Metonidazole inhibition of nitrogenase activity in Azotobacter vinelandii

354

Biochimica et Biophysica Acta 964 (1988) 354-360 Elsevier

BBA 22892

M e t r o n i d a z o l e inhibition of n i t r o g e n a s e activity in A z o t o b a c t e r vinelandii J a y B. P e t e r s o n Botany Department, Iowa State University, Ames, IA (U.S.A.) (Received 24 September 1987)

Key words: Nitrogenase; Enzyme inhibition; Metronidazole; (A. vinelandii)

Five electron acceptors were compared for their effects on respiration and nitrogenase activity in A zotobacter vinelandii. Metronidazole, menadione bisulfite, nitroblue tetrazolium, methyl viologen and benzyi viologen all inhibited nitrogenase activity. The electron acceptors did not significantly decrease oxygen uptake when nitrogenase activity was inhibited by about 40%. Concentrations of ATP, ADP and AMP were not altered by incubation with metronidazole or menadione bisulfite. Menadione bisulfite and nitroblue tetrazolium inhibited oxygen uptake by a cell fraction containing respiratory membrane particles. The fraction catalyzed the reduction of methyl viologen, henzyi viologen and nitroblue tetrazolium but metronidazole reduction was not detected. These results indicate that metronidazole is the most selective of the acceptors for low-potential processes, but during steady-state conditions other electron acceptors can he equally selective. This selectivity apparently arises from reductant production that is in excess of that needed for respiration.

Introduction Metronidazole (2-methyl-5-nitroimidazole-1ethanol) is a synthetic one-electron acceptor. Its midpoint redox potential, measured relative to 9,10-anthraquinone-2-sulfonate and duroquinone, is - 4 8 6 mV at pH 7.0 [1,2]. Metronidazole is used for treatment of infections with anaerobic bacteria [3,4]. In the absence of oxygen, two reduced metronidazole anions disproportionate and further accumulation of more electrons results in the breakdown of one metronidazole molecule ([2,5]; Fig. 1). Some of the intermediates in the degradation may be cytotoxic [5]. In the presence of oxygen, reduced metronidazole passes its electron Abbreviations: NBT, nitroblue tetrazolium; MBS, menadione sodium bisulfite; DMSO, dimethyl sulfoxide. Correspondence: J.B. Peterson, Botany Department, Iowa State University, Ames, IA 50011-1020, U.S.A.

to 02, forming superoxide [5,6] and regenerating the metronidazole (Fig. 1). These routes of metabolism may explain the toxicity to anaerobes and the resistance of aerobes. Metronidazole has been used frequently to inhibit the nitrogenase activity of bacteria [7-12]. It presumably inhibits by accepting low-potential electrons from iron-sulfur or flavoprotein electron carriers, thus preventing electron transport to nitrogenase. Concentrations of metronidazole that inhibit nitrogenase activity do not inhibit shortterm oxygen uptake in aerobes [8,10-12], suggesting that it does not affect respiration. It is therefore thought to be a selective inhibitor for lowpotential processes. Metronidazole might, however, accept an electron from the respiration electron transport chain and pass it directly to 02 , decreasing ATP synthesis and nitrogenase activity without any noticeable effect on O 2 uptake. Furthermore, it is not known whether toxic intermediates in metronidazole degradation are formed,

0304-4165/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

355

NBT

:o/:,. NBT"

M I ~

MV <-"---~

le-

~

4e-

•

~bflO;m;;t~?

