The isolation of a new, low-potential, iron-containing electron-transfer protein from rumen bacteria

Vol.71,No.

1,1976

BIOCHEMICAL

THE ISOLATION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF A NEW, LOW-POTENTIAL,

IRON-CONTAINING

ELECTRON-TRANSFER

PROTEIN FROM'RUMEN BACTERIA. Roy S. Bush and Frank D. Sauer Research Institute, Agriculture Ottawa, Ontario, Canada, KlA OC6

Animal

Received

May

Canada

5,1976

SUMMARY: A new low-potential iron-containing elect,ron-transfer protein has been isolated from cell free extracts of rumen bacteria. This protein, like some bacterial ferredoxins, can 1) transfer electrons from illuminated spinach chloroplasts to NADP; 2) catalyze ferredoxin dependent reductive carboxylation reactions; 3) act as electron acceptor in the pyruvate lyase catalyzed reaction in which pyruvate is oxidized to acetyl-CoA and COP. Unlike ferredoxin, this low potential iron protein contains no acid-labile sulfide; contains tightly bound iron that is released only after prolonged hydrolysis with 6 N HCl at 10S°C and with an optical spectrum different from that associated with the bacterial ferredoxins. INTRODUCTION:

Cell

free

acetate

to pyruvate

in

system,

an electron

donor,

as ferredoxin, late but

is

activity

ferredoxins

mechanism

replace

We have

deals

(LPIP)

ferredoxin

(Pyruvate: the acid-labile

generating

system

(1,

extract

is

cell

free

shown

by the that

of the

bacteria

chloroplast

photoreducing

electron 2).

over

of.either

rumen bacteria

carrier

The ability

passed

addition

appropriate

carboxylate

to carboxy-

DEAE cellulose bacterial

can synthesize

precursor

acid

such

or plant 2-oxo-acids

by a similar

4).

report

protein

electronegative

the

carboxylation (3,

This

if

of a spinach

a strongly

can be restored

(1).

by reductive

iron

lost

of some anaerobic

the presence

and an ATP

acetate full

extracts

with

from in

ferredoxin sulfide

the isolation

cell

free

the pyruvate

low

extracts

of rumen microorganisms

synthase

assay

oxidoreductase, and iron

of a non-ferredoxin,

or in

CoA acetylating, that

is

characteristic

the pyruvate EC 1.2.7.1), of the

potential which

can

lyase but

lacks

ferredoxins

Rumen bacteria were collected from fistulated MATERIALS AND METHODS: previously (3). Bacterial Holstein cows, sedimented and washed as described pellets (50 g) were suspended in 150 ml of 50 mM potassium phosphate buffer Abbreviations:

LPIP,

low-potential

Copyright Q 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

iron

protein, 53

DPIP,

dichlorophenolindophenol.

assay

(5).

Vol. 71, No. 1,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 1. Electrophoresis of LPIP in 15% polyacrylamide gel. was continued for 1.5 h at 4 mA in 0.38 M Tris-glycine buffer, Gels were stained with 1% (w/v) Amide Black in 7% (v/v) acetic points to LPIP band.

Electrophoresis pH 8.9 (4). acid. Arrow

(pH 7.5) containing 50 mM 2-mercaptoethanol and disrupted in a Ribi Cell Fractionator (Model R7-1) Norwalk, Conn., at 27,000 p.s.i. while cooled to 2-4'C. The suspension was centrifuged first at 27,000 g for 20 min to remove then at 166,000 g for 1 h in a Spinco Ultracentrifuge heavier particles, (Model L-2 65B). The supernatant contained LPIP, pyruvate lyase and pyruvate synthase. LPIP was adsorbed on a DEAE cellulose column (2.4 x 4.5 cm) prepared as described by Mortenson et al. (6), and washed with 10 column vol. -of 0.01 M Tris buffer (pH 7.5). The active protein was eluted with a linear gradient of Tris buffer, pH 8.0 (500 ml, 0.1 to 0.7 M) and collected in 10 ml fractions. The active fractions were pooled. The pooled sample was diluted with 2 vol. of water and concentrated by passage through a DEAE cellulose column (1.0 x 7.0 cm) and eluted with 0.7 M Tris buffer, pH 8.0. LPIP was used either directly or purified further by passage through Sephadex G-75. LPIP showed as a single major band after electrophoresis in 15% polyacrylamide gel at pH 8.9 (Fig. 1). Traces of contaminating protein, present as two diffuse bands, were not removed by this procedure. Throughout the purification procedure, fractions were tested for LPIP activity in the pyruvate lyase assay described in Fig. 2. Ferredoxin was isolated from Clostridium pasteurianum grown in 20 L flasks as described in Carnahan and Castle (7). Ferredoxin was isolated essentially as described by Buchanan -et al. (8) to step 5 in their purification procedure and had O.D. 390/280 ratios of approximately 0.40.

