David

Njus,

Vishram

ABSTRACT

ology the

Jalukar,

Ascorbic

but its reducing predominant

electron

donor

able neutral tively stable erful

donor

sufficient

to produce

ascorbate

rapidly

reduces

transfer

rather

than

This fast involving

electron

reduction of the cytochrorne anion but would circumvent mediates.

This

may

in the dianion

the

is an exceedingly

dianion

unfavor-

by outer-sphere

electron

the relais a pow-

acid

pH

corbate

observed. from

reaction concerted

rate

alone.

This

mechanism

ofascorbate

energetically

is in-

The

this

may be ratioproton-electron would

ofbiological

permit

monointer-

acid,

cytochrome

bS6l,

and

(Fig

volving

as an electron

metabolic

trons,

ascorbic

and

is oxidized

acid

reactions.

commonly

to the

free

Although

functions radical

donor

(reducing

it can

agent)

lose two

as a one-electron

semidehydroascorbate.

concerted

At

pH both ascorbate and semidehydroascorbate exist as monoanions (Fig I). Consequently, ascorbate oxidation involves the loss ofa proton (Hf) as well as an electron (e). There physiological

are three

mechanisms

by which

this

could

occur.

First, the ascorbate monoanion (AH) could lose an electron and the resultant free radical (AH . ) could then deprotonate to form the semidehydroascorbate anion (A). This mechanism may be ruled out because the midpoint reduction potential (E#{176}’ = +0.766 V) is prohibitively high (Table 1). Alternatively, the ascorbate monoanion may deprotonate to form the dianion (A =), which may then donate an electron to form the semidehydroascorbate anion. This outer-sphere mechanism

(Fig

2, A) accounts

for

reduction

by ascorbate

involving

compounds at neutral

or

of electron

biologically

pro-

suggests

that

transfer

between

pH.

We propose

significant

ascorbate-

using ascorbic acid as a reducing monoanion via a mechanism in-

deprotonation

ascorbate

donor

concerted

evidence

and electron

b56 I is a transrnembrane

intravesicular

levels

to cytochrome

(

transfer.

protein

found

in the

Its function is to reduce intraback to ascorbate to maintain 1 3- 15). The

extravesicular

dcc-

is probably cytosolic ascorbate. cytochrome b56 1 mediates transmembrane dccfrom cytosolic ascorbate to intravesicular semi-

Consequently,

transfer

b561

(Fig

3).

In

this

way,

it provides

reducing

ascorbate-consuming enzymes such and peptidyl-glycine a-amidat-

ing monooxygenase. Cytochrome

elec-

donor

the semidehydroas-

form

b56 1 at physiological

membranes ofsecretory vesicles. vesicular semidehydroascorbate

tron

acid functions vital

also

rate

to other

equivalents for intravesicular as dopamine /3-monooxygenase

Ascorbic in many

slowly

2, B). Kinetic

cytochrome

this also applies

dehydroascorbate Introduction

ascorbate

relatively

may

for the rapid

Cytochrome

proton-

pK2 is 11.34,

of total reduces

by a mechanism

requiring reactions: enzymes agent react with the ascorbate

ascorbic

because

fraction

ascorbate

transfer

directly

account

ascorbate

tron Ascorbic

Accordingly,

transfer

may

that

Moreover, small

monoanion

anion

adrenal

acid utilization: enzymes using ascorbic acid may react with the ascorbate monoanion via concerted proton-electron transfer. Am J C/in Nutr 199 1 ;54: 1 l79S-83S KEY WORDS electron transfer

ascorbate

ton-electron

med-

form.

pH (9, 12).