4e-

4'er°bes

O~ O~

I

02 02

Aerobes

Breakdown loss toxic

of A320 products

Fig. 1. The reactions of reduced metronidazole (MI), comparing similar reactions of NBT and methyl viologen (MV).

and what effect they might have on nitrogenase activity. This s t u d y was to d e t e r m i n e w h e t h e r metronidazole is a specific inhibitor of low-potential electron transport in nitrogen-fixing A. vinelandii. Comparative studies were done with the electron acceptors menadione bisulfite, methyl viologen, benzyl viologen and nitroblue tetrazolium. Experimental

Chemicals. Metronidazole, DMSO, menadione sodium (NBT),

bisulfite (MBS), nitroblue tetrazolium N a z A T P , N a 2 A D P , N a 2 A M P , N a 3phosphoenolpyruvate, xanthine, glycylglycine, N a 2 N A D H , N a 4 N A D P H , L-malic acid, disodium succinate, defatted bovine serum albumin, buttermilk xanthine oxidase, horse heart cytochrome c, firefly luciferase/luciferin, rabbit muscle pyruvate kinase and myokinase, and sodium dodecyl sulfate were from Sigma Chemical Company, St. Louis, MO. Calcium carbide (for generating C2H2), sodium acetate, and mannitol were from Fisher Scientific Co., Fairlawn NJ. Acetaldehyde was from K o d a k Chemical Company, Rochester, N Y and was distilled before use. Methyl viologen and benzyl viologen were from B D H Chemicals Ltd., Poole, U.K. Standard laboratory gasses were purchased from a local supplier. Metronidazole stock solutions were 0.5 M in DMSO.

Organism. Azotobacter oinelandii (ATCC 13705) was cultured in B6 m e d i u m [13] without nitriloacetic acid. For whole-cell assays, 100 ml culture medium was used in 300 ml flasks and the cultures were incubated on a reciprocating shaker at 76 rpm, 30°C. The cultures were harvested during exponential growth where the A58o was between 0.36 and 0.50. At this growth stage, the dissolved oxygen tension (measured by inserting an oxygen electrode 1 cm into the shaking culture) was 1.5% of that of culture media equilibrated with air. For cell washing and assays, the medium was the same except the phosphate was increased 5-fold to 50 raM. Mannitol was omitted in the wash medium and the carbon source in the assays was as noted. Cells were harvested by centrifugation, washed three times by re-suspending and centrifuging, and finally re-suspended in a small volume. The procedures were done at 0 - 4 ° C under a stream of hydrogen. The final suspension was equilibrated with Ar and stored on ice. To prepare respiratory membrane particles, the cells were grown under similar conditions, only in 1.5 liter batches. The following procedures were at 0 - 4 ° C . Cells were washed three times with 10 m M K-PO4, p H 7.5. The final pellet was suspended with a small volume of 50 m M K-PO4, p H 7.5. The suspension was sonicated for 5 × 20 s with the small probe of a Braunsonic sonifier at 90% power, 0 - 4 ° C . The electron transport fraction (R-3 fraction, see Ref. 14) was prepared by differential centrifugation. The disrupted cells were centrifuged for 30 min at 27 000 × g. The supernatant was then centrifuged for 2 h at 144000 × g. The resulting pellet was rinsed twice with small volumes of 50 m M K-PO 4, p H 7.5. It was then resuspended with the same buffer and stored at 0 - 4 ° C until used. Assays. Nitrogenase (C2H2) activity and longterm oxygen uptake by whole cells were measured simultaneously. The assays were performed in stoppered 37 ml serum vials with 3 ml bacterial suspension on a gyratory shaker. The shaker rate was 150 rpm and the temperature 23°C. Except where noted, the initial gas phase was 88% Ar, 2% 02, 10% C2I"I2, and the carbon source was 2 m M acetate. Inhibitors were added at 1 h, when acetylene reduction rates had reached or were close to m a x i m u m (Fig. 2). Oxygen use in these

356

0

-62 E

E)

o

0 ¢¢.

