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

Biosci. Biotechnol. Biochem., 69 (1), 149–159, 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, y Hirohide TOYAMA, and Kazunobu MATSUSHITA

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi 753-8515, Japan

Received August 30, 2004; Accepted November 4, 2004

Type II NADH dehydrogenase of Corynebacterium 13–14 different subunits and has FMN and several iron– glutamicum (NDH-2) was purified from an ndh over- sulfur clusters as the prosthetic groups. This enzyme is expressing strain. Purification conferred 6-fold higher able to pump protons from the cytosolic side to the specific activity of NADH:ubiquinone-1 oxidoreductase periplasmic side. NDH-2 is a single subunit enzyme and with a 3.5-fold higher recovery than that previously bears flavin but no iron–sulfur clusters.1) Although the reported (K. Matsushita et al., 2000). UV–visible and oxidation of NADH is extensively carried out by fluorescence analyses of the purified enzyme showed complex I in mammals, mitochondria from fungi con- that NDH-2 of C. glutamicum contained non-covalently tain an alternative NADH dehydrogenase, NDH-2, bound FAD but not covalently bound FMN. This together with complex I in Neurospora crassa2) or enzyme had an ability to catalyze electron transfer without complex I in Saccharomyces cerevisiae.3) Sim- from NADH and NADPH to oxygen as well as various ilarly to fungi, the bacterial respiratory chain has NDH-1 artificial quinone analogs at neutral and acidic pHs and NDH-2, or either one of these. Escherichia coli has respectively. The reduction of native quinone of C. glu- NDH-1 and NDH-2,4) which are encoded by the nuo tamicum, menaquinone-2, with this enzyme was ob- operon5) and the ndh gene6) respectively, but Paracoccus served only with NADH, whereas electron transfer to denitrificans has only NDH-17) and Bacillus subtilis has oxygen was observed more intensively with NADPH. only NDH-2.8) Although bacterial NDH-1 from This study provides evidence that C. glutamicum NDH-2 E. coli9,10) and P. denitrificans11,12) has been well char- is a source of the reactive oxygen species, superoxide acterized, NDH-2 has not been well studied, except for and hydrogen peroxide, concomitant with NADH and E. coli NDH-2.13,14) Prokaryotic NDH-2 has been NADPH oxidation, but especially with NADPH oxida- isolated from B. subtilis,15) Methylococcus capsulatus,16) tion. Together with this unique character of NADPH Acidianus ambivalens,17) and Sulfolobus metallicus.18) oxidation, phylogenetic analysis of NDH-2 from various These enzymes lack iron–sulfur clusters and contain a organisms suggests that NDH-2 of C. glutamicum is flavin, non-covalently bound FAD in most cases13,15,16) more closely related to yeast or fungal enzymes than to but covalently bound FMN in other cases.17,18) There is other prokaryotic enzymes. no evidence that NDH-2 from these microorganisms contains a metal binding motif. Only E. coli NDH-2 has Key words: type II NADH dehydrogenase; NAD(P)H– been reported to contain a Cu(I)–thiolate ligation quinone oxidoreductase; superoxide; bacte- domain.19) rial respiratory chain; Corynebacterium glu- The Gram-positive coryne-form bacterium Coryne- tamicum bacterium glutamicum is an amino acid producing strain used industrially for the production of L-lysine and L- NADH:quinone oxidoreductase, found in bacterial glutamate. The respiratory chain of this bacterium respiratory chains, can be divided into three different consists of several different primary dehydrogena- types: a proton-translocating type I NADH dehydrogen- ses,20,21) such as NADH dehydrogenase, succinate ase (NDH-1), type II NADH dehydrogenase lacking an dehydrogenase, L-lactate dehydrogenase, and malate: energy coupling site (NDH-2), and Naþ-translocating quinone oxidoreductase, and at least three terminal 22,23) NADH:quinone oxidoreductase. NDH-1, which is ho- oxidases, CN-sensitive cytochrome aa3, CN-resist- mologous to mitochondrial complex I, is composed of ant bypass oxidase,20) and cytochrome bd.24) The NADH

