Hydrogenase system in legume nodules: A mechanism of providing nitrogenase with energy and protection from oxygen damage

Vol. 86, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

January 30, 1979

Pages

259-264

HYDROGENASE SYSTEM IN LEGUME NODULES: A MECHANISM OF PROVIDING NITROGENASE WITH ENERGY AND PROTECTION FROM OXYGEN DAMAGE Tomas

Ruiz-Ar&esoT

W.

Emerich

and Harold

J.

Laboratory for Nitrogen Fixation Research State University, Corvallis, Oregon 97331

Oregon Received

David

December

Evans

USA

8,1978

SUMMARY Some strains of rhizobia possess a hydrogenase system which the oxidation of the H2 that is evolved from nitrogenase during N2 Oxidation of H2 by a hydrogen uptake positive strain of Rhizobium provides energy for support of the N2 fixation reactions and nitrogenase from 02 damage

catalyzes fixation. japonicum protects

INTRODUCTION The been

evolution

identified

as a source

symbiosis.

Energy

legumes

was

nitrogenase possess (3-7).

of H2 from

losses

estimated

a system extent

to which

the energy

lost

provides

other

benefits

through

hydrogenase

the utilization

ATP

bacteroids.

legume

nodules

These would

from

be expected

*Present address: Departamento A gronomos, Madrid-3 (Spain).

of the nodulated

supplied

by nitrogenase

protection explain to benefit

to symbionts in nodules

hydrogenase

H2 production

with

inoculation

complex for

the oxidation

of

Increases with

here

and

established.

pea nodules.

We report

hydrogenase

results

has

a few nodulated

associated

from

(6,9).

most

has not been

synthesis

obtained

nodules

of H2 by the nodule

legumes

of soybeans

by the bacteroid H2, mediated activity and provided respiratory nodule

the H2 produced

preparations

content

has been

as H2,

via nitrogenase-dependent

bacteroid

and nitrogen

from

of the energy

energy

to nodulated

root

in the Rhizobium-legume

H2 evolution

of losing

(8) has demonstrated

H2 by cell-free yield

of inefficiency

that recycles

recovers

Dixon

The

in legume

at 20 to 40 percent

Instead

(l-4).

nitrogenase

that

in

strains

containing

the oxidation

supported

the nitrogenase

of

nitrogenase in soybean

why the hydrogenase

system

the nitrogen-fixing

process.

de Microbiologia,

in

E. T. S. de Ingenieros

0006-291X/79/020259-06$01.00/0 Copyright

259

,4//

riohtc

@ I979 nf

rrrnr-mfnr~rinn

by Academic in

Press, nn\i

fnrm

Inc. r”rorlmA

Vol. 86, No. 2, 1979

MATERIAL

BIOCHEMICAL

AND

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

METHODS

The bacteroids used in this study were prepared from nodules of 30 day-old soybean plants (Glycine max. cultivar Wilkin) inoculated with a ~--__ ~ colony derivative (DES) of -.--~ Rhizobium japonicum strain USDA 122 which was isolated in this laboratory. Nodules produced by this strain evolved little or no H2 in air and took up H2 from external sources. Nodules for control experiments were produced by inoculation with the H2-uptake negative strain USDA 11’7 of R. japonicum (3,‘7). Plants were grown, nodules removed and bacteroids prepared by the methods described by Klucas --et al. (lo), with the exception that the bacteroids were washed twice in Mg-phosphate buffer (50 mM K2HP04, 2. 5 mM MgC12, pH = 7.0) and resuspended finally in N-2-hydroxyethylpiperazine-NT-2-ethane sulfonic acid (HEPES)-Mgphosphate buffer at pH 7. 5 (50 mM HEPES, 1 mM K2HP04, 1 mM MgC12). H2 and 02 uptake rates were measured simultaneously by use of the amperometric method described by Hanus et al. (11). H2 and 02 were added to the electrode chamber (2.8 ml) as H2 and 02 saturated HEPES-Mgphosphate buffer solutions. Nitrogenase activity was determined by the rate C2H4 formation was measured on a Hewlett-Packard of C2H2 reduction. HP-5830A gas chromatograph equipped with a 1.8 m x 3. 2 mm diameter column of Porapak R. Other details of the assays are given in legends. RESULTS

AND

DISCUSSION

Washed a capacity these

for

R. japonicum 02 dependent

H2 uptake

produced

an approximate

bacteroids

respiration

above

A similar

increase

concentration

bacteroids

that

resulting

of 10 mM,

which

of both

H2 and succinate

than with

either

substrate

throughout

rates

and the absence rate

observed also C2H2

was

only

increased

of H2,

endogenous

showed of H2 by

in the rate

for

of

substrates. succinate

at a

02 uptake.

of 02 uptake

In the was higher

were

at higher

of bacteroids

or reductant

support

260

in the presence 1.

three-times utilized.

