Selenium as an acid labile sulfur replacement in putidaredoxin

Vol.

30,

No.

BIOCHEMICAL

3, 1968

SELENIUM

AS

AN

ACID

J.C.M.

Received

January

hydroxylates

lar

z

undergoes

iron

acid

(TCA)

in

with

mercurials 33S,

isotope,

natural

an

two

active

longer

wavelength

duced

state

wi II be

PUTIDAREDOXIN*

Gunsalus

Illinois,

Urbana

61801

(+)-camphor

in

This

enzyme

1966).

of acid-labile

oxidation-reduction

with

57 Fe

removed

from

have

sulfur

been

an

E’d

-185 by

As

introduced,

in

and

1968)

1966).

putida,

contains

et.al.,

which

Pseudomonas

putidaredoxin

(Tsibris,

Rabinowitz,

of a system

a molecu-

iron

and

mVoits**.

The

treatment

or

by

with

treatment

replacement

elements,

usually

with

a radioactive

with

selenium

59Fe.

replaced

center and

indicative

the and

product

acid-labile with

sulfur

80Se

is biologically as

indicated

by

an

of

rhombohedml

enzyme.

reported

component

each

and

or

iron-sulfide

of

atmosphere

and

The

five

be

abundance

75Se,

the

argon

now

isotopic

I.C.

protein

atoms

can

%

have

of

Gunsalus,

(Malkin

selenide

in

and

56Fe,

We

tope

carbon

atoms

e.g.*

and

iron-sulfide

a one-electron and

32S,

is the

12,000,

sulfur

Namtvedt,

IN

COMMUNICATIONS

20, 1968

(Cushman

weight

RESEARCH

REPLACEMENT

University

methylene

Cl

BIOPHYSICAL

SULFUR

M.J.

Department,

Putidaredoxin

strain

LABILE

Tsibris,

Chemistry

AND

labeled active

by

electron

Changes

a shift spin

with and

in

the

observed

visible

an

axial

in

other

iso-

altered

spectrum

spectrum

than

the

radioactive

possesses

resonance

rather

the

of

in

symmetry physical

irorr to

the

re-

as seen properties

subsequently.

METHODS

Putidaredoxin, method

of

* Supported **Measurements

specific

Cushman,

in

part of

Tsai,

by E’,

activity and

5,

Gunsalus

was (1967).

prepared The

by labile

a modification iron

and

Grant AM00562, National Institutes of Health. by Dr. G. S. Wilson, University of Arizona.

323

of the sulfur

Vol.

30,

No.

were

removed

by

after

dropwise

nium-80

New

England

cedure

of

the

and

of

(enrichment with

Nitsche

(12,000

8.3,

to

The

two

enzymatic

method

method

atoms

activity

of

of

each

Cushman,

Katagiri,

mole

was

measured

et.al.

et.al.,

ment of

properties

of sulfide the

the

two

by

apo-protein

their

restomtion

sulfide.

by

Upon

with

32S,

restored

with

32S

the

principal

about

20

mp

the

33S

to

of

the

the

2.5

and

fixed

time

IOmM

described

this

atoms

table.

the

selenide

as

see

stage

per

the

mole

50 mM

Tris

sulfide

content

of

buffer,

(product

kinetic

native

as shown

mole

of

of native

the

was

forma-

DPNH-oxidation

bands

at

415 of

replacement

wavelength activity assay

Katagiri

(1968).

iron

are et.al

maxima

of

--the

the At

higher

1968). oxidized

maximum to

recent

the

324

formation

of

in

sulfide-

mp,

the

lack

and

without

the

UV

selenide to In

sample

spectrum

is

contents the the

of added

reconstituted

of

obtained

selenide

sample,

enzyme

are

shifted

is shifted

only

about

sulfide

the

the

values

enzyme

substmte-dependent

concentration,

removal

decrease

the

and and

., the

325

more

on

comparable

is comparable and

from

remain

The

(Tsibris,

absorption

formation

counts

table

4.55

replace-

The

8-mercaptoethanol + 75

enzyme. the

and

after

table.

by

indicated

and

the enzyme

as

75Se

in

in

95%,

presence

form

native

of selenide-80

shown

longer

biological

product

oxidation

of

4%

visible

‘Ib

5 mp.

Fe*in

enzyme and

than

absorption

than

to that

in

per

displacement

less

reconstructed

of

by

treated

DISCUSSION

are

contained

absence

AND

putidaredoxin

is greater

the

the

was

At

iron

pro-

of sulfide-free

versus

enzyme, by

the

(1968).

selenide-80Se

sulfides

35S,

the

of

dialysis

and

X1967),

RESULTS The

of

from

by

recovered

and

the

per

obtained

of ammonium

presence

2.6

B-mercaptoethanol,

was

mc/mg

material.

5 hours

sele-

Laboratory)

selenide

was

respectively

tempemture

apo-putidaredoxin

the

reconstituted

After

National

excess

in

room Elemental

(I

the

molar iron

iron

were

IO mM

of

putidaredoxin

protein).

containing

decreased

tion)

gm

Ridge

to ammonium

sample

ferrous

and

contents

Oak

COMMUNICATIONS

at

agitation.

selenium-75

IO-fold

selenide

sulfide

selenide

enzyme pH

the

from

RESEARCH

atmosphere

constant

reduced

a of

The

with

5 mg

with

. for

and

excess

argon

elemental

A

atmosphere

previously and

of

Co.)

molar

TCA

BIOPHYSICAL

an

obtained

(1957).