For example,

by the abundant ascorbate formation of unfavorable

be a general

pH

pH.

b56l

transfer

in bi-

at physiological

at neutral

cytochrome

chromaffin vesicles. by a mechanism

reductant

dianion forms radical anion and

rates

M Kelley

is a poor

its concentration

the reaction

1

monoanion)

to the

ascorbate

Patrick

At physiological

(the

it oxidizes

but

and

is an essential

of ascorbate

because

Zu,

is paradoxical.

free radical. The semidehydroascorbate

electron

ullary nalized

acid

power

form

Jian

between

b56 1 contains

a single

noncovalently

heme (17, but see reference 18 for a contrasting protein, cloned and sequenced (19), consists with

a molecular

weight

of 30 061.

The

view).

of273

primary

bound

The bovine amino acids structure

in-

that the cytochrome is a very hydrophobic protein with possibly six transmembrane regions and very little extramembranous protein. A relatively large fraction of aromatic amino dicates

acids

(16%)

ficiently

may

across

Furthermore,

clusters

the membrane ascorbate sequence

the

single long

heme distance

of cationic

may facilitate

amino

interaction

to transfer (across

electrons

ef-

the membrane).

acids on either ofthe cytochrome

side of with

and semidehydroascorbate. Cytochrome bS6l has little homology to other cytochromes, indicating that it may

represent perform

allow a relatively

a new class its unique

of cytochrome

independently

evolved

to

function.

of such

compounds

as ferricyanide and cytochrome c. The ascorbate dianion is a good electron donor since the midpoint reduction potential (E#{176} = +0.076 V) is quite favorable (Table 1). The rate of electron transfer via this pathway is highly pH dependent, however, because it reflects the pH dependence of the fraction Am J C/in Nuir

199l;54:l

1795-835.

Printed

in USA.

© 1991 American

I From the Department ofBiological Sciences, Wayne State University, Detroit. 2 Address reprint requests to D Njus, Department of Biological Sci-

ences, Society

Wayne for Clinical

State

University,

Nutrition

Detroit,

MI 48202.

I 179S

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Concerted proton-electron transfer ascorbic acid and cytochrome

1 1 80S

NJUS H2COH

H,COH

H2COH

pK1

HCOH

pK2

HCOH

1,o...,_o

-H’

H2COH HCOH

HCOH

pK2

-

sS

E01{

I

41.c

Cyt c

E02

H2S:OH

H2SxIH

PKr

E02

HcOH

HH -H’

Cyt c red

+11’ HO

0

H2OH

AH’

HCOH

FIG 1 . Interconversion ofascorbate species. Protonation reactions are shown horizontally and electron transfer reactions are shown vertically.

Because

electron

transfer

b56 1 , its electron-transfer catalytic

functions.

model

For

and

have

investigating

been

pared

Results The

bovine

adrenal

of cytochrome

complicated

by other

cytochrome

ofelectron

enzymes. kinetics

b56 1 in resealed

from

the

the kinetics the

function

are not

reason

ascorbate-using

ofcytochrome

is a superb

transfer

With

that

in mind,

of electron-transfer

membrane

chromaffin

H2cOH HcOH

we

reactions

vesicles

medullary

B)

between

(ghosts)

Cyt b561

pre-

vesicles.

and discussion simplest

Cyt b561 red

kinetic

tron-transfer the

that

for investigating

ascorbate

is the only reactions

analysis

reaction

of the

studied

cytochrome

the kinetics

b56 1 and

both

Cyt0,, + AH Cyt

+ FeCy0

Cyt

+ A;ut Cytox

two

H

AHUt

the rate

to a rate

reactants.

of the

and

+ A, +

Cyt0

+ AHUt

Cyt

+

this

dccand

+ H out

+ H

to

approach,

reactions

external

FeCy

Cyt0

ofan

constant

Using

following

internal

Cyt out

+

+

that

is proportional

concentrations

we have

assumes

between

HCOH

substrates:

(Reaction

1)

(Reaction

2)

(Reaction

3)

(Reaction

4)

FIG 2. Mechanisms transfer.

