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.