I

0... "1-

0

0

I

2 TIME

3

4

(h)

Fig. 2. The effects of oxygen concentration and metronidazole on acetate-supported nitrogenase activity in A. vinelandii. The initial oxygen concentrations were: II, 1%; O, 2%; v, 4%. Assay with 2% 02 and 2 m M metronidazole: O. The assays contained 194 btg bacterial protein in 3 ml. Values are averages of four replicates. After the 30 min measurements, all standard errors were less than 6% of the averages. D M S O by itself had no detectable effect on nitrogenase activity when added in place of the metronidazole/DMSO.

assays was measured with gas chromatography by injecting 1 ml of the gas phase onto a MS5A column equipped with a thermal conductivity detector. The column was run at 50 ° C with Ar as carrier gas. The gasses (N 2, 02, H2) were separated and the peak height of 02 was proportional to its concentration over the range 0.2-2%. Acetylene, ethylene and CO2 were not detected exiting from the column during the time required for the entire experiment. Adenylate levels were measured essentially as described for A. oinelandii by Upchurch and Mortenson [15]. The nitrogen fixation assays were terminated by injecting 0.6 ml cold 35% HC103 while the vials were shaking. Adenylates in each extracted sample were measured three times with the luciferin/luciferase enzyme system in a scintillation counter. Enzymatic conversions of A D P and A M P to ATP were as described [15] and corrections were made for percent conversion and recovery of adenylates. Reduction of metronidazole was measured as the decrease in absorption at 320 n m due to destruction of the parent compound [2,16]. For whole-cell experiments, assays were terminated by

centrifuging the cell suspensions for 10 min at 17000 × g at room temperature, exposed to the air. The supernatant was removed, and the /1320 measured. Comparative studies were done with nitroblue tetrazolium (NBT) as electron acceptor. Reduction of N B T was measured by accumulation of a blue formazan precipitate. Whole-cell assays with N B T were terminated and formazan solubilized with addition of 0.3 ml 22% SDS. After mixing, the vials were placed in a 45 o C water bath for 15 rain to complete extraction and solubilization. Results are reported as absorptions, recorded at 40 o C. Oxygen uptake by membrane particles was measured with a Clark-type 02 electrode at 23 ° C. Reduction of electron acceptors by the particles was measured anaerobically using purified Ar [11] or H 2 as gas phase. Reduction of viologen dyes was measured following the increase in /1602 in an anaerobic cuvette. The buffer was 50 m M K - P O 4, p H 7.5. The reduction of metronidazole by xanthine oxidase was measured by a modification of the assays described for xanthine-oxidase-catalyzed superoxide production [17] and metronidazole reduction [3]. The buffer was 50 m M K-PO 4 containing 0.1 m M EDTA, p H 7.8. The xanthine oxidase was dialyzed overnight at 0 - 4 ° C against this buffer before use. Reducing substrates were either 0.5 m M xanthine or 2.0 m M acetaldehyde. The other conditions were as described above for particle-catalyzed metronidazole reduction except that 0.1 m M metronidazole was used. Comparative studies measuring xanthine-oxidase-catalyzed 02 reduction to superoxide were performed as described [17] with 1.25 m M cytochrome c to detect superoxide production. Proteins were measured by the Lowry method [18] after trichloroacetic acid precipitation. Bovine serum albumin was the protein standard. Results

Nitrogenase activity and long-term oxygen uptake in intact A. vinelandii cells were studied in experiments to observe the effects of the electron acceptors (Table I). The assays were in stoppered serum vials with an initial oxygen concentration of 2% (Fig. 2). The ratios of oxygen uptake to C2H 2

357 TABLE 1 T H E EFFECTS OF ELECTRON ACCEPTORS ON N I T R O G E N A S E ACTIVITY (C2H2) A N D RESPIRATION (02 UPTAKE) BY A. V I N E L A N D H CELLS All values are means of four replicates. Assays contained 155-180 #g bacterial protein per 3 ml. n.d., not determined. Experiment

nmol C 2 H 4 produced a

/tmol 0 2 taken up

(treatment)

1~ 2 h

2~ 3h

1 --, 2 h

2--, 3 h

Control 2 mM metronidazole

679 451

809 306

4.50 4.65

4.48 3.94

Control 5 mM MBS

667 438

755 355

4.72 4.57

4.16 4.45

Control 0.1 mM methyl viologen 1.0 mM methyl viologen 0.1 mM benzyl viologen

639 344 12 13

743 255 5 5

2.92 2.78 2.10 n.d.