The

reduction

Addition

could

of H2 rate

and allowed

02 partial

maximal

the rate

for the maximal

to which

reduction for

of 02,

of H2 was

C2H2

bacteroids

in Figure

pressure

to function

of H2 on C2H2

122 (DES)

presented

substrates

Hp stimulated

ATP

the rate

pressures

are

Adding

in a suspension

of either

partial

02 partial

effect

the system

in the presence

in bacteroids

possible

oxidation

by adding

by USDA

the optimal

reduction.

The supply

of increasing

reduction

when

nitrogenase

reduction

of 0.1 atm

of C2H2

The

of endogenous

produced

(10 mM)

DES)

alone.

of C2H2

a series

122,

increase

oxidation

saturates

USDA

1). 3-fold

was

presence

The

(Table

from

in respiration

(strain

pressures

no H2 was

of the than

supplied.

be due to an increased

of nitrogenase

activity.

The

Vol.

86,

No.

BIOCHEMICAL

2, 1979

Table

1.

Effect

of

soybean

AND

substrates bacteroids

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

on H2 and 02 uptake

by washed

from

USDA

-R.

japonicum

122

DESa

Substrate

H2 uptake nmoles x h-l

added

02 uptake umoles x h-l

none H2,

0. 36 27 pIIl

Succinate,

1.57 10 mWI

Succinate, 10 mM H2, 27 VM

aAssays were of bacteroid concentration

0.94 0. 87

and 1.22

1.39

conducted in an electrode suspension (0. 24 mg dry of 02 was 22.1 $I.

chamber weight per

Hz

Partial

pressure

of

0,

concentratlan

containing ml). The

2. 8 ml initial

(atmi

(atm)

Fig. 1. Effect of 02 and H2 on the rates of C2H2 reduction by soybean nodule bacteroids (R. japonicum USDA 122 DES). Assays were conducted in 20 ml vials cont&iz2.4of HEPES-Mg-phosphate buffer. The gas phase consisted of 0. 1 atm C2H2, 0. 1 atm H2 as indicated and sufficient argon to obtain 1 atm. The reactions were initiated by injecting 0. 1 ml of bacteroids (5.7 mg dry weight) into each vial. Vials were incubated ina shaking bath (150 strokes/min) at 230 and 0. 5 ml gas samples were removed for C2H4 determination by gas chromatography after 15 and 30 minutes of incubation. Data presented are the averages of two replicate experiments + standard error of the mean. The inserted graph represents the maximal rates of C2H2 reduction and the optimal 02 partial pressures for maximal C2H2 reduction obtained from experiments identical to that depicted in the main graph except that H2 at the partial pressures indicated was added.

261

BIOCHEMICAL

Vol. 86, No. 2, 1979

initial

positive

response

of 02 either

with

Direct prepared

bacteroids

synthesis

(9).

have

R. leguminosarum

provided

that

were

of 02 above

atm of H2 was added,

at 0.012

atm

02.

Addition

activity

the oxidative

that

capacity

the nitrogenase the nitrogenase-inactivated

lowered

from

0. 6 to 426 nmoles these

conditions,

C2H2 bacteroid

reduction

bacteroid

rates

H2 produced

strain

from USDA

H2-dependent in the bacteroid maximal reduction Protection at a succinate

reversible.

of nitrogenase concentration

rates

of O2

pressure

were

increased

of nitrogenase

by 02

of nitrogenase pressures

of 0.1

atm H2 (Fig.

on the rate

of

of H2) was

partial

pressure

reduction

of C2H2

by the H2-uptake

decreased

increased

(Fig.

concentrations

observed

the partial

protection

increasing

formed

suspension

in

of H2 in 1,

reduction

by

negative

117.

C2H2

suspensions

at succinate was

nodules

The

the optimum

(in the absence

was

no effect

nitrogenase.

the bacteroid

reduction

was

in O2

above

When

1).

reduction

and the excess

inactivation

partial

(Fig.

an increase

into

The

with

suppressed

of C2H2

pressures

and respiratory

increased

suspensions

R. japonicum

hour.

up to a saturating

graph).

the C2H2

of C2H2

inhibited

for

also

excluded.

completely

bacteroids

therefore,

suspensions

the gas phase inserted

per

H2 oxidation

were

of the bacteroids,

0. 01 to 0. 002 atm,

Rhodopseudomonas

the rates

the rate

(18, 19).

with

substrates

the 02 input

system

02 over

atm,

protection

at 02 partial

by assuming

that

of H2 caused

respiratory

and therefore,

inactivated

under

was almost

0.1

may be explained

from

0.002

1).

ATP

obtained

has not been

by endogenous

reduction

(12) (Fig.

(13-15)

possibility

nitrogenase

supported C2H2

The

limited

in aerobically

those

algae

amounts was

of H2 increased

with

blue-green

(17).

activity

of ATP

the addition

consistent

(8),

pressures

in nitrogenase

exceeded

are

to increasing

phosphorylation

content

when

consumption decline

results

for bacteroid

and at 0. 02 atm,

maximal

oxidative

that

and Azotobacter

At partial

In contrast,

shown

bacteroids

reductant

reduction

through

reduction

that nitrogenase

of the steady-state

These

(16)

of C2H2

H2 suggests

of energy,

measurement

capsulata

in the rates

or without

by the generation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

below

at succinate from

as the succinate 2).

