (Fig. 2). The peak around 450 nm was reduced by the addition of dithionite, indicating the presence of a typical flavin cofactor, but the flavin content estimated from the spectrum might have been underestimated because a large portion of flavin was detached during dialysis, as can be seen in the finding that NADH:Q 1 reductase activity was reduced to 34% (from 273 to 93 U/mg) after the dialysis. The decreased activity during dialysis could not be recovered by further addition of FAD or FMN, although the reason is not known. Thus a flavin moiety attached to the dialyzed enzyme was examined by fluorescence analysis at various phs. 32) The flavin moiety extracted from the dialyzed enzyme by heat treatment exhibited a fluorescence spectrum with a single emission at 530 nm when excited at 452 nm (Fig. 3A and B). The emission maximum was observed at acidic ph (Fig. 3C), suggesting that NDH-2 of C. glutamicum contains a noncovalently bound FAD. Although, since NDH-2 of E. coli has been shown to contain copper (Cu(I)), 19) luminescence spectroscopy was performed with the purified enzyme, no bands corresponding to Cu(I) were observed (data not shown). In addition, metal analysis using induced-coupled plasma atomic emission spectrometery indicated that purified NDH-2 did not contain any copper (data not shown). Kinetic properties of the purified enzyme C. glutamicum NDH-2 oxidizes NADPH at acidic ph with or without Q 1 in addition to oxidizing NADH with Q 1 at neutral ph. 24) In this study, we examined both

5 Type II NADH Dehydrogenase of Corynebacterium glutamicum 153 Fig. 4. ph-dependent NAD(P)H Oxido-Reductase Activities with Various Artificial Electron Acceptors or Oxygen. The closed and open circles represent oxidation of NADH and NADPH respectively. NADH and NADPH oxidations kinetically with various electron acceptors in more detail. NADH oxidation coupled with various artificial electron acceptors exhibited maximal activity at neutral ph between 6 and 7, whereas NADPH oxidation had an optimum ph under acidic conditions at about ph 5 (Fig. 4). Only NADH:Q 1 oxidoreductase activity had two optimum phs. Therefore the kinetic properties of NDH-2 for NAD(P)H oxidation coupled with the reduction of several electron acceptors were further examined at their optimum ph (Table 2 and 3). As expected, the purified enzyme exhibited a higher affinity for NADH than for NADPH when oxygen or Q 1 was used as the electron acceptor, although the K m for NADH with oxygen could not be determined due to a low value of less than 2 mm (Table 2). The highest affinity for the electron acceptor of the purified enzyme was observed with MQ 2 coupling for the oxidation of NADH, while the reduction of MQ 2 was not observed with NADPH oxidation (Table 3). MQ 2 Table 2. Substrate Kinetic Parameters of NAD(P)H for Purified NDH-2 NADH NADPH K m (NADH) V max K m (NADPH) V max mm (units/mg) mm (units/mg) NAD(P)H oxidase <2 (2.13) NAD(P)H:Q 1 oxidoreductase 11.6 (105) Measured at ph 7. Measured at ph 5. The V max value for NADH:Q 1 reductase is not accurate due to the relatively low Q 1 concentration (50 mm) used for this assay. reductase activity was measured as FR reduction activity via MQ 2, in which the final electron acceptor was required to maintain the higher activity. The MQ analog MD was reduced by the enzyme even with NADPH, although the affinity and activity were lower than those for NADH (Table 3). A higher affinity for the artificial

6 154 N. NANTAPONG et al. Table 3. Kinetic Parameters for Electron Acceptors of Purified NDH-2 As described in Materials and Methods, all the activities were measured by following the decrease in absorbance at 340 nm, except for NAD(P)H:FR, :MD and :MQ 2 oxidoreductase activities, in which FR reduction was followed without and with MD or MQ 2 respectively. NAD(P)H was used at a concentration of 400 mm in this experiment. Substrate NADH NADPH K m (acceptor) V max K m (acceptor) V max mm (units/mg) mm (units/mg) NAD(P)H:Q 1 oxidoreductase (37.7) NAD(P)H:Q 2 oxidoreductase (69.0) NAD(P)H:FR oxidoreductase (34.3) NAD(P)H:MD oxidoreductase (118) NAD(P)H:MQ 2 oxidoreductase ND Measured at ph 7 except for FR reductase activity, which was measured at ph 5. Measured at ph 5. The V max values of these activities are not accurate because the NADPH concentration used was not saturated. ND, not detected. electron acceptors, ubiquinone analog (Q 1 or Q 2 ) and FR, was observed for NADPH oxidation rather than for NADH. It should be noted that purified NDH-2 did not react with deamino-nadh (100 mm) as in the case of E. coli enzyme (less than 