y To whom correspondence should be addressed. Tel: +81-83-933-5858; Fax: +81-83-933-5859; E-mail: [email protected] Abbreviations: DEPMPO, 5-diethoxyphosphonyl-5-methyl-1-pyrroline N-oxide; EGTA, O,O0-Bis<2-aminoethyl>ethyleneglycol-N,N,N0-N0-tetra acetic acid; FR, ferricyanide; KPB, potassium phosphate buffer; MD, menadione; MQ2, menaquinone-2; NDH-1, type I NADH dehydrogenae; NDH- 2, type II NADH dehydrogenase; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Q1 or Q2, ubiquinone-1 or -2 150 N. NANTAPONG et al. dehydrogenase of C. glutamicum is NDH-2, which is (EGTA), and 4 mM MgCl2 at a final concentration of able to work partly as an NADPH oxidase.25) In spite of 10 mg protein per ml. The suspension was subsequently the fact that there is only NDH-2 and no NDH-1 in used for purification. C. glutamicum, the disruption of NDH-2 does not lead to severe growth defects.26) In this organism, NAD- Purification of NDH-2. All steps were performed at dependent malate and/or lactate dehydrogenases are 0–4 C. The membrane suspension was incubated on ice active and are coupled with malate:quinone oxidore- for 30 min in the presence of 2% Triton X-100 and then ductase and/or L-lactate dehydrogenase as part of an ultracentrifuged at 40,000 rpm for 30 min. The super- NADH oxidizing system.26,27) Thus NDH-2 of C. glu- natant was directly applied on a DEAE-Toyopearl tamicum has unique character compared with NDH-2 column (1 ml of bed volume per 2–3 mg of protein from other bacterial species. applied) which had been equilibrated with 50 mM KPB, In this study, NDH-2 was purified from the over- pH 6.5, containing 1 mM EGTA and 4 mM MgCl2. The expressing strain of C. glutamicum and subsequently column was washed with 3 bed volumes of column characterized. The results show that it contains non- buffer (50 mM KPB, pH 6.5, containing 1 mM EGTA, covalently bound FAD, which has an ability to catalyze 4mM MgCl2, 0.1% Triton X-100, and 20 mM FAD), electron transfer from NADH or NADPH to quinone and followed by washing with 3 bed volumes of the column oxygen, the latter reaction being a source of superoxide buffer containing 0.1 M KCl. Then the enzyme was as well as hydrogen peroxide. eluted by a linear gradient consisting of 3 bed volumes each of the column buffer containing 0.1 M KCl and Materials and Methods 0.3 M KCl. Enzyme activity appeared at 0.2 M KCl from the column. The pooled active fractions were dialyzed Materials. Ubiquinone-1 or -2 (Q1 or Q2) and against 30-fold volumes of column buffer at 4 C for menaquinone-2 (MQ2) was kindly supplied by Eizai 12 h. The dialyzate was applied on a Heparin–Sepharose Co., Japan, and by Professor N. Sone and Dr. J. column (1 ml bed volume per mg of protein) which had Sakamoto of the Kyushu Institute of Technology, previously been washed with 50 mM KPB, pH 6.5, respectively. FAD, FMN, deamino-NAD, and horse- containing 1 mM EGTA and 4 mM MgCl2. After the radish peroxidase were from Sigma. 5-Diethoxyphos- column was washed with 3 bed volumes of column phonyl-5-methyl-1-pyrroline N-oxide (DEPMPO) was buffer containing 0.15 M KCl, the enzyme was eluted from Oxis (Portland, OR, U.S.A.). All other materials with 3 bed volumes of column buffer containing 0.35 M were of reagent grade and were obtained from commer- KCl. The active fractions were then combined and used cial sources. for further experiments.

