The ascorbate reduction of denatured ferricytochrome c

Vol. 49, No. 1, 1972

BIOCHEMICAL

THE ASCORBATE REDUCTION Laurence

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF DENATURED FERRICYTOCHROME

S. Kaminsky

and Victor

J.

Department of Physiology and Medical University of Cape Town Medical Cape Town, South Africa Received

August

c*

Miller

Biochemistry, School,

18,1972

Summary: The reduction of ferricytochrome c by ascorbate in solutions of the denaturants, 1-propanol ana urea,proceeds under conditions where the 695 nm absorbance band is completely quenched, thus indicating that the heme iron-methionine 80 bond is not an essential requisite for the reduction of the iron of ferricytochrome c by ascorbate. The implications of these results for the elucidation of the mechanism of action of cytochrome c are discussed. The denaturant induced autoxidation of ferrocytochrome c was used in monitoring the reduction indirectly with an oxygen electrode.

Elucidation involves enter

of the the

determination

and leave

and reduction object

the

bond

absorbance monitored

such measurements

denaturant

becomes

autoxidisable (5) and,

ferrocytochrome the

reduced

under

state

(6).

*Supported by grants from Council and the University Copyright AN rights

0 19 72 by Academic Press, Inc. in any form reserved.

of reproduction

such a

methionine of the

are

80

695 nm

usually

are

for

suspect

the

any reduction the

although

We report

two reasons:

ferrocytochrome

conditions

2 can disappear

that

In solutions

masking

certain

of

at 550 nm.

concentrations thus

reported

type

this

investigated

presence

this

spectrophotometrically

at high

in

of

oxidation

with

reduction

heme iron

by the

c

electrons

of the

has been well

when the

Reductions

of denaturants

occur

proceed

the

(4) have

as evidenced

band.

whereby

2 have been performed

and Greenwood

intact,

of cytochrome

pathways

and in particular

can only

is

action

Many studies

c by ascorbate

Wilson

reduction

heme iron.

(11,

ferricytochrome

of

of the

of cytochrome

in view

(2,3);

mechanism

here

c which

550 nm band

the heme iron our

may

studies

of remains

on the

the South African Medical Research of Cape Town Staff Research Fund 252

BlOCHEMlCAL

Vol. 49, No. 1, 1972

reduction

of cytochrome

denaturants

which

a system

which

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2 by ascorbate

completely

quench

in the the

overcomes

the

problems

Materials

and Methods

presence

of

695 nm band,

of

the

using

spectrophotometric

assay.

Horse

heart

0.425%Fe)

cytochrome

2 (Miles-Seravac,

was oxidised

by passage

through

ascorbic

acid

immediately

with

use.

as previously

for

and pH shifts.

Oxygen

consumption

Instrument bath the

Clark

buffered

were mixed oxygen reaction

solutions

of

absorbance

the

by the c

solution

changes

at

DBGT spectrophotometer. absorbance the

was distilled

electrode

a stable

(10

PM)

solutions

and deionized.

a Yellow

in

Springs

a thermostatted

water

were made up as follows: and ascorbate

base

addition and the

in the

presence

line

was obtained

rate

of change

Denaturant

695 nm were determined The residual from

(O.OlM)

of an aqueous

was recorded.

band was subtracted

of

(5) and were corrected

equilibrated

After

solutions

and urea

using

of denaturant

was initiated

content

Water

solutions

and thermally

of ferricytochrome

described

oxygen

Reaction

electrode.

were prepared

was determined

type

(2 0.02').

Reagent) alcoholic

1,

and purified

Aqueous

Buffered

were prepared

ferricyanide

Grade

G-25 column.

Analytical

before

shrinkage

potassium

a Sephadex

(Merk,

95% pure,

the the

solution of

oxygen

induced

with

absorbance the

of

reported

a Beckman of the

530 nm

readings

of

695 nm absorbance. Results

The rates chrome

of oxygen

and Discussion consumption

c and ascorbate'incorporating

of

solutions varying

253

of

ferricyto-

concentrations

of

Vol. 49, No. 1, 1972

1-propanol of

BIOCHEMICAL

or urea

are

ferricytochrome

denaturants

c in

(Figures

0

0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

compared

with

the

absorbance

solutions

of

the

same concentrations

1 and 2).