1% of NADH) and also that it could not be inhibited with 10 mm rotenone. Antimycin A inhibition upon the oxidation of NAD(P)H by purified NDH-2 Since NDH-2 of C. glutamicum has an ability to donate electrons to menaquinone or ubiquinone, sensitivity to several quinone inhibitors was examined to get more insight into the reactivity of the enzyme towards quinone and oxygen. First, various different inhibitors were used to determine whether they inhibited NAD(P)H:quinone reductase activity. As for the result, the reduction of quinones with purified NDH-2 was shown to be inhibited only by antimycin A (data not shown). As shown in Fig. 5, NADH:MQ and NAD(P)H:Q 2 reductase activities were intensively inhibited with 25 mm antimycin A, but NAD(P)H oxidase activities were not. This result also indicates that NADPH:Q 2 reductase activity was slightly more sensitive to antimycin A than was NADH reductase. Production of superoxide and hydrogen peroxide by purified NDH-2 The NADH oxidase system of C. glutamicum can produce superoxide anions. 20) Moreover, type II NADH dehydrogenase (rotenone-insensitive NADH dehydrogenase) of Trypanosoma brucei 33) and S. cerevisiae 34) produce superoxide in the presence of NADH. Therefore, in this study, superoxide generation dependent on NADH or NADPH oxidation was examined using the purified NDH-2 of C. glutamicum. The production of superoxide was quantitatively determined by following the reduction of horse heart cytochrome c at various ph conditions and comparing this with the NAD(P)H oxidase activity of the enzyme (Table 4). Superoxide production was observed at phs 5 and 6 with NADPH as the substrate, and at ph 5 to 8 with NADH. At the higher ph, it was higher when compared with O 2 uptake. The addition of FAD increased superoxide formation as well as O 2 uptake with NADPH, but not with NADH (data not shown). Fig. 5. Antimycin A Inhibition of NAD(P)H Oxidase, and NAD(P)H:Q 2 and NADH:MQ 2 Reductase Activities. Enzyme activities with NADH and with NADPH were measured in 50 mm KPB, ph 6.5, and in 50 mm Na acetate buffer, ph 5.5, respectively, as described in Materials and Methods., NADH oxidase;, NADPH oxidase;, NADH:Q 2 reductase;, NADPH:Q 2 reductase;, NADH:MQ 2 reductase.

7 Table 4. NAD(P)H-Dependent Superoxide (O 2 ) and Hydrogen Peroxide (H 2 O 2 ) Formation by NDH-2 Oxidase ph Oxidase activity O 2 formation H 2 O 2 forsystem U/mg U/mg (%) mation (%) NADPH (0) (8) (18) 57 NADH (1.8) (14) (14) (24) Type II NADH Dehydrogenase of Corynebacterium glutamicum 155 To confirm the production of superoxide during NAD(P)H oxidation, the formation of superoxide with NDH-2 was detected directly by EPR, in which superoxide was determined by the formation of DEPMPOadduct under aerobic conditions. The EPR signal with hyperfine coupling constants of a N ¼ 13:0, a H ¼ 11:1, a P ¼ 50:0 in Gauss was obtained in both the NADH and NADPH oxidations in the presence of purified NDH-2 (Fig. 6). The EPR signals were attributed to DEPMPO- OOH, which derived from superoxide but not from the hydroxyl radical ( OH), and was not observed without NAD(P)H or NDH-2. A higher signal intensity was obtained with NADPH than with NADH at phs 5.5 and 6.5, and also a higher signal was obtained at ph 5.5 than at ph 6.5 with NADPH. Together with the observations for cytochrome c reduction, we concluded that NDH-2 produces a superoxide radical concomitant with NADPH oxidation at acidic ph, and to a lesser extent with NADH oxidation. Although, judging from the cytochrome c reduction, the highest efficiency of superoxide formation with NADPH appeared to occur at neutral ph, EPR analysis indicated that superoxide formation occurs parallel to oxidation activity. Since generated superoxide appears to be converted spontaneously into hydrogen peroxide under acidic conditions, we further examined the production of hydrogen peroxide from NADH and NADPH at the ph that induced the highest superoxide production and oxidase activity (Table 4). The results indicate that the oxidation of NADH and NADPH resulted in the generation of hydrogen peroxide, which may be produced from superoxide at least at ph 5 with NADPH, because superoxide generation was not very high in spite of being expected to be very high by EPR analysis under these conditions. Discussion Fig. 6. EPR Determination of Superoxide Radical Produced by Purified NDH-2. Approximately 1.2 mm purified NDH-2 was incubated for 15 min with 1 mm NADPH (A and B) or NADH (C and D), 0.2 mm FAD, and 45 mm DEPMPO in 50 mm Na acetate buffer, ph 5.5, (A and C) or in 50 mm KPB, ph 6.5, (B and D). Recently, NDH-2 was purified from a lysozymesensitive strain of C. glutamicum and shown to exhibit NADPH oxidase activity in addition to NADH dehydrogenase activity. 