Báo cáo Y học: Inactivation of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus by reactive oxygen species pot

Highest Na+-NQR activity was observed in anaerobically prepared membranes that exhi-bited 1 : 1 coupling of NADH oxidation and Q reduction activities 1.6 UÆmg1.. It is shown that the [2F

Inactivation of the Na+-translocating NADH:ubiquinone

Julia Steuber1, Miche`le Rufibach1, Gu¨nter Fritz2, Frank Neese3,* and Peter Dimroth1

1

Mikrobiologisches Institut der Eidgeno¨ssischen Technischen Hochschule, ETH-Zentrum, Zu¨rich, Switzerland;

2

Biochemisches Institut, Universita¨t Zu¨rich, Switzerland;3Mathematisch-naturwissenschaftliche Sektion, Fachbereich Biologie, Universita¨t Konstanz, Germany

The Na+-translocating NADH:quinone oxidoreductase

(Na+-NQR) from Vibrio alginolyticus was inactivated by

reactive oxygen species Highest Na+-NQR activity was

observed in anaerobically prepared membranes that

exhi-bited 1 : 1 coupling of NADH oxidation and Q reduction

activities (1.6 UÆmg)1) Optical and EPR spectroscopy

documented the presence of b-type cytochromes, a [2Fe)2S]

cluster and an organic radical signal in anaerobically

pre-pared membranes from V alginolyticus It is shown that the [2Fe)2S] cluster previously assigned to the Na+-NQR ori-ginates from the succinate dehydrogenase or the related enzyme fumarate reductase

Keywords: electrochemical sodium gradient; reactive oxygen species; Vibrio; Na+ transport; NADH: quinone oxido-reductase

The marine bacterium Vibrio alginolyticus possesses a

Na+-translocating NADH:Q oxidoreductase (Na+

-NQR) that maintains an electrochemical sodium gradient

required for nutrient uptake and motility [1–5] This

respiratory sodium pump contains one Fe–S cluster,

noncovalently bound FAD, covalently bound FMN and

ubiquinone-8 as redox cofactors [2,6–8] that are likely to

participate in NADH oxidation, electron transfer, Na+

transport and Q reduction Sequence analysis of the six

subunits of the Na+-NQR from V alginolyticus (NqrA–

NqrF) showed that only the NqrF (or b-) subunit

comprises a cysteine motif (Cys69, Cys75, Cys78 and

Cys110) required for ligation of one Fe–S cluster [3,4]

Tightly bound ubisemiquinones were proposed to play a

central role during the redox-linked Na+transport by the

Na+-NQR [6,9,10] The translocation of Na+through the

protein matrix is thought to be dependent on ion-pair

formation of a positively charged Na+ cation with a

negatively charged ubisemiquinone anion generated in a

medium of low dielectric [4,10] This hypothesis was

supported by the detection of an NADH-induced radical

signal in aerobic samples of the Na+-NQR from

V alginolyticus by EPR spectroscopy [6] However, the

high concentration of the radical (13 lM) compared to the [2Fe)2S] cluster (3 lM), together with the observation that the Na+-NQR produced superoxide radicals in the presence of NADH and O2, strongly suggested that the overstoichiometric formation of radicals was due to oxidative damage of the enzyme The identification of the individual redox cofactors of coupled Na+-NQR is a prerequisite to understand the mechanism of redox-linked

Na+ transport Here we show that the Na+-NQR is already inactivated in the membrane-bound state if the cells are disrupted in the presence of dioxygen, resulting in

an uncoupling of NADH oxidation and Q reduction activities It is also demonstrated that the [2Fe)2S] cluster previously assigned to the Na+-NQR [6] originates from the succinate dehydrogenase or the related fumarate reductase

M A T E R I A L S A N D M E T H O D S Preparation of membrane vesicles for EPR spectroscopy

V alginolyticus (DSM 2171T, Braunschweig, Germany) was grown aerobically in a 300-L fermenter as described previously [6] Frozen cells (20 g) were washed with anaerobic extraction buffer (10 mMHepes/KOH, pH 7.5,

