BIOCHEMICAL

Vol. 82, No. 2, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

May 30,1978

Pages

727-730

ATP SYNTHETASE ASSOCIATED WITH THE NITROCENASE OF AZOTOBACTER VINELANDII Lucille Department Yale Rece.ived

April

S. Morowitz of Molecular University,

11,

and Harold Biophysics New Haven,

J. Morowitz

and Biochemistry Conn. 06520

1978

SUMMARY: Preparations of nitrogenase from Azotobacter vinelandii show an ATP synthetase activity when incubated in the presence of ADP, phosphate, ammonium chloride and an oxidizing agent. The synthesis is linked to an oxidation-reduction and the activity parallels nitrogenase activity through purification and in a step gradient sedimentation. The reductive dephosphorylation of nitrogen fixation may possibly be reversed to yield an oxidative phosphorylation. All nitrogen the

nitrogenase fixation

reaction

tion.

or other

agent

or C,H,.

laboratory such as sodium

ATP formation

as well

tion

we included

hexokinase

ADP from ATP formed

genase

showed

This

synthesis

complex

that

in solution

reaction

consists

reported

is is

ability redox involved

paper

in the

to form

system.

All

as to whethphosphoryla-

the enzyme with substrate

ATP,

the enzyme with To assay direc-

6-phosphate

and rege-

preparations under

to be catalyzed

a

such as N,

the appropriate

glucose active

in

we may regard

oxidative

we mixed in

ATP synthesis

and appears

in nitrogen

ATP (1)

as an oxidant.

the reaction

and glucose

linked

phase

and ferricyanide

to catalyze

context

of mixing

in this

for

the question

and an oxidized

as to drive

nerate

the

In this

backward

ammonium chloride,

for

requirement

and raise

dithionite

In the experiments

ADP, phosphate,

activity.

dephosphorylation

can be driven

The usual

show an absolute

reductive

as reductive

er the system reducing

preparations

these

of nitroconditions.

by the

same enzyme

fixation.

MATERIALS

AND METHODS

The organism used was Azotabacter vinelandii (ATCC $113705). The cells were grown under vigorous aeration in a medium consisting of 0.2 g KHaPO,, 0.0025 g NaaMoO, l 2H,O, 0.3 g FeCl,, 0.045 g CaCl, (Anhydrous), 0.2 g MgSO,, 0.8 g KeHPO,, 20 g sucrose and 1000 ml HaO. This method of enzyme purification was essentially that described by Burns and Hardy (2) and involves: cell removal of cell fragments by centrifugation, prebreakage in a French press, cipitation of nucleic acids by protamine sulfate, heat treatment and centrifugation to remove denatured proteins, precipitation of nitrogenase with protamine sulfate and resolubilization. The supernatant from the heat inactivation stage is designated A60 and the resolubilized protamine sulfate precipitate is designated PS-2. These two fractions were used in this study. Assays 'Abbreviations aminoethane

are

carried

used: Sulfonic

out

in TES buffer.l

TES buffer, 0.05 Acid, BSA, bovine

Nitrogenase

activities

mol N-tris [Hydroxymethyl] serum albumin.

are

deter-

methyl-2-

0006-291X/78/0822-0727$01.00/0 727

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved

BIOCHEMICAL

Vol. 82, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mined by the reduction of acetylene to ethylene. ATP generator solution consists of 5 mg BSA, 27.6 mg ATP, 63.7 mg phosphocreatine, 1 mg creatine phosphokinase (Sigma from rabbit muscle), 1.5 ml TES buffer, 0.5 ml of 0.1 mol in TES buffer. To a 5 ml serum bottle are added 0.2 ml of ATP generator tic12 solution and 0.4 ml of TES buffer. The bottle is sealed, evacuated, and filled The reacto 0.8 atmospheres with argon and 0.2 atmospheres with acetylene. tion is initiated by adding 0.2 ml of enzyme solution and 0.2 ml of sodium dithionite solution (174 mg of sodium dithionite dissolved in 10 ml of TES satThe bottle is placed ina30°C shaking water bath for 30 minurated with Ha). utes following which the reaction is terminated by the addition of 0.3 ml of Samples of the gas phase are then examined chromatographically for 5N HaSO,. the ratio of ethylene to acelylene. We used a l/8 inch x 6 foot column of The ATP synthetase assay uses the following solutions: carbosieve B (Supelco). Solution 20 10 10 0.9 0.1

A

Solution

mg hexokinase (Sigma type mg glucose mg KaHPO, ml TES buffer ml 0.1 mol MgCl, in TES

IV from yeast)

15 mg potassium 10 mg NH,Cl 7.5 mg ADP 1 ml TES 5 ucurie [""P]

B ferricyanide

phosphate

Assays are performed by first mixing 0.2 ml of solution A, 0.2 ml of solution B, and 0.4 ml of TES in a 5 ml serum bottle which is then sealed, evacuated, and filled to one atmosphere with argon. The reaction is initiated with the addition of 0.2 ml of enzyme solution. After 30 minutes at 3O'C, 3.8 ml of 0.19 M tricholoracetic acid are added and the mixture is centrifuged at 5000 rpm for ten minutes. Samples from the supernatant are then analyzed for the ratio of [32 P] glucose 6-phosphate to c3"P] phosphate using the method of Watkins and Lehninger (3). Protein determinations are carried out with the biuret reaction. RESULTS AND DISCUSSION To establish PS-2 preparation per minute vity

per

mg of protein.

as one nanomole

was run without for

the ATP synthetase activity, the assay and showed an activity of 7.2 nanomoles

ten minutes.

terial treated

less

enzyme

heat

fixed behavior

were

The next is normally

consistent step agent

showed

treated while

at

with

units.