25) In this study, in order to characterize this enzyme in more detail, NDH-2 of C. glutamicum was purified from an over-producing strain by an improved purification procedure in which FAD was added throughout the purification process. After purification, NDH-2 was enriched 6.2-fold with 15% yield based on NADH:Q 1 reductase activity, of which the specific activity and recovery were enhanced by 6-fold and 3.5-fold respectively, compared with the values previously reported. 25) Similarly to NDH-2, found in other organisms, the spectrum of the purified enzyme showed that C. glutamicum NDH-2 contains a flavin moiety. This is consistent with the sequence information on this enzyme, which shows that it contains an FAD

8 156 N. NANTAPONG et al. binding motif. In this study, we also presented evidence that C. glutamicum NDH-2 contains non-covalently bound FAD but not covalently bound FMN. Although E. coli NDH-2 contains thiolate-bound Cu(I) and is predicted to have two conserved cysteine residues, 19) C. glutamicum NDH-2 does not contain Cu and has neither a heavy-metal-associated domain nor conserved cysteine residues in its polypeptide sequence (data not shown). NDH-2 of E. coli contains the predicted transmembrane domain at the C-terminal region, 19) and the external alternative NADH dehydrogenase of N. crassa also contains a transmembrane domain at the N-terminal region. 35) On the other hand, NDH-2 of A. ambivalens contains 3 possible putative amphipatic helices located around amino acid residues 15 27, and , which are also present in E. coli NDH-2. 36) Therefore the secondary structure of C. glutamicum NDH-2 was predicted using programs provided by the TMHMM ( hmmtop/) and Columbia University ( bioc.columbia.edu) servers. The results indicated that there is a single putative transmembrane region in the C- terminal region (around amino acid residues ), and also in similar possible amphipatic regions (residues , and ). Thus we cannot provide strong evidence to show how this enzyme is located on the membrane of C. glutamicum. When the oxidation of NAD(P)H was examined with various electron acceptors, reduction of MD, Q 1,Q 2, and FR was observed with both NADH and NADPH, whereas reduction of MQ 2 was observed only with NADH. But undetectable NADPH MQ 2 reductase activity does not necessarily imply that this enzyme cannot reduce MQ 2 with NADPH. Since the oxidation of NADPH by purified enzyme is very weak compared with NADH oxidation, it is conceivable that the oxidation of NADPH with MQ 2 might be below the detection limit in the in vitro assay. The K m and V max for MQ 2 reduction of C. glutamicum NDH-2 with NADH (K m ¼ 5 mm; V max ¼ 82 U/mg) were reasonably low and high respectively, whereas the affinity (K m ¼ 41 mm) for ubiquinone analogue, Q 1, is significantly lower than that in E. coli NDH-2 (K m ¼ 5:9 mm). 14) Also the K m values for Q 1,Q 2, and FR reduction due to NADH oxidation were higher than those due to NADPH oxidation, and thus these artificial electron acceptors are more favorable electron acceptors for coupling with NADPH oxidation. The results suggest that menaquinone is a more favorable electron acceptor for C. glutamicum NDH-2 than other quinones at least for NADH oxidation. Furthermore, since the purified enzyme exhibits reasonably higher oxidase activity when coupled with NADPH, the physiological electron acceptor coupled with NADPH oxidation of C. glutamicum NDH-2 is probably oxygen, although the actual affinity for oxygen was not determined in this study. Taken together with these results, it can be concluded that NDH-2 in C. glutamicum oxidizes NADH tightly coupled with a natural electron acceptor menaquinone within the membrane, while it may oxidize NADPH partly coupled with oxygen reduction. However, the results obtained with antimycin A inhibition provided evidence that C. glutamicum NDH-2 donates electrons from both NADH and NADPH to quinone in the same manner, or at the same site, yet with different efficiencies, whereas the oxygen reacting site may be different from the quinone site. The results obtained in this study also suggest that the binding of NADPH and/or NADH might cause a conformational change in the enzyme structure, which causes a change in the binding site for the electron acceptor, including oxygen, or that it might directly overlap the acceptor binding site so that NADPH binding instead of NADH changes the binding of the acceptors. Various redox