5 mM Mg2SO4, 0.2M K2SO4) and resuspended in the presence of a trace of DNAse (2–4 mL extraction buffer per

g cells) The cells were broken by a single passage through a French press at 80 MPa The crude extract eluting from the French press was collected under a stream of N2 All subsequent manipulations were carried out in the anaerobic chamber under an atmosphere of N2/H2(95/5%) 50 mM

EDTA (potassium salt) was added to the crude extract, and the membrane vesicles were sedimented by ultracentrifuga-tion Membranes were washed once in extraction buffer containing 10 mMEDTA Subsequently, complexed Mn2+ was removed by a second and a third washing step with

10 mM Hepes/KOH, pH 7.5 containing 0.2M K2SO4 EDTA treatment did not diminish the Na+-NQR activity

of the membrane vesicles that were stimulated by Na+

Correspondence to J Steuber, Mikrobiologisches Institut der

Eidgeno¨ssischen Technischen Hochschule, ETH-Zentrum,

Schmelzbergstr 7, CH-8092 Zu¨rich, Switzerland.

Fax: + 41 1 6321148, Tel.: + 41 1 6323830,

E-mail: fritz-steuber@micro.biol.ethz.ch

Abbreviations: Q1, ubiquinone-1; Na + -NQR, Na + -translocating

NADH:ubiquinone oxidoreductase; TTFA,

2-thenoyltrifluoro-acetone; MTT, 3-(4,5-dimethyl 2-thiazolyl) 2,5-diphenyl

tetrazolium bromide.

*Present address: Max-Planck Institut fu¨r Strahlenchemie,

D-45470 Mu¨lheim an der Ruhr, Germany.

Dedication: Dedicated to Achim Kro¨ger on the occasion

of his 65th birthday.

Note: A web site is available at http://www.micro.biol.ethz.ch

(Received 7 November 2001, accepted 8 January 2002)

ions by a factor of three (specific Q reductase activity in

100 mM KCl, 0.6 lmolÆmin)1Æmg)1; in 100 mM NaCl,

2.1 lmolÆmin)1Æmg)1) The membrane vesicles were

resus-pended in extraction buffer to a final protein concentration

of 42 mg mL)1 Membrane vesicles (0.3 mL) were mixed

with substrates (NADH, succinate, fumarate in H2O,

potassium salts) or inhibitor (thenoyltrifluoroacetone in

ethanol) as indicated and transferred to EPR tubes in the

anaerobic chamber

Analytical methods and spectroscopy

Na+was determined by atomic absorption spectroscopy

with a Shimadzu AA-646 spectrometer Protein was

deter-mined by the bicinchoninic acid method [11] If not

mentioned otherwise, NADH:Q oxidoreductase assays [8]

were performed at 25°C in sealed cuvettes filled in the

anaerobic chamber and flushed with N2 In order to

investigate the influence of dioxygen and NADH on the

activity of the Na+-NQR, membrane vesicles were prepared

(A) in the presence of dioxygen without additions (B) with

300 UÆmL)1superoxide dismutase and 40 UÆmL)1catalase

(C) with 10 mM pyruvate and 20 UÆmL )1L-lacate

dehy-drogenase (D) under exclusion of dioxygen without further

additions Succinate dehydrogenase activity was followed

by the reduction of 3-(4,5-dimethyl 2-thiazolyl) 2,5-diphenyl

tetrazolium bromide (MTT) at 560 nm (e560¼

12.1 mM )1Æcm)1) in the presence of phenazine methosulfate

[12] Visible spectra of membranes were recorded on a

Shimadzu UV-3000 spectrophotometer in the difference

spectrum mode X-band EPR spectra were obtained with a

Bruker ESP300 spectrometer with peripheral equipment

and data handling as previously described [13] The spectra

were simulated with the program EPR [14] Quantification

of the EPR signals was carried out by comparison with a

Cu2+standard [13]

