EPR evidence for nickel-substrate interaction in carbon monoxide dehydrogenase from Clostridium thermoaceticum

Vol. 108, No. 2, 1982 September 30, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 658-663

EPR EVIDENCE FOR NICKEL-SUBSTRATE

INTERACTION

IN CARBON MONOXIDE DEHYDRCZENASE FROM CLOSTRIDIUM Steve

THERMOACETICUM

W. Ragsdale, Lars G. Ljungdahl Daniel V. DerVartanian

and

Department of Biochemistry University of Georgia Athens, Georgia 30602 Received

August

6, 1982

SUMMARY, Carbon monoxide dehydrogenase from Clostridium thermoaceticum EPR resonances with g-values at contas different Fe4S4 rhombic-type The enzyme after 2.04, 1.94, 1.90 and 2.01, 1.86, 1.75, respectively. in EPR signal at g = 2.07 and reacting with CO or HCO3-/CO2 also reveals readily observed at 95K, is attributed to a Ni(II1) 2.02. This signal, interaction with a radical species formed from CO or HCO3-/CO2. INTRODUCTION Nutritional carbon

studies

monoxide

Clostridium

is

(2).

incorporated

electrophoresis

dehydrogenase

a partially

a constituent

it

a protein

of

anaerobic

bacteria

and Clostridium

has been demonstrated,

that

CO dehydrogenase

band obtained extracts

from

by polyacrylamide

contains

nickel

and CO

(4).

homogeneity

440,000

is hexamerous giving

per

mol 2 nickel,

the

metal

from C. thermoaceticum

the

content anaerobic

The

(5). and it

enzyme

consists

structure

may vary

of three

different

in the presence absorption

spectrum,

0006-291X/82/180658-06$01.00/0 0 1982 by Academic Pres, of reproduction in any form

a molecular

Inc. reserved.

658

weight

to

of about

each of two different a6,

and 14 acid-labile

between

conditions

with

has now been purified

The dimer,

(aB)j. 12 iron

3 zinc,

enzyme has a porphyrin-type

Copyright All rights

is

of the

purified

of C. pasteurianum

apparent

Under

nickel

formicoaceticum

use of 63Ni

and that

The CO dehydrogenase

subunits

Clostridium

the

into (3)

that

(CO dehydrogenase)

(1) With

C. thermoaceticum gel

implied,

dehydrogenase

pasteurianum

thermoaceticum nickel

have

apparently sulfur.

preparations of the with

contains However,

of the substrate,

peaks

at 600,

enzyme. CO,

the 393,

Vol. 108, No. 2, 1982 380,

typical

with centers

AND BIOPHYSICAL

310 and 277 nm and shoulders

enzyme activity,

sulfur

BIOCHEMICAL

and converts

iron-sulfur centers CO.

as well

In this

at 670 and 550 nm.

the

protein.

spectrum

Thus,

and a possible

nickel

of the

Oxygen destroys

enzyme to that

the enzyme appears

as an additional

communication

RESEARCH COMMUNICATIONS

chromphore

we report chromophore

which

on a EPR-study of the

the

of a

to contain

iron-

seems to react of the

iron

CO dehydrogenase

sulfur

from C.