4.16 3.62 2.34 n.d.

Control 0.1 mM NBT

574 357

865 247

4.14 3.81

3.82 1.88

" Ethylene was measured at 1 h and 3 h. The 2 h data were estimates made from separate experiments. TABLE III

reduced (calculated from data in Table I) range from 4.6:1 to 7.2:1 for the controls during the second hour of assay. These values are slightly lower than those reported with another strain of A. vinelandii in oxygen-limited batch cultures [19]. Concentrations of the electron acceptors were used that gave about 40% inhibition of nitrogenase during the first hour. A high concentration (1.0 mM) of methyl viologen, which almost completely inhibits nitrogenase activity, was also tested. Inhibitions increased during the second hour (Ta-

R E D U C T I O N OF M E T R O N I D A Z O L E CATALYZED BY A. V I N E L A N D H M E M B R A N E PARTICLES OR BY X A N T H I N E OXIDASE

TABLE II

Enzyme a

Metronidazole concentration (mM)

Reductant

AA320

Particles Particles Particles Particles Particles

2 2 2 2 2

1 mM N A D H 1 mM N A D P H 0.1 atm H 2 10 mM succinate 10 mM e-malate

+ 0.004 -0.001 0.000 -0.005 -0.002

Xanthine oxidase Xanthine oxidase

0.1

0.5 mM xanthine

- 0.003

0.1

2.0 mM acetaldehyde

-0.040

T H E I N H I B I T I O N OF A. V I N E L A N D H MEMBRANE PARTICLE-CATALYZED O X Y G E N UPTAKE BY VARIOUS C O M P O U N D S Assays were in duplicate. Particles were added to give a final concentration of 32 #g protein per ml. 1 mM N A D H was the reductant in each case. Inhibitor

0 2 uptake:

percent change 2.0 mM metronidazole DMSO control a 5.0 mM MBS 5.0 mM NaC1 control 0.1 mM methyl viologen 1.0 mM methyl viologen 0.1 mM NBT

- 13 - 2 -32 +2 - 1 +5 -98

a The same amount of DMSO as added with metronidazole.

Protein concentrations were 99 # g / m l for A. vinelandii particles and 9 2 / ~ g / m l for xanthine oxidase. Incubations were for 1 h. With 2 mM metronidazole, samples were diluted 1:20 before the A320 measurement. When NAD(P)H was the reducing substrate, the reaction mixture was exposed to air and shaken for 1 h, to oxidize the NAD(P)H prior to the absorption measurement. Controls without metronidazole were run and the A320 subtracted. Values are means of four replicates. Absorption of 0.1 mM metronidazole at 320 nm is approx. 0.890.

a Particle-catalyzed oxygen uptake rates, in # m o l . ( m g protein) -1. min-1, were: N A D H , 0.83; NADPH, 0.70; L-malate, 0.55; succinate, 0.006. Xanthine-oxidase-catalyzed oxygen reductions were measured as the A A s s o / m i n of cytochrome c reduction. The rates were 0.18 and 0.05 with xanthine and acetaldehyde, respectively. Assays contained 23 # g / m l xanthine oxidase.

358 TABLE IV R E D U C T I O N OF NBT CATALYZED BY A. V I N E L A N D H MEMBRANE PARTICLES Assays were performed in triplicate. Assay vials contained 35 ~tg/ml protein and 0.1 mM NBT. Reductant

Incubation time (min)

AA 560

None 1 mM 1 mM 1 mM 1 atm 10 mM 10 mM

20 10 20 20 20 20 20

0.004 0.688 0.863 0.519 0.035 0.230 0.142

NADH NADH NADPH H2 succinate e-malate

ble I). During the first hour of incubation, only 1.0 mM methyl viologen had a significant effect on O 2 uptake. Inhibitions of 0 2 uptake occurred during the second hour with metronidazole and NBT. The second-hour oxygen uptake rate increased with methyl viologen but it was still less than the control. Acetylene reduction activity was more sensitive to benzyl viologen than methyl viologen (Table I). The effects of metronidazole and MBS on cell