Bacterial strains and growth conditions. An NDH-2 Enzyme assays. All enzyme assays were performed at over-expressing strain derived from C. glutamicum 25 C. NAD(P)H oxidase activity was spectrophoto- KY971427) was used in this study. For cultivation, the metrically measured at 340 nm by following the de- strain was inoculated into Luria–Bertani (LB) medium crease in NAD(P)H concentration. The reaction mixture supplemented with 25 mg/ml kanamycin and incubated (1 ml) contained an appropriate amount of enzyme, at 30 C under 200 rpm shaking overnight. The LB- 0.2 mM NAD(P)H, and 50 mM KPB or Na–acetate grown cells were then transferred with the 1% inoculum buffer. To see full enzyme activity, 20 mM FAD was into 1-liter of glucose minimum medium20) containing also added to the reaction mixture when indicated. The 25 mg/ml kanamycin in a 3-liter Erlenmeyer flask and amount of enzyme that oxidized 1 mmol of NAD(P)H grown at 30 C until the late exponential phase. per min was defined as 1 unit, where a millimolar extinction coefficient of 6.2 or 6.3 was used for the Membrane preparation. The cells were harvested by calculation of NADH or NADPH oxidase activity centrifugation and washed twice with 20 mM potassium respectively. Q1 or Q2 reductase activity was spectro- phosphate buffer (KPB), pH 7.5. The washed cells were photometrically measured by following the decrease in resuspended in the same buffer at 2.5 ml per g wet cells absorbance at 340 nm in a 1 ml reaction mixture in the presence of 0.5 mg/ml of lysozyme and then consisting of an appropriate amount of enzyme, incubated at 30 C under 80 rpm rotation for 1 h. Then 0.2 mM NAD(P)H, 50 mM Q1 or 30 mM Q2, and 50 mM the lysozyme-treated cells were disrupted by passing KPB or Na–acetate buffer. A unit of activity was defined them twice through a French pressure cell press at as 1 mmol of NAD(P)H oxidized per min, calculated 16,000 psi, and the cell debris was removed by centri- using a millimolar extinction coefficient of 6.81. fugation at 6,000 rpm for 20 min at 4 C. The super- Ferricyanide (K3Fe(CN)6; FR) reductase activity was natant was ultracentrifuged at 40,000 rpm for 90 min at spectrophotometrically measured by following the de- 4 C to separate the soluble fraction from the precipitate. crease in absorbance at 420 nm. The reaction mixture The membrane precipitated was resuspended with consisted of an appropriate amount of enzyme, 0.2 mM 0 50 mM KPB, pH 6.5, containing 1 mM O,O -Bis<2- NAD(P)H, 1 mM K3Fe(CN)6, and 50 mM KPB. A unit of 0 0 aminoethyl>ethyleneglycol-N,N,N -N -tetra acetic acid activity was defined as 2 mmol of K3Fe(CN)6 reduced Type II NADH Dehydrogenase of Corynebacterium glutamicum 151 per min (equivalent to 1 mmol of NAD(P)H oxidized), 1.0 Gauss. A flat quartz cell (JEOL LC11, inner volume where a millimolar extinction coefficient of 1 was used 60 ml) was used for the EPR measurement. The EPR for the calculation. MQ2- or menadione (MD)-depend- sample contained 1.2 mM purified NDH-2, 0.2 mM FAD, ent FR oxidoreductase activity was also spectrophoto- 1mM NAD(P)H, and 45 mM DEPMPO in 50 mM KPB, metrically measured by following the decrease in pH 6.5, or 50 mM Na–acetate buffer, pH 5.5. The sample absorbance at 420 nm. The reaction mixture consisted was incubated for 15 min at room temperature. Hydroxyl of an appropriate amount of enzyme, 0.2 mM NADH, radical ( OH) and superoxide radical (O2 ) can be 1mM K3Fe(CN)6,30mM MQ2 or 50 mM MD, and 50 mM trapped and stabilized by DEPMPO, and the adducts KPB or Na–acetate buffer. A unit of activity was defined show different characteristics of the EPR spectrum.29) as 2 mmol of K3Fe(CN)6 reduced per min, and FR reduction activity without MQ2 or MD was subtracted to SDS–PAGE analysis. Sodium dodecyl sulfate–poly- lead MQ2- or MD-dependent FR reductase activity. Km acrylamide gel electrophoresis (SDS–PAGE) was per- 30) and Vmax values were