thermoaceticum. EXPERIMENTAL C. thermoaceticum (ATCC 39073) was grown at 58OC in a 400-liter fermenter in a previously described medium (6) modified by the addition of 10d6M NiC13. The enzyme from 180 g of wet weight cells was purified to an activity of 450 (about 40-fold) umol of CO oxidized min-' mq-' when assayed at 50°C with 10 mM MV as electron acceptor (7). The purification, which is described in detail elsewhere (5) was performed inside an anerobic chamber in an atmosphere of N2. .H 2 (95:5) using as buffer 50 mM Tris/HCl, pH 7.4 containing 2mM sodium dithionite. The purification steps included preparation of cell extract using a French pressure cell, heat treatment at 67OC for 30 min, ammonium sulfate fractionation, (45-60% saturation), chromatography on and gel filtration with BioDE32-cellulose, Bio-Gel HTP and DEAE Sephacel, Gel A.1.5 M and Ultrogel AcA22. The purified enzyme was prepared for EPR studies by applying a solution containing 40 mg to a DE32-cellulose column (1.5 x 5 cm), previously equilibrated with 50 mM Tris/HCl, pH 7.6 and 2 mM sodium dithionite. The column was washed with 5 volumes of oxygen-free 50 mM Tris/HCl, pH 7.6, without the dithionite. The enzyme was then eluted in 6 ml with 0.5 M Tris/ HCl, pH 7.6, and concentrated to 2 ml using a PM 30 ultrafiltration membrane The final and an Amicon ultrafiltration cell (Amicon Lexington, Mass.). protein concentration was 20 mg ml-l. Samples for EPR were prepared anaerobically. in 260 ~1 0.5 M Tris/HCl, pH 7.6, and 4.8 mg of CO to individual tubes were as follows: none, sodium potassium ferricyanide, 0.5, 1 or 2 mM; and sodium Tubes were also prepared with 100% CO (99.99% pure, or air as gas phase. Prepared tubes were stored in analyzed with EPR spectroscopy.

Each EPR tube contained dehydrogenase. Additions dithionite, 2 mM; bicarbonate, 80 mM. Matheson, Morrow, GA.) liquid nitrogen until

EPR spectroscopy was performed with a Varian E-109 spectrometer interfaced with a Hewlett-Packard HP-85 microcomputer. Measurements in the liquidhelium temperature range were performed with an Air Products APD-E automatic temperature controller. Other EPR experimental conditions are found in Figure Legends. Nickel and iron were determined using plasma emission spectroscopy (8). RESULTS AND DISCUSSION The EPR spectrum under

anaerobic

The spectrum

at 10K of ~C

conditions indicates

thermoaceticum

and without reduced

multiple

any addition iron-sulfur

CO dehydrogenase is

shown in Fig.

centers.

These

as isolated 1A. signals

Vol. 108, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMJNICATIONS

/

0

2

Fiqure 1. EPR Spectra of Carbon Monoxide Dehydrogenase Purified from Clostridium thermoaceticum. A.) Purified CO dehydrogenase (42 pM in protein in 500 mM Tris buffer, pH 7.6) maintained under anaerobic conditions (argon) . EPR conditions: microwave power, 20 uW; microwave frequency, 9.162 GBz; modulation amplitude, 10 G; temperature, 10 K; scanning rate, 500 G per min; time constant, 0.1 set; Gain = 1.25 x 104. B.) Purified CO dehydrogenase as in (A) but reacted under anaerobic conditions with carbon monoxide for 1 hour. EPR conditions as in (A) except that Gain = 5 X 103. C.) Purified CO dehydrogenase as in (A) but reacted under anaerobic conditions with 80 mM sodium bicarbonate for 1 hour. EPR conditions as in (A) except that Gain = 8 x 103. Figure 2. EPR Spectum of Carbon Monoxide Dehydrogenase at the Redox State of Figure 1 B. EPR conditions as in Fig. 1 B except that the scan range was two-fold expanded; microwave power, 10 mw; microwave frequency, 9.161 GHs; temperature, 95 K; scanning rate, 250 G per min and Gain = 5 x 103.

are

not

observed

when

EPR measurements

be two Fe4S4 rhombic-type 1.94

and 1.90,

Quantitation

while

EPR resonances;

the other

has g-values

by double-integration using

dependence

on transition

probability

(9)

yields

1.8

per

with

the

ferricyanide

cupric-EDTA

of this

conditions

electrons

appearance using

are

as the

mol

spectroscopy

at

95K.

one exhibits of 2.01,

g-values 1.86

dimeric

results

660

enzyme.