2

6

In the

6 IO MOLE % PAOPANI

presence

12

at 695 nm of

of both

16

Fig. 1. The effect of l-propanol on the rate of reduction by ascorbate and the 695 nm absorbance of ferricytochrome rates of reduction measured indirectly using 0, P,As oxygen consumption rates; @,a & 659 nm absorbance. Cytochrome c 10 PM, 0.1 M acetate buffer, 250.

0

0

2 UREA

I MOLARITY

Fig. 2. The effect of urea on the ascorbate and the 695 nm absorbance Other details as in Fig. 1.

254

c.

6

rate of reduction of ferricytochrome

by c.

BIOCHEMICAL

Vol. 49, No. 1, 1972

denaturants at

at pH 5, 6, and 7 the

concentrations

produce

of denaturant

complete

quenching

When the

protein. either

ascorbate

consumption where

ferricytochrome

in

the

of our

earlier

susceptible

to direct

observed

autoxidation

in

of

these

(Figures

(5) and not

from

rates

at very

from

a decreased

most

rates

system

2.

surrounding methionine

the

result

oxygen from

As a consequence

by ascorbate

the

to yield of denaturants

ferricytochrome

from

a failure

high

slow

c is

completely

denaturant rate

extensive

denaturation

of the protein

solvent 80 sulphur

either

(7),

or through

atom

the

of ferricytochrome

shown to have

liganded

255

in oxidation

the

(6). pathway c.

conjugated

protein

to the

iron.

of

The two heme

an edge exposed the

results

may be a consequence

concerning

through

rates

probably

which

are

has been

The decline

of reduction

heme iron

routes

autoxidation

concentrations

into

the

concentrations

or zero

to reduce.

much speculation

which

must

c

(autoxidizable)

that

concentrations

has been

likely

of

ferrocytochrome

at low denaturant

1 and 2) result

electrons

relatively

1 and 2).

The slow oxidation

There

of urea

presence

is clear

ferrocytochrome

695 nm band of the

more

the

experiments

2 even at high

of the

no oxygen

by oxygen

and urea,it

ferrocytochrome

(Figures

in

oxidation

c must be reduced

quenched

without

became

(5),that

ferricytochrome

the

of the

concentrations

rates

findings

of propanol

consumption

where

which

were constituted

of ascorbate

from

those

695 nm absorbance

at high

proceeds

2.

solutions

the

than

c or both

of autoxidation

and was subtracted

becomes

the

mixtures

except

of oxygen

greater

or ferricytochrome

rate

In view

consumption far

of

reaction

was noted

the

fast

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

to the

itself

via

The results

the

BIOCHEMICAL

Vol. 49, No. 1, 1972

of Wilson more

likely

cleavage the

absorbance

for

in vitro of the

the the

imply

f4)

with

as evidenced

(81,does presence

not of

passage

latter

bond

however

indicate

by the prevent

reduction,

from

is

the

since

appeared

to prevent

that

quenching

the methionine

of electrons

pathway

as reductant

heme iron

Our results

bond,

the

ascorbate

methionine

reduction.

of this

that

and Greenwood

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cleavage

of the

695 nm band

and demonstrate

sulphur ascorbate

is not into

essential

the

heme iron.

References 1. E. Margoliash (1966)

and A. Schejter,

Advan.Prot.Chem.,

21,

263

2. K. Skov and G.R. Williams, in Structure and Function of Cytochromes, Ed. K. Okunuki, M.D. Kamen and I. Sekuzo, University of Tokyo Press, Tokyo, 1968, p.349 3. C. Greenwood 4. M.T.

Wilson

and G. Palmer,

J.Biol.Chem.,

and C. Greenwood, R.L.

Wright

and A.J.

6. L.S. Kaminsky, 1354 (1972)

F.C.

Yong and T.E.

King,

Biopolymers,

258

22,

Davison,

7. R.E. Dickerson, T. Takano, D. Eisenberg, L. Samson, A. Cooper and E. Margoliash, 246, 1511 (1971) and P. Saludjian,

3660

Eur.J.Biochem.,

5. L.S. Kaminsky, 10, 458 (1971)

8. E. Schechter

237,

(1965)

11 (1971)

Biochemistry,

J.Biol.Chem.,

247,

O.B. Kallai, J.Biol.Chem., 2,

788

(1967)