determined from a Hofstee plot of formed according to the method of Laemmli followed several electron acceptors, oxygen, Q1,Q2, FR, MD, and by staining with Coomassie brilliant blue R250. The MQ2, for NDH-2 in the presence of NADH or NADPH protein standard marker used consisted of phospho- as an electron donor. rylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor Detection of superoxide and hydrogen peroxide. (21.1 kDa), and lysozyme (14.4 kDa). Production of the superoxide radical was quantitatively determined by the reduction of cytochrome c.The Protein determination. The concentration of protein reaction was performed in an appropriate buffer along was determined by a modified Lowry method,31) and with 0.2 mM NAD(P)H, 30 mM horse heart cytochrome c, bovine serum albumin was used as a standard. 2mM EDTA, and purified NDH-2 with a total volume of 1 ml. Cytochrome c reduction was monitored at 550 nm Results at 25 C, where a millimolar extinction coefficient of 19.0 was used. The production of hydrogen peroxide by Purification of NDH-2 NDH-2 was measured using a horseradish peroxidase- Although NDH-2 has previously been purified from based o-dianisidine assay.28) The initial reaction was C. glutamicum KY9714,25) the enzyme was unstable and carried out with 60 mM NAD(P)H and purified NDH-2 in recovery during purification was low, suggesting that an appropriate buffer (total volume 1 ml) by incubation FAD might have been lost. In this study, we attempted at 25 C until NAD(P)H was completely oxidized. After to purify the enzyme from an NDH-2 overexpressing the reaction, 100 to 200 ml of the initial solution was strain derived from KY9714 and tried to improve its transferred to a new 1 ml-cuvette (final volume 1 ml) recovery by adding FAD throughout the purification containing 50 mM KPB, pH 7.0, 0.15 mM o-dianisidine, steps. Purification was performed by two-step column 7.5 U horseradish peroxidase, and 0.025 mM EDTA. The chromatography using DEAE-Toyopearl and Heparin– absorbance change at 460 nm was followed, and the Sepharose, as described in ‘‘Materials and Methods’’. As hydrogen peroxide concentration of the initial solution summarized in Table 1, NADH:Q1 reductase activity was determined from a standard curve. was enriched 6.2-fold with a 15.6% yield. Enzyme purity was confirmed on SDS–PAGE, where a single Spectroscopic analyses. To remove FAD and also polypeptide of 55-kDa corresponding to NDH-2 was exchange Triton X-100 with dodecylmaltoside in the observed (Fig. 1). purified enzyme, the purified enzyme (about 1.5 mg protein) was dialyzed against 50 mM KPB, pH 6.5, and Spectral analysis of the purified enzyme then applied on a DEAE-Toyopearl column (1 ml bed To avoid interference in spectral analysis, excess volume) equilibrated with the same buffer. The column FAD supplemented during the purification was removed was washed with 10 ml of the same buffer containing by dialysis and Triron X-100 was exchanged with 0.1% dodecylmaltoside, and the enzyme was eluted with dodecylmaltoside, as described in ‘‘Materials and Meth- the same buffer containing 0.1% dodecylmaltoside and ods’’. The purified enzyme thus prepared exhibited an 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 Table 1. Summary of NDH-2 Purification (Hitachi 557) at 25 C. Fluoresence spectra of purified Total Specific Total Purification Yield NDH-2 were recorded at 25 C with a Hitachi 650-10S protein activity activity steps (%) fluorescence spectrophotometer. (mg) (U/mg) (U) EPR spectra were recorded on a Bruker ELEXSYS E Membrane 280 44.0 12,320 100 500 spectrometer operating at 9.85 GHz at room temper- Triton supernatant 92.8 105 9,785 79.4 ature. The EPR conditions were typically a microwave DEAE-Toyopearl 25.2 128 3,226 26.2 power of 10 milliwatts and a modulation amplitude of Heparin–Sepharose 7.02 273 1,917 15.6 152 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.