Titration in

appear

to

at 2.04,

and 1.75.

under

non-saturating

and by correcting

by the procedure of

There

EPR spectrum

standard

of two Fe4S4 centers. EPR

made

for

g-value

of Aasa and vanngard This

is

consistent

of the enzyme with

diminishment

of

the

signal

Vol. 108, No. 2, 1982 intensities (based

BIOCHEMICAL

of the iron-sulfur

ferricyanide oxygen

is

essentially

increase

(12%)

centers.

(2 mM), that

of the in the

anaerobic

at 95K, when the

interfering

Addition

very

signals

conditions

it

formed

nor

suggests

at 95K.

Since is

of nickel nickel

does

in the is

substantial

values

not

saturate

nickel

is

amount

at 2.3, different

new signal

form

the

intense only

of the

of nickel

with over

from

any Ni(II1)

of

range

power

it

with

enzyme).

present

is

probable

mol which

paramagentic

to have a Ni(II1)

EPR signal

obtained

The

strongly This

complex.

of 30 mW when measured

metal

The EPR signal

is

ferricyanide

sensitivity metal

reduced

Neither

(besides that

Double-integration per

anaerobic

CO, or the

the

a transition

transition

and 2.02

when the enzyme is

and temperature

661

the

increase

under

substrate,

inactivates

Fe4S4 centers),

(10,ll).

that

at g = 2.07 formed

a wide

latter

in this

the

absent

1C) has an

CO or HC0T/C02.

at a microwave

is

only the

reacted

have now been shown 2.2 and 2.0

is

observed

0.4 electrons

(Fig.

and that

new signal

not

in the new signal. for

are

5%.

enzyme with

comes from

the only

enzymes

with

observed

enzyme to CO, except

It

behavior

appears

centers

less

(the

signal

iron-sulfur

iron-sulfur

titrated

of oxygen

a slight

of the

is

is

and then

accounts

of hydrogenases

quite

The signal

involved

signal

by

is readily

by the

of the

saturation the

Enzyme inactivated

EPR signal

significance.

when the enzyme is

that

is

represented

dithionite

new signal

which

and 2.02

biological

power

highest

This

of exposing

species

in the presence

microwave

with

of the reduced

to the anaerobic

centers

HCOg/CO,.

sodium

the

a new intense

signals

by interaction

product,

70% occurs

signals

1B).

iron-sulfur

The enzyme is of obvious

(Fig.

to that

at g = 2.07 of the

of the

of HCO;/CO,

similar

new signal

A maximum decrease

was used.

more interestingly, and 2.02

effect

shown).

enzyme to CO at 10K yields

intensity

at 2.07

2).

RESEARCH COMMUNICATIONS

EPR silent.

However,

g-values

(not

of approximately

concentration

The exposure

with

centers

on double-integration)

(Fig.

AND BIOPHYSICAL

iron,

a form

of this indicates

form

(20%).

EPR signal in Fig. with

that

proposed a

Anumber with

g-

2 is obviously hydrogenase.

In

Vol. 108, No. 2, 1982 fact

the

doublet lyase

new signal

reported

was attributed

possibility would

by Babior

does

it

by Babior

exhibit

species Hu --et al

with

(12);

these

which

is

line

low-field

CO dehydrogenase

the enzyme may contain results which like

appear

to indicate

is associated factor.

This

with

that

nickel

factor

the

acetate system

C-l

species

in turn

possibly

appears

not

F43G from Methanobacterium

the

enzyme

lacks

system

of Factor

an absorption

is

not

and 2.02

from

CO

centered signal

at g = 2 described

nickel

(III)-

synthesized from

and they

have proposed

is

level in

CO

C. - thermoaceticum that

of for-mate.

the form

associated

with

to the

a porphyrin-

nickel-containing (14)

The structure

been elucidated

our

of a radical,

thermoautotrophicum

at 430 nm (5).