carriers of the electron transport chain are possible sources of reactive oxygen species formation. In mammalian mitochondria, the main sites for superoxide and hydrogen peroxide production are NADH:quinone reductase, complex I, and ubiquinolcytochrome c reductase. Type II NADH dehydrogenase of E. coli, 28) T. brucei 33) or S. cerevisiae 34) is also a source of endogenous superoxide production. Our study also indicates that NDH-2 of C. glutamicum is a potential source of superoxide and hydrogen peroxide formation. The oxidation of NADH and NADPH with oxygen, especially NADPH oxidation, with NDH-2 led to the formation of superoxide and hydrogen peroxide. Since, unlike E. coli enyzme, NDH-2 of C. glutamicum has the ability to oxidize NADPH in addtion to NADH, it is possible that superoxide was generated from NADPH oxidation with NDH-2. E. coli enzyme can also generate superoxide from NADPH, although not as high an amount as observed for that from C. glutamicum (unpublished results). In contrast, a similar amount of superoxide formation from NADH using NDH-2 between C. glutamicum and E. coli detected by EPR was observed in our laboratory (unpublished). However, similarly to the C. glutamicum enzyme, NDH-2 of T. brucei and S. cerevisiae produce a relatively high superoxide from NAD(P)H and NADH respectively. 33,34) NDH-2 of C. glutamicum exhibits a unique characteristic, NADPH oxidation, that occurs more often in eukaryotic enzymes than in prokaryotic ones. The external alternative NADH dehydrogenases NDE-1 and NDE-2 of N. crassa oxidize NADPH at acidic ph, similarly to the C. glutamicum enzyme. 35,37) Therefore, a phylogenetic tree of NDH-2 from various organisms including C. glutamicum was constructed using the Clustal W program (Fig. 7). As expected, NDH-2 of Corynebacterium sp. as well as its counterpart Mycobacterium sp. were found to be more closely related to eukaryotic enzymes than to other prokaryotic ones. Thus NDH-2 of C. glutamicum appears to have evolved from the same ancestor as the enzymes found in yeast and fungi.

9 Type II NADH Dehydrogenase of Corynebacterium glutamicum 157 Fig. 7. Phylogenetic Relationship for NDH-2 from C. glutamicum and Other Organisms. This tree was constructed using the Clustal W program provided by the European Bioinformatics Institute ( index.html), and is displayed as a phylogram. The polypeptide sequences used for the construction came from the NCBI Entrez database with accession numbers for each organism as follows: AAK19737 (Azotobacter vinelandii NDH-2), NP (Pseudomonas aeruginosa), AAF97237 (P. fluorescens NDH-2), NP (E. coli NDH-2), NP (Salmonella typhimurium NDH-2), BAC59234 (Vibrio parahaemolyticus), NP (V. cholerae), NP (Haemophilus influenzae), AAD56918 (Zymomonas mobilis), BAB07126 (B. halodulans), CAD33806 (Acidianus ambivalens), NP (Sulfolobus solfataricus), NP (C. glutamicum), NP (C. efficiens), NP (C. diphtheriae), NP (Mycobacterium tuberculosis), NP (M. bovis), AAC46302 (M. smegmatis), CAB77710 (Candida albicans), NP (S. cerevisiae NDE-1), NP (S. cerevisiae NDE-2), XP (N. crassa NDE-2), CAA07265 (Yarrowia lipolytica NDH-2), CAA43787 (S. cerevisiae NDI-1), EAA32649 (N. crassa NDE-1), XP (N. crassa NDI-1), AAM95239 (T. brucei), NP (Synechocystis sp.) and NP (Nostoc sp.). NDH-2 of C. glutamicum donates electrons to membranous menaquinone at least when it reacts with NADH, while it might preferentially transfer electrons to oxygen with NADPH concomitant with the production of superoxide or hydrogen peroxide. C. glutamicum cells contain a relatively high amount of superoxide dismutase as well as catalase, and the superoxide dismutase appears to be localized in both the cytoplasm and the periplasm (unpublished data). Thus NDH-2 of C. glutamicum might work as an NADH dehydrogenase as a primary dehydrogenase of the NADH oxidase respiratory chain and also as an NADPH oxidase, or partly as an NADH oxidase, not coupled with the respiratory chain. The latter direct oxidation reactions with NDH-2 appear to correspond to non-energy producing bypass electron transfer routes originally considered to be the bypass oxidase system in the respiratory chain of C. glutamicum. 20,27) Our knowledge of the function of NDH-2 in C. glutamicum and other organisms is still limited. However, our study together with others will help us to understand the biology of the utilization of NAD(P)H within the cell. Acknowledgments This work was supported in part by a Grant-in-Aid for scientific research (No ) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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