finding that NADH:Q1 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 fluo- rescence 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), sug- gesting that NDH-2 of C. glutamicum contains a non- covalently bound FAD. Fig. 2. UV–Visible Spectrum of Purified NDH-2 (4 mM)ofC. glu- Although, since NDH-2 of E. coli has been shown to tamicum in the Presence of 0.1% Dodecyl Maltoside. contain copper (Cu(I)),19) luminescence spectroscopy The purified enzyme was treated as described in ‘‘Materials and was performed with the purified enzyme, no bands Methods’’ before this measurement. The oxidized (curve 1) and dithionite-reduced (curve 2) spectra are shown. corresponding to Cu(I) were observed (data not shown). In addition, metal analysis using induced-coupled plasma atomic emission spectrometery indicated that absorption spectrum with a maximum at 273 nm and a purified NDH-2 did not contain any copper (data not broad shoulder between 350450 nm (Fig. 2). The peak shown). around 450 nm was reduced by the addition of dithion- ite, indicating the presence of a typical flavin cofactor, Kinetic properties of the purified enzyme but the flavin content estimated from the spectrum might C. glutamicum NDH-2 oxidizes NADPH at acidic pH have been underestimated because a large portion of with or without Q1 in addition to oxidizing NADH with 24) flavin was detached during dialysis, as can be seen in the Q1 at neutral pH. In this study, we examined both 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 Table 2. Kinetic Parameters of NAD(P)H for Purified NDH-2 electron acceptors in more detail. NADH oxidation coupled with various artificial electron acceptors NADH NADPH Substrate exhibited maximal activity at neutral pH between 6 Km (NADH) Vmax Km (NADPH) Vmax and 7, whereas NADPH oxidation had an optimum pH mM (units/mg) mM (units/mg) under acidic conditions at about pH 5 (Fig. 4). Only NAD(P)H NADH:Q1 oxidoreductase activity had two optimum oxidase <2 (2.13) 105 7.5 pHs. Therefore the kinetic properties of NDH-2 for NAD(P)H:Q1 NAD(P)H oxidation coupled with the reduction of oxidoreductase 11.6 (105) 394 101 several electron acceptors were further examined at Measured at pH 7. their optimum pH (Table 2 and 3). As expected, the Measured at pH 5. purified enzyme exhibited a higher affinity for NADH The Vmax value for NADH:Q1 reductase is not accurate due to the relatively low Q1 concentration (50 mM) used for this assay. than for NADPH when oxygen or Q1 was used as the electron acceptor, although the Km for NADH with oxygen could not be determined due to a low value of reductase activity was measured as FR reduction activity less than 2 mM (Table 2). via MQ2, in which the final electron acceptor was The highest affinity for the electron acceptor of the required to maintain the higher activity. The MQ analog purified enzyme was observed with MQ2 coupling for MD was reduced by the enzyme even with NADPH, the oxidation of NADH, while the reduction of MQ2 was although the affinity and activity were lower than those not observed with NADPH oxidation (Table 3). MQ2 for NADH (Table 3). A higher affinity for the artificial 154 N. NANTAPONG et al. Table 3. Kinetic Parameters for Electron Acceptors of Purified donate electrons to menaquinone or ubiquinone, sensi- NDH-2 tivity to several quinone inhibitors was examined to get As described in ‘‘Materials and Methods’’, all the activities were measured by following the decrease in absorbance at 340 nm, except more insight into the reactivity of the enzyme towards for NAD(P)H:FR, :MD and :MQ2 oxidoreductase activities, in which quinone and oxygen. First, various different inhibitors FR reduction was followed without and with MD or MQ2 respectively. were