B-mercaptoethanol

iron

100%

amount

Nuclear

a 5-fold

with

96.87%

a small

argon

AND

equilibmtion

addition

mixed

in

BIOCHEMICAL

3, 1968

selenide

in

both

DPNH putida-

Vol. 30, No. 3, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3235

3319

3355

3415

I

3271 EPR Spectra

of

Reduced

33193 J430

Putidaredoxin.

32

Native form, curve S; selenide exchanged The two curves represent equal numbers of

of EPR spectroscopy: microwave power, modulation amplitude 3 Gauss; scanning constant I set; temperature, 830K.

Conditions 9,223 MHz min; time

Ordinate is an arbitrary measure curve. Field markers (abscissa) egursion of the 8o Se-putidaredoxin ( S) putidaredoxin.

redoxin

is inhibitory The

in

electron

the

spin

is shown

superimposed

figure.

The

protein

induces

on an of

a dramatic

active

center

ted

in

Se-enzyme

of

***Measurements by Research, University

of are

DPNH

the

of the

earlier

spectrum

of

2 atoms

of 80Se

(spin

native

in

the

material,

to a rhombohedml

Drs Beinert and of Wisconsin.

frequency Gauss per

assay.

spectrum***

change

27 mWatt; rate, 100

the first derivative of the EPR absorption in Gauss; upper set refers to the major curve and the lower set to the native

oxidation

resonance

substitution

The

the

8o Se . spins.

sulfide-32s

g = 2.01

Orme-Johnson,

into

of and

with

putidaredoxin enzyme,

0 isotope)

EPR spectrum

complex

325

selenide

the

the reduced

g = 1.93, gz = 2.04,

Institute

see

for

native species.

is distorgy = 1.98,

Enzyme

Vol. 30, No. 3, 1968

and g, = the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1.93.

This

patamagnetic

they

complex

replace

and

substitution. nium

enzyme

in

EPR spectrum

the

by

atoms

the

in

the

therefore

been

done

analogy

prove

iron

(Tsibris,

Selenide-80

complex

may

result

in

Nevertheless,

this

the

spin with

= 3/2 the

primary to

an

in

et.al.,

lagands

to the

isotope

in

77Se establish

g = 1.94

the

on

(spin

from the

et.al.,

namely,

acid-labile this

sele-

part

Tsibris, 33S,

of

the

effect

by

nuclei

compare

effort

profound

earlier

which

accompany

They

found

in

atoms

unexplained.

was

and

sulfur

of

interesting

participate

the

the

positioned

ends

as

in

as

not

field

interacts

spectm

way

of low

are

high

what

participate

S) atoms

with

yet

atoms

same

the

32.S with

g = 1.94

80Se-putidaredoxin by

near

strongly of

It should

(and

as

the distortions

preparation.

substitution

which

complex.

are

selenium

exactly

bands

underlines

electron

the

asymmetric

minor

the

that in

spectrum

32S

(1968),

that

The

residual

suggests

that sulfur

pammagnetic = l/2)

with

many

Se

how center,

as

has

1968).

Putidaredoxin:

Properties

and

Activity

Enzyme

Acid

!I

I. 4.

labile

Fe

Activity Product Coupled

formation4 DPNH

Absorption

spectra

Fischer, Cushman,

units/mg

Price, 1966.

1964. 5.

protein

5 4

oxidation5

mlJ 455 415 325 280

3.1 5-7

EmM 9.6 IO. 15. 22.5

2. Fogo, Popowsky, Katagiri, nmoles DPNH

326

q

1949. 3. oxidized,

EmM

477

8.6

433 330

8.9 16.

Radiochemical min-lml’l.

15Se.

Vol.

30,

No.

3, 1968

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGEMENTS

We

wish

Institute

for

ments from these

on

to thank Enzyme

Drs

H.

Beinert

Research,

earlier

data.

potential

its

mutual We

also

paper, wish

measurements

dehydrogenase

on

Professor and

Orme-Johnson,

of Wisconsin,

for

thank

32S-

H.

superposition

and

to

and

assays

W.

University

80Se-putidaredoxin,

our

and

on

for the

discussions

Dr.

G.

M.

Katagiri

sulfide-

and

EPR measure32S

spectrum

interpretations

S. Wilson for

the

for

his

of

additional

the

coupled

-27,

I05

DPNH

80Se-putidaredoxins.

REFERENCES

Beinert,

H . , and

Palmer,

Cushman,

D.

Cushman, Commun.,

D. W., Tsai, R. L., 26, 577 (1967).

Katagiri, 000 (1968)

M., Ganguli, April.

Fischer, Fogo, Malkin, Nitsche, Tsibris, Hansen,

D. J.

K., R., R.,

W.,

and

G.,

and

S.,

and and

Gunsalus,

B.,

Price, Popowsky,

Rabinowitz,

Angew.

Adv.

Chemie,

in

Enzymology,

I. C., and

and

D.

Gunsalus,

Gunsalus,

C.,

Anal. C.,

69, -

I. C.,

1966,

86.

Biochem.

Federation

Chem.,&, Chem.,g,

B&hem. 333

Proc.

I. C.,

Clinical

M., J.

Bacterial.

(1965).

Proc.

Res.

27,

21 (1964). 732

Biophys.

Biophys.

(1949). Res.

Commun.,

23,

822

(1957).

J. C. M., Tsai, R. L., Gunsalus, I. C., and Orme-Johnson, R. E., and Beinert, H., Proc. Nat. Acad. Sci., 59, 000 -

327

W. (1968).

H.,

(1966).