adenylate levels were determined in experiments analogous to those in Table I. Cell adenylate concentrations were not significantly different than controls 5 min and 1 h after their addition (data not presented). Inhibition of nitrogenase activity was not, therefore, a result of altered ATP or ADP levels. The electron transport fraction was examined to determine whether the electron acceptors could be acting directly on respiratory electron transport. Metronidazole had only a slight inhibitory effect on oxygen uptake but MBS and NBT were strongly inhibitory (Table II). Methyl and benzyl viologen had no detectable effect (Table II). This might be expected if they accept an electron and pass it directly to O 2 (forming superoxide), thereby supporting 02 uptake. Reduction of metronidazole, NBT, benzyl viologen, and methyl viologen can be detected by changes in their absorption. The anaerobic reduction of metronidazole catalyzed by xanthine oxidase was demonstrated (Table III). Acetaldehyde but not xanthine served as reducing substrate in the reaction (Table III), although xanthine supported 02 reduction better than acetaldehyde (footnote, Table III). Particle-catalyzed reductions were studied under anaerobic conditions.

TABLE V M E T R O N I D A Z O L E R E M A I N I N G IN THE ASSAY M E D I U M AFTER I N C U B A T I O N WITH A. V 1 N E L A N D H CELLS D U R I N G ACETYLENE R E D U C T I O N ASSAYS Values are averages of four replicates. Samples were diluted 1:20 for measurement. Incubations were for 2 h. Assays contained approx. 200 /tg bacterial protein in 3 ml. These data are from two separate experiments, one with acetate and the other with mannitol. Assay conditions carbon source

oxygen conch.

AC 2 H 4:

A 320 (metronidazole)

percent inhibition a

of diluted supernatant

2 mM acetate 2 mM acetate (expected) b No carbon, no cells

0 2% 2%

46% -

0.889+0.005 0.894_+ 0.006 (0.835) 0.903 + 0.002

2 mM mannitol 2 mM mannitol (expected) b No carbon, no cells

0 2% 2%

68% -

0.882 ± 0.006 0.882_ 0.003 (0.823) 0.888 + 0.001

a Differences between control and metronidazole-inhibited ethylene production. No ethylene production was detected with zero 0 2. b Expected absorption changes calculated assuming reductant from C2H 2 reduction entirely re-directed to metronidazole and four equivalents used per metronidazole reduced.

359 T A B L E VI A C C U M U L A T I O N OF F O R M A Z A N F R O M DUCTION IN A. V I N E L A N D I I CELLS A C E T Y L E N E R E D U C T I O N ASSAYS Values are were for 1 tained 174 acetate and

NBT REDURING

averages from three replicates_S.E. Incubations h, NBT concentration was 0.1 mM. Assays con# g and 200 /~g bacterial protein in 3 ml for the mannitol experiments, respectively.

Assay conditions carbon source

AC2H4:

Formazan a

oxygen concn, percent produced inhibition (A580)

None(endogenous) 0 2 mM acetate 0 2 mM acetate 2%

39%

0.014+0.003 0.020__+ 0.003 0.089 + 0.002

None (endogenous) 0 2 mM mannitol 0 2 mM mannitol 2%

36%

0.025 _+0.005 0.077 + 0.028 0.096 + 0.002

a Absorptions of extracted cells from identical assays without NBT were subtracted to give these values.

Metronidazole reduction could not be detected (Table III). NBT is thought to be a general dehydrogenase acceptor, and was reduced by many substrates (Table IV). A. cinelandii membrane NADH dehydrogenase can reduce viologens [20]. The reduction of 0.1 mM benzyl viologen was measured in the presence of 24/zg/ml particle protein. The A A 6 0 2 / m i n w a s 0.030 and 0.005 with 1 mM NADH and 1 mM NADPH, respectively. The corresponding rates with 0.1 mM methyl viologen as acceptor were 0.003 and 0.001. Rates were zero without NAD(P)H. Metronidazole metabolism in nitrogen-fixing cells was studied by observing the reductive loss of absorption at 320 nM. Comparative studies were done with NBT, since it undergoes a similar series of internal interconversions ([21]; Fig. 1). The two compounds differ, however, in that metronidazole breakdown requires anaerobic conditions [2,5] whereas formazan production occurs in the presence of 02 (Table II). The results (Tables V and VI) showed that reductive loss of metronidazole was not observed whereas the internal conversion of reduced NBT to formazan was observed. The experiments were also performed with mannitol as carbon substrate, since it gave high levels of NBT