used to determine whether they inhibited NAD(P)H was used at a concentration of 400 mM in this experiment. NAD(P)H:quinone reductase activity. As for the result, the reduction of quinones with purified NDH-2 NADH NADPH was shown to be inhibited only by antimycin A (data Substrate Km (acceptor) Vmax Km (acceptor) Vmax not shown). As shown in Fig. 5, NADH:MQ and mM (units/mg) mM (units/mg) NAD(P)H:Q2 reductase activities were intensively in- NAD(P)H:Q1 hibited with 25 mM antimycin A, but NAD(P)H oxidase oxidoreductase 41 256 6.6 (37.7) activities were not. This result also indicates that NAD(P)H:Q2 oxidoreductase 32 841 5.5 (69.0) NADPH:Q2 reductase activity was slightly more sensi- NAD(P)H:FR tive to antimycin A than was NADH reductase. oxidoreductase 133 23.8 36 (34.3) NAD(P)H:MD Production of superoxide and hydrogen peroxide by oxidoreductase 31 284 86 (118) purified NDH-2 NAD(P)H:MQ2 oxidoreductase 5.0 81.5 ND The NADH oxidase system of C. glutamicum can produce superoxide anions.20) Moreover, type II NADH Measured at pH 7 except for FR reductase activity, which was measured at pH 5. dehydrogenase (rotenone-insensitive NADH dehydro- 33) 34) Measured at pH 5. genase) of Trypanosoma brucei and S. cerevisiae The Vmax values of these activities are not accurate because the NADPH produce superoxide in the presence of NADH. There- concentration used was not saturated. ND, not detected. fore, in this study, superoxide generation dependent on NADH or NADPH oxidation was examined using the purified NDH-2 of C. glutamicum. The production of electron acceptors, ubiquinone analog (Q1 or Q2) and superoxide was quantitatively determined by following FR, was observed for NADPH oxidation rather than for the reduction of horse heart cytochrome c at various pH NADH. It should be noted that purified NDH-2 did not conditions and comparing this with the NAD(P)H react with deamino-NADH (100 mM) as in the case of oxidase activity of the enzyme (Table 4). Superoxide E. coli enzyme (less than 1% of NADH) and also that it production was observed at pHs 5 and 6 with NADPH could not be inhibited with 10 mM rotenone. as the substrate, and at pH 5 to 8 with NADH. At the higher pH, it was higher when compared with O2 Antimycin A inhibition upon the oxidation of uptake. The addition of FAD increased superoxide NAD(P)H by purified NDH-2 formation as well as O2 uptake with NADPH, but not Since NDH-2 of C. glutamicum has an ability to with NADH (data not shown).

Fig. 5. Antimycin A Inhibition of NAD(P)H Oxidase, and NAD(P)H:Q2 and NADH:MQ2 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:Q2 reductase; , NADPH:Q2 reductase; , NADH:MQ2 reductase. Type II NADH Dehydrogenase of Corynebacterium glutamicum 155 Table 4. NAD(P)H-Dependent Superoxide (O2 ) and Hydrogen Peroxide (H2O2) Formation by NDH-2

Oxidase Oxidase activity O formation H O for- pH 2 2 2 system U/mg U/mg (%) mation (%) NADPH 4 4.5 0 (0) — 5 7.1 1.1 (8) 82 6 3.9 1.4 (18) 57 NADH 5 1.3 0.05 (1.8) — 6 1.5 0.40 (14) — 7 2.5 0.69 (14) 87 8 1.3 0.61 (24) —

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 super- oxide was determined by the formation of DEPMPO- adduct under aerobic conditions. The EPR signal with N H hyperfine coupling constants of a ¼ 13:0, a ¼ 11:1, aP 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 Fig. 6. EPR Determination of Superoxide Radical Produced by may be produced from superoxide at least at pH 5 with Purified NDH-2. NADPH, because superoxide generation was not very 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, high in spite of being expected to be very high by EPR and 45 mM DEPMPO in 50 mM Na–acetate buffer, pH 5.5, (A and C) analysis under these conditions. or in 50 mM KPB, pH 6.5, (B and D).