R E S U L T S A N D D I S C U S S I O N

Inhibition and uncoupling of the Na+-NQR

activity by dioxygen

In the presence of NADH and dioxygen, the purified Na+

-NQR from V alginolyticus was inactivated with a half time

of approximately 3 min This decrease in enzymatic activity

correlated with the formation of superoxide radicals,

indicating a superoxide-mediated destruction of the enzyme

[6] We reasoned that the Na+-NQR might be already

inactivated during the aerobic disruption of the cells due to

the presence of NADH in the crude extract In order to test this hypothesis, French press cell rupture and preparation of membrane vesicles of V alginolyticus cells were performed under exclusion of dioxygen In addition, the effect of enzymes that detoxify reactive oxygen species (superoxide dismutase and catalase) on the Na+-NQR activity during aerobic cell rupture was investigated In another experiment, the concentration of NADH in the aerobically prepared crude extract was diminished by the action of lactate dehydrogenase plus pyruvate Both the NADH dehydro-genase and the ubiquinone-1 (Q1) reduction activities of membranes were followed in order to determine the degree

of uncoupling of the enzyme The term ÔuncouplingÕ describes the observation that the electrons derived from the oxidation of NADH by the Na+-NQR are not comp-letely transferred to the substrate, Q1 Instead, dioxygen seems to act as electron acceptor at an unspecified site of the enzyme to yield superoxide radicals The highest specific Q reductase activity was found in anaerobically prepared membranes that exhibited 1 : 1 coupling of NADH dehy-drogenase and Q reduction activities (1.6 lmolÆminÆmg)1; Table 1) The lowest Q reductase activity was observed in membranes prepared aerobically without any additions (0.6 lmolÆmin)1Æmg)1) Removal of superoxide or NADH

in aerobically prepared crude extracts and membranes resulted in increased Q reductase activities, but the uncoup-ling of NADH oxidation from Q reduction could not be prevented by these measures (Table 1) It is concluded that the Na+-NQR is already inactivated in the membrane-bound state if the cells are disrupted in the presence of dioxygen, resulting in an uncoupling of NADH oxidation and Q reduction activities The uncoupling of the

Na+-NQR activity in membranes by aerobic cell rupture was not restricted to the French press procedure, but was also observed with the more gentle osmotic shock protocol described by Tokuda & Unemoto, with NADH oxidation and Q reduction activities of 2.45 and 0.99 lmolÆmin)1Æmg)1, respectively [15]

Characterization of redox cofactors in membranes fromV alginolyticus

Based on the results of Table 1, the Na+-NQR is partially inactivated during cell rupture in the presence of dioxygen Superoxide radicals that are generated during the oxidation

of reduced electron carriers by O2 apparently cause this inhibition For example, the fumarate reductase from Escherichia coli produces superoxide in air [16] Optical and EPR spectroscopic investigations were performed to

Table 1 Inhibition and uncoupling of the Na + -NQR activities in membranes from V alginolyticus by dioxygen and NADH The enzymic activities of membranes were determined as described in Materials and methods with Q1 as electron acceptor in the presence of NaCl With membranes prepared under aerobic conditions, the assay was performed aerobically With anaerobically prepared membranes, the activity was determined under exclusion of air ND, not determined.

Conditions of cell rupture

NADH oxidation (lmolÆmin)1Æmg)1)

Q1 reduction (lmolÆmin)1Æmg)1)

Ratio of NADH oxidation/ Q1 reduction activity

a The high NADH oxidation activity is due to the presence of residual -lactate dehydrogenase and pyruvate.