reduction (Table VI). Little or no metronidazole reduction was detected (Table V). Under anaerobic conditions, no reductive degradation of metronidazole was observed (Table V). Discussion

Metronidazole and other electron acceptors inhibited nitrogenase activity in A. vinelandii cells. They had little or no detectable effect on oxygen uptake or (for metronidazole and MBS) cell adenylate levels during the first hour of incubation. Concentrations of the electron acceptors were used that inhibited nitrogenase activity by about 40%. By these criteria, it appears that electron acceptors with a range of midpoint redox potentials can be 'specific' for low-potential electron transport. The available midpoint redox potentials (pH 7.0) are: metronidazole, -486 mV [1,2]; methyl viologen, -440 mV [22]; benzyl viologen, - 3 6 0 mV [22]; menadione (parent compound of MBS), - 5 mV [23]. The high reactivity of the NBT with respiratory electron transport indicates a relatively high redox potential. The inhibition of oxygen uptake by several of the acceptors during the second hour of incubation, and by high levels of methyl viologen that completely inhibit nitrogenase activity, indicates that the selective inhibition of low-potential processes depends on the experimental conditions. The 'selectivity' of metronidazole compared to other electron acceptors was examined in vitro with the isolated respiratory electron transport particles. Particle-catalyzed acceptor reduction and effects of the acceptors on particle-catalyzed oxygen uptake were measured. The only effect of metronidazole was a slight inhibition of oxygen uptake. The other compounds had large effects on one or both of these parameters. The selectivity of the other acceptors in the physiology studies is probably due to the relative insensitivity of the respiration system to them in vivo. Under steadystate conditions there is apparently a level of reductant in excess of that needed for respiration. The inhibition of nitrogenase activity by metronidazole does not appear to arise from the formation of 'toxic products' proposed to form from reductive interconversions under anaerobic conditions [5]. If any breakdown of metronidazole

360 to toxic p r o d u c t s occurred, it was very slight. F u r t h e r m o r e , the lack of an effect o n 02 u p t a k e a n d adenylate levels indicates that the cell respiration remains normal. Like metronidazole, reduced N B T goes t h r o u g h reductive i n t e r c o n v e r s i o n s (Fig. 1). T h e p r o d u c t s are colored formazans [21,24]. I n contrast to metronidazole, the reductive i n t e r c o n v e r s i o n s were detected. This reaction of reduced N B T is app a r e n t l y resistant to oxygen, because N B T almost completely inhibits particle-catalyzed oxygen uptake. The l o n g - t e r m i n h i b i t o r y effect of N B T m a y be due to toxicity of the precipitated formazan. N B T a d d e d to growing cultures resulted in a t e r m i n a t i o n of 02 uptake in several hours (data n o t presented). These results have i m p l i c a t i o n s for m e t a b o l i s m central to n i t r o g e n fixation; particularly how the organism fixes n i t r o g e n i n the presence of oxygen. T h e nitrogenase enzymes are extremely oxygen-labile [25]. It has b e e n suggested [26] that 'switch-off' of nitrogenase activity b y exposure to high oxygen arises from diversion of electrons away from the nitrogenase enzymes. The relative insensitivity of respiration in vivo to the electron acceptors, ascribed herein to an excess of reductant, w o u l d s u p p o r t that conclusion.

Acknowledgement This work was supported b y a g r a n t from the A c h i e v e m e n t F o u n d a t i o n of Iowa State U n i v e r sity.

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