Discussion cation, NDH-2 was enriched 6.2-fold with 15% yield Recently, NDH-2 was purified from a lysozyme- based on NADH:Q1 reductase activity, of which the sensitive strain of C. glutamicum and shown to exhibit specific activity and recovery were enhanced by 6-fold NADPH oxidase activity in addition to NADH dehy- and 3.5-fold respectively, compared with the values drogenase activity.25) In this study, in order to character- previously reported.25) Similarly to NDH-2, found in ize this enzyme in more detail, NDH-2 of C. glutamicum other organisms, the spectrum of the purified enzyme was purified from an over-producing strain by an showed that C. glutamicum NDH-2 contains a flavin improved purification procedure in which FAD was moiety. This is consistent with the sequence information added throughout the purification process. After purifi- on this enzyme, which shows that it contains an FAD 156 N. NANTAPONG et al. binding motif. In this study, we also presented evidence natural electron acceptor menaquinone within the that C. glutamicum NDH-2 contains non-covalently membrane, while it may oxidize NADPH partly coupled bound FAD but not covalently bound FMN. with oxygen reduction. However, the results obtained Although E. coli NDH-2 contains thiolate-bound with antimycin A inhibition provided evidence that Cu(I) and is predicted to have two conserved cysteine C. glutamicum NDH-2 donates electrons from both residues,19) C. glutamicum NDH-2 does not contain Cu NADH and NADPH to quinone in the same manner, and has neither a heavy-metal-associated domain nor or at the same site, yet with different efficiencies, conserved cysteine residues in its polypeptide sequence whereas the oxygen reacting site may be different from (data not shown). NDH-2 of E. coli contains the the quinone site. The results obtained in this study also predicted transmembrane domain at the C-terminal suggest that the binding of NADPH and/or NADH region,19) and the external alternative NADH dehydro- might cause a conformational change in the enzyme genase of N. crassa also contains a transmembrane structure, which causes a change in the binding site for domain at the N-terminal region.35) On the other hand, the electron acceptor, including oxygen, or that it might NDH-2 of A. ambivalens contains 3 possible putative directly overlap the acceptor binding site so that amphipatic helices located around amino acid residues NADPH binding instead of NADH changes the binding 15–27, 181–194 and 332–347, which are also present in of the acceptors. E. coli NDH-2.36) Therefore the secondary structure of Various redox carriers of the electron transport chain C. glutamicum NDH-2 was predicted using programs are possible sources of reactive oxygen species forma- provided by the TMHMM (http://www.enzim.hu/ tion. In mammalian mitochondria, the main sites for hmmtop/) and Columbia University (http://www.cubic. superoxide and hydrogen peroxide production are bioc.columbia.edu) servers. The results indicated that NADH:quinone reductase, complex I, and ubiquinol- there is a single putative transmembrane region in the C- cytochrome c reductase. Type II NADH dehydrogenase terminal region (around amino acid residues 384–439), of E. coli,28) T. brucei33) or S. cerevisiae34) is also a and also in similar possible amphipatic regions (residues source of endogenous superoxide production. Our study 179–196, 329–346 and 442–459). Thus we cannot also indicates that NDH-2 of C. glutamicum is a provide strong evidence to show how this enzyme is potential source of superoxide and hydrogen peroxide located on the membrane of C. glutamicum. formation. The oxidation of NADH and NADPH with When the oxidation of NAD(P)H was examined with oxygen, especially NADPH oxidation, with NDH-2 led various electron acceptors, reduction of MD, Q1,Q2, to the formation of superoxide and hydrogen peroxide. and FR was observed with both NADH and NADPH, Since, unlike E. coli enyzme, NDH-2 of C. glutamicum whereas reduction of MQ2 was observed only with has the ability to oxidize NADPH in addtion to NADH, NADH. But undetectable NADPH–MQ2 reductase it is possible that superoxide was generated from activity does not necessarily imply that this enzyme NADPH oxidation with NDH-2. E. coli enzyme can cannot reduce MQ2 with NADPH. Since the oxidation