The

1 mM.

concentrations

02 demage,

of 50 mM.

The

however, additional

effect

concentration

of H2 was

No H2-supported higher was

than

C2H2 1 mM.

provided

respiratory

by H2

BIOCHEMICAL

Vol. 86, No. 2, 1979

-0.01 :.Fl

.f ;: z

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I I

Ok

0

I 2

Succinate

3

4

concentration

5

(log FM)

with and without H2 on the maximal rates of Fig. 2. Effect of succinate C2H2 reduction and on the optimal 02 partial pressures for maximal C2H2 A series of experiments analogous to that in Figure 1 (major reduction. graph) were conducted at different succinate concentrations. From the curves obtained the maximal rate of C2H2 reduction and the optimal 02 partial pressures for maximal rates of C2H2 reduction were determined and plotted against the succinate concentration. The assays were carried out as described in the legend of Figure 1 except that succinate was included in the HEPES-Mg-phosphate buffer as indicated an the pH was adjusted to 7. 5 with KOH. protection 50 mM

resulting succinate

increased (Table for

from

is consistent

the rate 1).

R. japonicum

metabolic

interaction

and provide on N2 fixation

a rational

by bacteroids

even

provide between basis

in nodulated

containing

the observation

functions

bacteroids results

H2 to suspensions

with

of O2 uptake

H2 obviously

These

supplying

that the addition

supplied

with

effectively

as a respiratory

at saturated

concentrations

direct

evidence

hydrogenase for expecting

for

effect

or

of Hz

succinate substrate of succinate.

the existence

and nitrogenase a beneficial

10 mM

of a

in bacteroids of hydrogenase

legumes.

ACKNOWLEDGEMENTS The authors express appreciation to Mrs. Flora Ivers for typing the manuscript. D. W, E. acknowledges the receipt of a Post-Doctoral Fellowship from the Rockefeller Foundation and T. R. A. thanks the Program of Cultural Cooperation between the United States and Spain for support. This research was supported by NSF Grant PCM 77-08784 and the Oregon Agricultural Experiment Station from which this is Paper No. 4889.

263

Vol. 86, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2.

3. 4. 5. 6. 7. 8. 9.

10. Il. 12. 13. 14. 15. 16. 17. 18. 19.

Schubert, K. R., and Evans, H. J. (1976) Proc. Nat. Acad. Sci., USA 73, 1207-1211. Evans, H. J., Ruiz-Argueso, T., Jennings, N. T., and Hanus, J. (1977) in Genetic Engineering for Nitrogen Fixation (Hollaender, A . 9 ed.) pp. 333-354, Plenum Press, New York. Carter, K. R., Jennings, N. T., Hanus, J., and Evans, H. J. (1978) Can. J. Microbial. 24, 307-311. Ruiz-Argiieso, T., Hanus, J., and Evans, H. J. (1978) Arch. Microbial. 116, 133-118. Dixon, R. O.D. (1967) Ann. Bot. N. S. 31, 179-188. Schubert, K. R. , Jennings, N. T. , and Evans, H, J. (1978) Plant Physiol. 61, 398-401. McCrae, R. E., Hanus, J., and Evans, H. J. (1978) Biochem. Biophys. Res. Commun. 80, 384-390. Dixon, R. O.D. (1972) Arch. Mikrobiol. 85, 193-201. Evans, H. J., Emerich, D. W., Ruiz-Argiieso, T., Maier, R. J., and Albrecht, S. L. (1978) in Proceedings of the Steenbock-Kettering International Symposium on Nitrogen Fixation. Madison, Wisconsin, USA, June 12-16, 1978. In press. Klucas, R. V., Koch, B. , Russell, S. A., and Evans, H. J. (1968) Plant Physiol. 43, 1906-1912. Hanus, F. J., Carter, K. R., and Evans, H. J. (1978) in Methods in Enzymology (San Pietro, A. , ed. ). In press. Appleby, C.A., Turner, G. L., and Macnicol, P.K. (1975) Biochim. Biophys. Acta 387, 461-474. Benemann, J. R., and Weare, N. M. (1974) Arch. Microbial. 101, 401-408. Bothe, H., Tennigkeit, J. , and Eisbrenner, G. (1977) Arch. Microbial. 114, 43-49. Tel-Or, E., Luijk, L.W., and Packer, L. (1977) FEBS Letters 78, 49-52. Kelley, B. C., Meyer, C. M., Gandy, C., and Vignais, P.M. (1977) FEBS Letters 81, 281-285. Walker, C. C., and Yates, M. G. (1978) Biochimie 60, 225-231. Drozd, J., and Postgate, J. R. (1970) J. Gen. Microbial. 60, 427-429. Haaker, H., Kok, A. de, and Veeger, C. (1974) Biochim. Biophys. Acta 357, 344-357.

264