characterize the redox enzymes present in V alginolyticus

membranes and to determine their redox state The optical

difference spectrum of washed membranes from V

algino-lyticus prepared under exclusion of dioxygen (as isolated

minus air-oxidized, Fig 1) revealed the presence of reduced

b-type cytochromes with absorption maxima at 526, 531,

557 and 561 nm No further reduction of the b-type

cytochromes upon addition of dithionite was achieved In

addition, reduction of air-oxidized membranes with NADH

or dithionite did not increase the amount of reduced b-type

cytochromes compared to membranes prepared under

exclusion of dioxygen (not shown) From their characteristic

a- and b-absorption maxima, the redox complexes succinate

dehydrogenase, or fumarate reductase (557 nm and

526 nm) [17] and the cytochrome bo-type ubiquinol oxidase

(561 nm and 531 nm) [18] were assigned Based on the

extinction coefficient of the succinate dehydrogenase from

E coli[12], the amount of b-type cytochromes associated

with succinate dehydrogenase or fumarate reductase in

V alginolyticusmembranes was 1.3 lmolÆmg protein)1 The

succinate dehydrogenase activity of membranes was

0.2 lmolÆmin)1Æmg)1 The presence of a succinate

dehy-drogenase or fumarate reductase in membranes from

V alginolyticuswas also confirmed by EPR spectroscopy

showing the typical resonances of partially reduced center I,

the [2Fe)2S] cluster of succinate dehydrogenase, or

fuma-rate reductase, with gx,y,z¼ 1.92, 1.93, 2.03 (Fig 2, traces A

and B) No further reduction was achieved upon the

addition of succinate (Fig 2, trace B) Furthermore, an

organic radical signal at g¼ 2.005 was detected Upon

addition of NADH to membranes prepared under exclusion

of dioxygen, there was a twofold increase in intensity of a

near-axial signal with resonances at gx,y¼ 1.92, 1.93 (Fig 2,

trace C) Addition of 80 mMNa+did not alter the

NADH-induced EPR signals measured at 40 K and 2 mW (Fig 2,

trace D) Note that the residual Na+ concentration of

washed membrane vesicles was 3–4 mM Addition of Na+

had no obvious effect on the NADH-induced radical signal

measured at 70 K and 4 lW (not shown) No additional

signals due to [4Fe)4S] clusters were observed at 4 K (see

below) These results are very similar to those reported for membranes from V alginolyticus prepared in air [6] In this study, the NADH-induced signal at g 1.93 was assigned

to the [2Fe)2S] cluster of the Na+-NQR, and the strong radical signal to ubisemiquinone bound to the Na+-NQR However, addition of NADH in the presence of thenoyltri-fluoroacetone (TTFA), a specific inhibitor of succinate dehydrogenase and fumarate reductase, prevented the increase in signal intensity in the g 1.93 region (Fig 3) This result clearly demonstrates that the NADH-induced

g 1.93 EPR signal observed by Pfenninger-Li et al [6] was due to the reduced center I, the [2Fe)2S] cluster in succinate dehydrogenase, or in fumarate reductase

We also investigated the EPR spectrum of the g¼ 2.005 signal at 70 K and 4 lW in the presence of TTFA and fumarate Under these conditions, the addition of NADH did not lead to any detectable effect (not shown), suggesting that the radical signal is not due to ubisemiquinone bound

to the Na+-NQR, as proposed previously [6] The radical might be associated with cofactors of other respiratory complexes, such as the flavosemiquinone in succinate dehydrogenase or fumarate reductase [17] TTFA blocks the electron transfer between the Fe–S centers of succinate dehydrogenase, or fumarate reductase, and the quinone pool Both complexes exhibit almost identical EPR spec-troscopic properties [17] As V alginolyticus is capable of anaerobic growth, the fumarate reductase is likely to be

Fig 1 Optical difference spectrum of membranes from V alginolyticus.

The spectrum shows the difference in absorbance of membranes

pre-pared under exclusion of dioxygen (Ôas isolatedÕ) minus the air-oxidized

membranes The protein concentration was 10 mgÆmL)1.