54

Vol. 71, No. 1,1976

BIOCHEMICAL

AND BIOPHYSICAL

1 1

OL 0

I 2

I 3 Time

RESEARCH COMMUNICATIONS

I 4

I 5

Im~n)

Fig. 2. Acetyl-CoA formation from pyruvate by pyruvate lyase in the presence Each cuvette contained the and absence of a suitable electron acceptor. following (mM) in a final vol. of 1.0 ml: Tris buffer pH 8.0, 50; coenzyme A, 0.18; 2-mercaptoethanol, 25; sodium malate, 7.5; sodium pyruvate, 5; NAD, 0.38; malate dehydrogenase, 15 pg; citrate synthase, 15 pg; LPIP as indicated. After 2 min, the reaction was started by the addition of 100 pg of ferredoxin-free (6) Curve I, reaction in the absence pyruvate lyase isolated from 5. pasteurianum. of LPIP; Curve II, reaction with 70 ug LPIP. 1 The iron content of LPIP or ferredoxin was measured by the o-phenanthroline method, exactly as described by Lovenberg et Alternately -- al. (9). the samples were deproteinized at 8O“C in 0.1 N KC1 (or hydrolyzed as indicated) and the free iron measured by atomic absorption spectrophotometry. Acid-labile sulfide was measured exactly as described by Brumby --et al. was measured by the Lowry phenol reagent method (11) with (10) * Protein No correction factor was crystalline bovine serum albumin as standard. used for the protein determination of ferredoxin (9). RESULTS AND DISCUSSION: requires,

in addition

enzymes,

an electron

potential.

not

chloroplasts specific

(EA = -0.432 spinach (Table

for

and an electron system

ferredoxin

drives

therefore

the reductive

completely

dependent

LPIP,

carrier used

which,

in the

carboxylation

pyruvate

of added LPIP

of ferredoxins. by methyl

viologen by illuminated

of acetate

reduction synthase

to pyruvate

potential. assay

(Table

to pyruvate. chloroplasts

or ferredoxin

illuminated The requirement

photoreduced

of acetate

55

oxidation-reduction

of DPIP,

carboxylation

on enzyme and illuminated

absence

consists

when

a low oxidation

to pyruvate

and the appropriate

of low

can be replaced

the reductive

must have ferredoxin

measures

in the

commonly

which

V at pH 7) (2).

LPIP replaces

activity

donor

of acetate

system

and any one of a number

chloroplasts l),

carboxylation

to an acetate-activating

The reducing

spinach is

The reductive

because

but

1) which

The system

is

retains

some

DEAF. cellulose

BIOCHEMICAL

Vol. 71, No. 1,1976

Table

1.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The requirement of reduced synthesis of pyruvate from

ferredoxins [1-14C]acetate

Incubation system

Complete + C. pasteurisnum

or LPIP in the and CO2. Pyruvate

53,070

ferredoxin

Complete + Spinach ferredoxin*

62,060

Complete

+ LPIP

25,020

Complete

- ferredoxin - LPIP

- chloroplasts

3,660

+ LPIP

290 200

- enzyme + LPIP The incubation medium, assay procedure exactly as described previously (4). from cell-free rumen bacterial extract through DEAE cellulose (6) immediately contained 1 mg of this protein. Other doxin, 170 ug; Spinach ferredoxin, 140 contained 10 umoles of [1-14Clacetate final volume of 3.0 ml.

*

Spinach Chemical

ferredoxin, Type III Co. and used without

chromatography

does not

LPIP substitutes

for

C. pasteurianum

ferredoxin

and responded

linearly

Ferredoxin which also

no NAD reduction

crude,

in Fig. cell

to,

the cleavage

an electron

described

but

or FAD serve

measures

free

acceptor in

remove

ferredoxin spinach

and hydrazone isolation were done The pyruvate synthase activity was freed of ferredoxin by passage before use. The reaction mixture additions: C. pasteurianum ferreug; LPIP, 175 pg. Each incubation (2.92 x lo5 d.p.m. per umole) in a

was a commercial product further purification.

completely

at pH 7) by illuminated

is

hydrazone (dpm)

in

the

the rate

LPIP concentrations

of pyruvate

1 was routinely

this

in

to acetyl-CoA assay

of a suitable used in

extract.

56

(Fig.

Sigma

ferredoxin.

LPIP was less

acceptors

from

of NADP (EA = -0.320

of NADP reduction

as electron

the absence

reduction

chloroplasts.

increasing

in

the endogenous

obtained

1).

electron the purification

active

than

was dependent (results

In this

of LPIP

shown).

lyase 12).

assay,

acceptor.

on,

not

the pyruvate and CO2 (4,

V

assay LPIP

there

The assay from

the

is

Vol. 71, No. 1,1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(b)

250

3co

Fig. 3a. C. pasteurianum to pH 1.5 ior 15 min then Fig. 3b. LPIP before pH 7.5 (---).