also generate superoxide from NADPH, although not as of NADPH by purified enzyme is very weak compared high an amount as observed for that from C. glutamicum with NADH oxidation, it is conceivable that the (unpublished results). In contrast, a similar amount of oxidation of NADPH with MQ2 might be below the superoxide formation from NADH using NDH-2 be- detection limit in the in vitro assay. The Km and Vmax for tween C. glutamicum and E. coli detected by EPR was MQ2 reduction of C. glutamicum NDH-2 with NADH observed in our laboratory (unpublished). However, (Km ¼ 5 mM; Vmax ¼ 82 U/mg) were reasonably low and similarly to the C. glutamicum enzyme, NDH-2 of high respectively, whereas the affinity (Km ¼ 41 mM) for T. brucei and S. cerevisiae produce a relatively high ubiquinone analogue, Q1, is significantly lower than that superoxide from NAD(P)H and NADH respective- 14) 33,34) in E. coli NDH-2 (Km ¼ 5:9 mM). Also the Km values ly. for Q1,Q2, and FR reduction due to NADH oxidation NDH-2 of C. glutamicum exhibits a unique character- were higher than those due to NADPH oxidation, and istic, NADPH oxidation, that occurs more often in thus these artificial electron acceptors are more favor- eukaryotic enzymes than in prokaryotic ones. The able electron acceptors for coupling with NADPH external alternative NADH dehydrogenases NDE-1 oxidation. The results suggest that menaquinone is a and NDE-2 of N. crassa oxidize NADPH at acidic pH, more favorable electron acceptor for C. glutamicum similarly to the C. glutamicum enzyme.35,37) Therefore, NDH-2 than other quinones at least for NADH oxida- a phylogenetic tree of NDH-2 from various organisms tion. Furthermore, since the purified enzyme exhibits including C. glutamicum was constructed using the reasonably higher oxidase activity when coupled with Clustal W program (Fig. 7). As expected, NDH-2 of NADPH, the physiological electron acceptor coupled Corynebacterium sp. as well as its counterpart Myco- with NADPH oxidation of C. glutamicum NDH-2 is bacterium sp. were found to be more closely related to probably oxygen, although the actual affinity for oxygen eukaryotic enzymes than to other prokaryotic ones. Thus was not determined in this study. Taken together with NDH-2 of C. glutamicum appears to have evolved from these results, it can be concluded that NDH-2 in the same ancestor as the enzymes found in yeast and C. glutamicum oxidizes NADH tightly coupled with a fungi. 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 (http://www.ebi.ac.uk/clustalw/ 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 253228 (Pseudomonas aeruginosa), AAF97237 (P. fluorescens NDH-2), NP 415627 (E. coli NDH-2), NP 460181 (Salmonella typhimurium NDH-2), BAC59234 (Vibrio parahaemolyticus), NP 231524 (V. cholerae), NP 438906 (Haemophilus influenzae), AAD56918 (Zymomonas mobilis), BAB07126 (B. halo- dulans), CAD33806 (Acidianus ambivalens), NP 344274 (Sulfolobus solfataricus), NP 600682 (C. glutamicum), NP 738203 (C. efficiens), NP 939574 (C. diphtheriae), NP 216370 (Mycobacterium tuberculosis), NP 855537 (M. bovis), AAC46302 (M. smegmatis), CAB77710 (Candida albicans), NP 013865 (S. cerevisiae NDE-1), NP 010198 (S. cerevisiae NDE-2), XP 331372 (N. crassa NDE-2), CAA07265 (Yarrowia lipolytica NDH-2), CAA43787 (S. cerevisiae NDI-1), EAA32649 (N. crassa NDE-1), XP 322239 (N. crassa NDI-1), AAM95239 (T. brucei), NP 441103 (Synechocystis sp.) and NP 488134 (Nostoc sp.).

NDH-2 of C. glutamicum donates electrons to mem- producing bypass electron transfer routes originally branous menaquinone at least when it reacts with considered to be the bypass oxidase system in the NADH, while it might preferentially transfer electrons respiratory chain of C. glutamicum.20,27) to oxygen with NADPH concomitant with the produc- Our knowledge of the function of NDH-2 in C. glu- tion of superoxide or hydrogen peroxide. C. glutamicum tamicum and other organisms is still limited. However, cells contain a relatively high amount of superoxide our study together with others will help us to understand dismutase as well as catalase, and the superoxide the biology of the utilization of NAD(P)H within the dismutase appears to be localized in both the cytoplasm cell. and the periplasm (unpublished data). 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