Fig 2 Electron paramagnetic resonance spectra of membranes from

V alginolyticus Trace A shows the EPR spectrum of anaerobically prepared membranes, as isolated Trace B, membranes plus 40 m M

succinate, 80 m M Na + , 360 m M K + trace C, membranes plus 8 m M

NADH, 4 m M Na + , 450 m M K + ; trace D, membranes plus

8 m M NADH, 80 m M Na+, 380 m M K+; (final concentrations) The membranes were incubated with substrates for 4–5 min prior to freezing in liquid N 2 The protein concentration was 42 mgÆmL)1 EPR parameters: microwave frequency, 9.652 GHz; microwave power,

2 mW; modulation amplitude, 1 mT; temperature 40 K.

present under limiting O2 concentrations at high cell

densities in batch culture Addition of NADH to

mem-branes shifts the redox potential towards more negative

values and increases the concentration of ubiquinol

compared to succinate-reduced membranes Higher quinol

concentrations allow the reduction of fumarate reductase

that unlike succinate dehydrogenase cannot be completely

reduced by succinate [17] As a consequence, an increase in

signal intensity of the reduced center I of fumarate

reduc-tase in NADH- compared to succinate-reduced membranes

might occur, as shown in Fig 2 By subtracting the EPR

spectrum of succinate-treated membranes from the

spec-trum obtained in the presence of NADH, center I of the

fumarate reductase (or succinate dehydrogenase) was

erro-neously assigned to the Na+-NQR [6]

The amount of center I of succinate dehydrogenase, or

fumarate reductase, in membranes from V alginolyticus

was estimated by simulation of the EPR spectrum of

NADH-treated membranes using the parameters

gx,y,z¼ 1.92, 1.935, 2.029, width (x,y,z) ¼ 1.2, 1.0, 1.2 mT

(Fig 4) These parameters are based on the EPR

spectro-scopic properties of center I, which are remarkably similar

in all succinate dehydrogenases and fumarate reductases

[17] The amount of center I (1.0 lmolÆmg protein)1) in

membranes from V alginolyticus determined by EPR

spectroscopy compares favorably with the amount of

b-type cytochromes associated with succinate dehydrogenase,

or fumarate reductase, as determined by optical

spectros-copy (1.3 lmolÆmg)1)

Detection of succinate dehydrogenase or fumarate

reductase in the partially purified Na+-NQR

The NADH-induced g 1.93 EPR signal that was

erro-neously assigned to the membrane-bound Na+-NQR was

also observed in the partially purified Na+-NQR obtained

by anionic exchange chromatography [6] This g 1.93 EPR signal found in the enriched Na+-NQR also origin-ated from reduced center I of succinate dehydrogenase, or fumarate reductase The presence of fumarate reductase in

an aliquot of the Na+-NQR preparation analysed by EPR spectroscopy [6] was confirmed by immunostaining using antiserum raised against the 66-kDa flavoprotein subunit (FrdA) of fumarate reductase from E coli [17] Figure 5 shows a Western Blot of the partially purified Na+-NQR analysed by EPR spectroscopy and membranes from E coli grown on glycerol and fumarate The antiserum raised against E coli FrdA detected a polypeptide with an apparent molecular mass of 66 kDa in the Na+-NQR and in the E coli membranes A second cross-reactive polypeptide with lower molecular mass in the E coli membranes probably represents a proteolytic fragment of the FrdA subunit [19] The stoichiometry and localization of the flavin cofactors of the Na+-NQR are a matter of debate [7,20] A contamination of the Na+-NQR with FrdA that contains a covalently bound FAD [8a-N(3)-histidyl-FAD] will result in an overestimation of covalently bound flavin in partially purified Na+-NQR preparations

A comparison of the EPR spectra of the Na+-NQR from different purification stages further supports the assumption that the NADH dehydrogenase activity and the Fe–S cluster are properties of two distinct proteins, the

Na+-NQR and the fumarate reductase or related succi-nate dehydrogenase The oxidation of NADH is catalysed

by the NqrF subunit of the Na+-NQR This subunit contains a FAD cofactor and a Cys-(X)5-Cys-(X)2-Cys motif that ligates a Fe–S cluster No additional cysteine-rich motifs that indicate the presence of Fe–S clusters are found on the remaining Nqr subunits, NqrA–NqrE [4]

An increase of the specific NADH dehydrogenase activity upon purification of the Na+-NQR is therefore expected

to be accompanied by an increase in the amount of

Fig 3 Electron paramagnetic resonance spectra of membranes from

V alginolyticus in the presence of TTFA TTFA is a specific inhibitor of

succinate dehydrogenase, or fumarate reductase, that prevents the

complete reduction of center I Upper trace, membranes plus 40 m M

fumarate, 2 m M TTFA; lower trace, membranes plus 40 m M fumarate,

2 m M TTFA, 8 m M NADH (final concentrations) The protein

concentration was 44 mg mL)1 EPR parameters as in Fig 2.