activity

The net are

ma-

to the unwas due A A60 of 1.59 units

synthesis

shown

100°C

the heated

compared

an ATP synthetase

and the results

(1.4 0.137

was to test present

had no activity

of the ATP synthesis

of 36 activity

of protein with

enzyme heat

This established that the phosphorylation catalyst associated with the nitrogenase.

labile

concentration

as compared

The oxidizing nite

two controls

one percent

activity

of protein contration

minutes

ADP, and with

was then made which

and a nitrogenase function

than

preparation.

to an ADP linked preparation

without

The first

showed

(We will define a unit of ATP synthetase actiper mg of protein.) For controls the assay

per minute

enzyme,

was carried out on a of ATP synthesized

was run

in Table

I.

mg),

0.260 pmol ATP were synthesized in 60 in 30 minutes. The time and concentration an enzyme-catalyzed reaction. umol if

the phosphorylation

in the assay

used as the reducing

solution

agent

728

in

was indeed is

ferricyanide

the forward

reaction.

as

At a

redox

linked.

while

dithioVarious

Vol. 82, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE I ATP synthesis Protein

mixtures

linked

nitrogenase ometry

which

operating

the preparation

the

of some material

that

stage

the

we did

reduction

with

were

shown in Table

idea

At this

and ferricyanide

in sample

in 30 min urn01 umol urn01 umol

dithonite

are

supports

in reverse.

of ATP formation

groups

and sodium

The results

further

of protein

ATP produced .133 .286 .337 .518

ferricyanide

on ATP synthesis.

redox

of amount

per sample 1.4 mg 2.8 mg 5.6 mg 8.4 mg

of potassium

the effect is

as a function

to ascertain The reaction

synthetase not

because

uncharacterized

used II.

may be the

pursue

the stoichi-

of the presence

oxidizing

in

and reducing

(4). The further

similarity

of an enzyme solution gradient

of the

over

two activities

3 ml of TES buffer

was made up in a cellulose

rpm for

7 hours

in 5 fractions

in a swinging each of which

shown

in the

plexes

while

The activity

the

at sample

ratio

is

(2)

that

observed

for

the nitrogenase

the variation.

a sigmoidal

equilibrium,

ferent

steps.

for

both

somewhat

across

assay reaction

the forward In any case

and this

sequence

and reverse the figure

The results

is

It

of this

pellet. has been

nature

in part, operating

may be rate-limited the

are com-

frequently

be responsible,

reactions

demonstrates

nitrogenase

gradient.

curve

might

collected

some of which

the

step

and spun at 40,000

activities.

due to aggregates,

2 ml

This

was then

made up of single

activity-concentration

In a complex

from

1 is

tube

The gradient

peak is

seen to vary

reported

centrifuge

head.

was assayed

The central

by layering

made up in 33% D20.

nitrate

bucket

figure. rise

was tested

close

for far

by dif-

association

of the

two activities. One clear a soluble

result

enzyme

which

system

emerges

of Azotobacter

of ATP from ADP and phosphate The synthetase

parallels

precipitation,

its

tion

behavior

synthetase indicate fixation,

in

resistance gradient.

the hydrolysis

tase

of oxidative

tive

dephosphorylation

the presence

which

in It

phosphorylation. involves

which

behavior

inactivation

enzyme being the

from

729

run

source

the action When run protons

is

the existence

catalyzes

toward

oxidizing

agent.

protamine

sulfate

to suggest

of the protons

of nitrogenase in reverse the ATP (5).

that This

in reverse.

of

the synthesis

at 60°C and its

seems reasonable

of ATP is relates

experiments of a suitable

its

to thermal

may be the nitrogenase a concept

these

vinelandii

nitrogenase

on a step

that

from

sedmentathe ATP would

in nitrogen

to the ATP synthereaction of reduc-

BIOCHEMICAL

Vol. 82, No. 2, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE II ATP synthesis

as a function

of amounts

Ferricyanide 9.0 mm01 4.5 mm01 0 0

Sample 1 2 3 4

of oxidant

and reductant

Dithionite 0 8.5 mm01 17.0 mm01 34.0 mm01

ATP produced .633 umol .159 pm01 -046 Umol 0 km01

. 1250-

.E E \m IOOOE z 2 5Q) 750Jz :: ra0

-300

.A0 0 \

\

500-

-0 5 0 n

\ . .

-200

r z

0

_

=I E z : -3

-

100

0

250

‘\

I I

.A0

I

I

I

I

2

3

4

5

sample

number

Figure 1 Fractions from a step gradient are assayed for nitrogenase and ATP synthetase. Fraction 1 is at the bottom of the tube fraction 5 at the top. ATP synthetase activities shownindots and nitrogenase activity shown in open circles.

would like to thank Barbara Gillette for the ACKNOWLEDGEMENTS: The authors gas chromatograms and Donald DeFranco for help in the enzyme preparations. We wish to thank Patrick Hallenbeck and Stephen Sligar for helpful discussions, and the National Science Foundation for support. REFERENCES 1. 2. 3. 4. 5.

Burns, R.C. And Hardy, R.W.F. (1975) Nitrogen Fixation in Bacteria and Higher Plants, Springer-Verlag. Burns, R.C. and Hardy, R.W.F. (1972) in Methods in Enzymology XXIV edited by A. San Pietro, Academic Press. Watkins, C. and Lehninger, A. (1963) in Methods in Enzymology VI edited by S.P. Colowick and N.O. Kaplan, Academic Press. Bullen, W. A. and LeCompte, J.R. (1972) in Methods in Enzymology XXIV edited by A. San Pietro, Academic Press. Morowitz, H.J. (1978) Proton Semiconductors and Energy Transduction in Biological Systems, in press, Am. J. Physiol. 235 #3-.

730