Table

Iron

2.

350

400

450

ferredoxin readjusting

(--)

and acid-labile

at acid

sulfide Iron

450

500

after (---).

after

of ferredoxin

*

and LPIP.

Acid-labile sulfide protein) 0.29

C. pasteurianum ferredoxin

0.53

0.47

LPIP

0.06

0.06

LPIP hydrolyzed in 6 N HCl for 24 h at 105°C

0.37

----

Iron measurements were done by both by atomic absorption spectrophotometry. methods were in excellent agreement.

Clostridial sulfide (Fig.

and iron 3a) with

ferredoxins, (13).

This

an irreversible

acidification

readjusting

0.18

*

ferredoxin

400

pH (-*-)

content

(Umoleslmg Spinach

350

before acidification(-), with 1N alkali to pH 7.5

acidification

Sample

500 250 300 Wavelength (nm)

the o-phenanthroline Values obtained

when acidified,

evolve

technique and by the two

H2S and lose

acid-labile

results

in a decrease

in absorbancy

at 390 nm

loss

of the shoulder

characteristic

of the

57

to

Vol. 71, No. 1,1976

spectra

for

with

LPIP

This

shows

ment

bacterial (Fig.

are noted

ferredoxins.

3b)

that,

the

on acidification

at acid

(0.12

10 min at 8O'C)

The iron

of LPIP

expressed

absorption cannot

in LPIP

(Table

In summary, associated

with

ferredoxin

in

quantitatively

be measured

its

optical

iron

that

Studies

on EPR spectra

spectral

in Table

releases

alterations

2.

Mild

the ferredoxin

acid

iron

by either

method

amounts

unless

method.

LPIP is

of acid-labile

treat-

when measured

or by the o-phenanthroline

No significant

first

sulfide

hydrolyzed were

2).

the bacterial

bound

at 412 nm observed

on neutralization.

no irreversible

LPIP has the electron

tightly

reappears

spectrophotometry

h in 6 N HCl at 105'C.

detected

the peak

of LPIP. is

atomic

pH but

ferredoxins,

The same result N HCl for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

By contrast,

disappears

unlike

by either

24

BIOCHEMICAL

transferring

and plant spectrum,

requires

ferredoxins.

absence acid

and the nature

properties

of iron

LPIP differs

of acid-labile

hydrolysis

generally

sulfide

of the protein binding

from

in LPIP

and with for

release.

are currently

in progress.

ACKNOWLEDGEMENTS: The authors acknowledge the expert technical assistance of Mr. Wm. Cantwell. The authors thank Dr. I.L. Stevenson for assistance growing 5. pasteurianum cultures. This is Contribution No. 629 from the Animal Research Institute. REFERENCES: 1.

Bachofen, Sci,

R., 3,

2.

Raebum, S. and Rabinowitz, (San Pietro, A. ed.) Springs, Ohio.

3.

Sauer,

F.D.,

Buchanan, 690-694.

Erfle,

B.B.

J.D.

and Amon, J.C. (1965) pp. 189-197.

and Mahadevan,

D.I.

(1964)

Proc.

Nat.

Acad.

in Non Heme Iron Proteins The Antioch Press, Yellow S.

(1975)

Biochem.

J, 150,

357-372. 4.

Bush,

R.S.

and Sauer,

5.

Orme-Johnson,

6.

Mortenson, L.E., Valentine, Biophys. Res. Comm, 1,

7.

Camahan,

J.E.

W.H.

F.D. (1973)

and Castle,

(1976) Ann.

Biochem. Rev. Biochem,

R.C. and Carnahan, 448-452. J.E.

(1958)

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J.

In press. 42,

159-204.

J.E.

(1962)

J. Bacterial,

75,

Biochem. 121-124.

in

BIOCHEMICAL

Vol. 71, No. 1,1976

8.

Buchanan, B.B., Acad. Sci,

9.

B.B. Lovenberg, W., Buchanan, Chem, 238, 3899-3913.

AND BIOPHYSICAL

Lovenberg, W. and Rabinowitz, 49, 345-353.

10.

Brumby, P.E., Miller, 2222-2228.

11.

Lowry,

12.

Raebum,

13.

Hong,

and Rabinowitz,

R.W. and Massey,

O.H., Rosebrough, N.J., Biol. Chem, 193, 265-275. S. and Rabinowitz,

J. and Rabinowitz, 246-252.

Farr, J.C.

J.C.

W. A.L.

(1971) (1967)

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J.C.

(1963)

Proc.

J.C.

(1963)

J. Biol.

(1965)

J. Biol'.

and Randall,

Nat.

Chem, 240,

R.J.

(1951)

Arch.

Biochem.

Biophys,

Biochem.

Biophys.

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Comm, 29,

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