Fig 4 Experimental (upper trace) and simulated (lower trace) electron paramagnetic resonance spectrum of center I in NADH-reduced mem-branes from V alginolyticus EPR parameters: microwave frequency, 9.652 GHz; microwave power, 0.126 mW; modulation amplitude,

1 mT; temperature 4 K Simulation parameters: g x,y,z ¼ 1.92, 1.935, 2.029, width (x,y,z) ¼ 1.2, 1.0, 1.2 mT.

[2Fe)2S] cluster However, purification of the Na+-NQR

led to a loss of the Fe–S EPR signal (Table 2) In the

Na+-NQR obtained after gel filtration that exhibited the

highest NADH dehydrogenase activity, no Fe–S cluster

could be detected by EPR spectroscopy We monitored

the succinate dehydrogenase activity in fractions enriched

in NADH:quinone oxidoreductase during the purification

of the Na+-NQR according to [6] In parallel with a decrease in signal intensity of the Fe–S cluster, there was a decrease in the specific succinate dehydrogenase activity (Table 2) No succinate dehydrogenase activity was observed in the Na+-NQR purified by gel filtration These data clearly show that the Fe–S cluster observed in fractions enriched in Na+-NQR [6] is identical to center I

of fumarate reductase (or succinate dehydrogenase)

C O N C L U S I O N S

To our knowledge, this study demonstrates for the first time that a purification procedure under strict exclusion of dioxygen will be an absolute prerequisite to obtain a realistic picture of the coupling of NADH:Q oxidoreduction to

Na+ transport by the Na+-NQR from V alginolyticus The succinate dehydrogenase and/or fumarate reductase are major respiratory complexes present in V alginolyticus membranes that impede the detection of the Fe–S cluster of the Na+-NQR in the membrane-bound state by EPR spectroscopy

A C K N O W L E D G E M E N T S

This work was supported by a grant from the commission of research, ETH, to J S We thank S Albracht, University of Amsterdam, for valuable discussions, and P Kroneck, for using the EPR facilities at the University of Konstanz Antiserum against the E coli fumarate reductase flavoprotein subunit was a generous gift from G Cecchini,

VA Medical Center, San Francisco.

R E F E R E N C E S

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5 Nakayama, Y., Hayashi, M & Unemoto, T (1998) Identification

of six subunits constituting Na + -translocating NADH-quinone reductase from the marine Vibrio alginolyticus FEBS Lett 422, 240–242.

Fig 5 Detection of fumarate reductase in the Na + -NQR analysed by

EPR spectroscopy The arrow indicates the FrdA flavoprotein subunit

of fumarate reductase present in E coli membranes (10 lg) and in the

Na+-NQR (10 lg) after Q-Sepharose chromatography.

Table 2 Succinate dehydrogenase activity and amount of reduced [2Fe )2S] cluster (center I) detected in the Na + -NQR at different purification stages The Na+-NQR was purified according to [6] The nearly axial g  1.93 EPR signal of a reduced [2Fe )2S] cluster (center I of succinate dehydrogenase or fumarate reductase) present in the Na + -NQR was quantified as described previously [6] ND, not detected.

Purification step

NADH dehydrogenase activity (lmolÆmin)1Æmg)1)

Succinate dehydrogenase activity (lmolÆmin)1Æmg)1)

Amount of reduced [2Fe )2S] (l M spin concentration)

6 Pfenninger-Li, X.D., Albracht, S.P.J., Belzen, R.V & Dimroth, P.

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