F430 has recently

from

obtained

to be identical

factor

porphinoid

formed

on the oxidation

porphinoid native

we

at 2.07

as does the

enzyme system

species

that

and the

shifted.

of this

an C-l

state

likely,

may be due to a unique

by an enzyme

a part

Co(U)

g-values

is

Their

the low-spin

quite

species

g = 2.3

have demonstrated

is

in

CO or HCO3 is with

ammonia

propanol.

with

CO dehydrogenase near

ethanolamine

Co(I1)

a radical

differences

and 5-methyltetrahydrofolate

for

isoelectronic

new EPR signal

shown for

a broad

(13)

is

the radical

of amino-2,l

involving

interacting

The signal

--et al

the

(12)

between

Ni(II1)

species that

species

or HC03/C02.

radical

Since

to propose

a Ni(II1)

--et al

to an interaction

species.

RESEARCH COMMUNICATIONS

in EPR properties

in the presence

of a radical

like

AND BIOPHYSICAL

strongly

and adenosylcobalamin

and a radical

nor

resembles

intermediate

signal

is

BIOCHEMICAL

since of the

(15).

ACKNOWLEDGEMENT The authors wish to thank Ms. Susan K. Holmes for the excellent technical assistance with the computer-assisted EPR spectroscopy, Dr. John Wampler for interfacting and programming the Varian E-109 spectrometer with the Hewlett-Packard 85 microcomputer for data acquisition and Drs. Isamu Yamamoto and Shiu-Mei Liu as well as Ms. Joan E. Clark for This work furnishing cell material and help with isolation of the enzyme. was supported by Public Health Service grant AM-27323 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, by project DE-ASO9-79 ER 10499 from U. S. Department of Energy and a Grant from the University of Georgia Research Foundation. REFERENCES 1. 2.

Diekert, G. B., Graf, E. G. and Thauer, R. K. (1979) 122, 117-120. zkert, G., and Thauer, R. K. (1980) FEMS Microbial. 662

Arch. Lett.

Microbial. 1,

187-189.

Vol. 108, No. 2, 1982 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Drake, H. L., Hu, S.-I., and Wood, H. G. (1980) J. Biol. Chem. 255, 7174 -2180. 149, 561-566. Drake, H. L. (1982) J. Bacterial. Ragsdale, S. W., Clark, J. E., LjwTahl, L. G., Lundie, L. L., and submitted. Drake, H. L. Manuscript Ljungdahl, L. G., and Andreesen, J. R. (1978) Methods Enzymol. 2, 360-372. Clark, J. E., Ragsdale, S. W., Ljungdahl, L. G. and Wiegel, J. (1982) J. Bacterial. 151, 507-509. Plant. Anal. 8, 349-365. Jones, J. B., Jr. (1977) Commun. Soil. Sci., T. (1975) J. Magn. Reson. 19, 308-315. Aasa, R., and Vanngard, Albracht, S. P. J., Graf, E.-G., and Thauer, R. K-71982) FEBS Lett. E, 311-313. P. O., Moura, I., Peck, H. D., Jr., Xavier, A. V., LeGall, J., Ljungdahl, Moura, J. J. G., Teixera, M., Huynh, B. A. and DerVartanian, D. V. (1982) Biochem. Biophys. Res. Conunun. 106, 610-616. Babior, B. M., Moss, T. H., Onne-Johnson, W. H. and Beinert, H. (1974) J. Biol. Chem. 249, 4537-4544. Au, S.-I., Drake, H. L., and Wood, H. G. (1982) J. Bacterial. 149, 440-448. Gunsalus, R. P., and Wolfe, R. S. (1978) FEMS Microbial. Lett. 2, 191. Pfaltz, A., Jaun, B., Fassler, A., Eschenmoser, A., Jaenchem, R., Gilles, H. H., Diekert, G., and Thauer, R. K. (1982) Helv. Chim. Acta. 65, 828-865.

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