Reduction

ofascorbate

ofcytochrome

concerted proton-electron b56 1) is indicated. TABLE 1 Thermodynamic

parameters

of ascorbic

Parameter E,,7(V)

(V)

+0.766

E#{176}t (V) pK1 pI(2

+0.076 4.04 11.34

pK,

-0.45

Eo

S

The

assigned

value

using the following Similar compilations

t The assigned Kr using

K2K1)/(l

Measured values

+0.330

the equation +

EH]/Kr)}.

Reference

transfer.

Reduction

electron

(B) Hypothesized

ofcytochrome

for defining

rate constants

reactions

at the inside

surface

1) to denote

reactions

0 (or -0)

to denote

bS6l

the subscript

and

subscript

outward

direction

in the inward direction. The (A for ascorbate-semidehydroascorbate

1 2

+0.340 +0.93 +0.85to+l.00

3 4 5

transfer reactant

4.04 11.34

6 6

the three experimental state electron transfer

b56 1 (Cyt

0.45

7

equation: E#{176}’ = E#{176} + (RI/F) have been given by others

E#{176}2, pK2

,

(ln 10) (pK2 (9-I 1).

value for E#{176}2 was calculated from E,,7 , K1 (8): E#{176} = E,,1 (RI/F) ln {( 1 + [W]/K2

vesicle

and pKr -

, K2 ,

and

indicates

a negative

is to use the subscript of cytochrome

electron subscript

at the outside transfer

indicates

superscript

in the electron

and

denotes the F for fern-

reactions

by using

ferrocyanide).

We have

from

-

A positive

+0.300

for E#{176}’ was calculated

1 (or

surface.

+0.330

-

-

c (Cyt c) is indicated.

acid

Our convention Assigned value

A (A) Outer-sphere

oxidation.

pK) and

+ [H]2/

ghosts

determine

the kinetics

rate

ferncyanide

as ferricyanide

nide concentration, tionality allows

ferricyanide

constants

to cytochnome

to external

of the above

in Figure 4. Steadywithin chromaffin-

schemes illustrated from ascorbate trapped

to external

the

ascorbate sured

studied

(Fig

for electron b56l

(k). reduction)

(ks’) The

and

rate

4, A) may transfer

from

be used

from

cytochrome

ofelectron

is proportional

at least at low concentrations. us to calculate the rate constant

transfer to the This k.

to

internal b561 (meaferricyaproporAt high

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AH2

ml

H2COH

A)

HCOH

a

HO

OH

AL

-H’

H)...( HO

ET

PROTON-ELECTRON

ferncyanide

concentrations

a maximum

rate that

concentration.

This

k’ . The

details

the

rate

of electron

is proportional allows

of this

us

method

transfer

reaches

to the internal

to

calculate

have

the

been

in secretory vesicles. 1, semidehydnoascorbate with permission from reference 16).

rate

constant

published

elsewhere

steady-state

determine

the

chrome

bS6l

addition medium

rate

experiment constant

to external

external

semidehydroascorbate.

electron

transfer

from

The external rate,

the

rate

and

constant

the

centration Finally,

state

spectrophotometrically

fin-vesicle

membranes

with

tion is proportional to the low ascorbate concentrations)

cyto-

In this case,

it can

internal

state

can then

cytochrome,

after ascorbate.

be inferred

ascorbate

of the the

from

Rate

constants

this

mixing initial

cyto-

stoppedb56l is

of chromafrate

of reduc-

ascorbate concentration (at least at and the slope is the rate constant

constants and

for the above

approaches,

are

for the

reactions

semidehydroascorbate

parent

that

electron relatively

they

reactions,

summarized

are too

determined

in Table

of cytochrome (ks’,

k,

k,

) are

transfer. Cytochrome c is reduced slow rate at physiological pH (12,

is highly

pH

dependent,

increasing

by using

2. When b56 1 with

fast to be compatible

the

with

rate

ascorbate

examined,

it is ap-

(ks’ and ofcytochrome

than

would

of

at a this 10 with

k1

not

reduction

Rates

nearly

as pH

reduction The

of cytochrome

of cytochrome

(Table

reduction

as would

2). Moreover,

by ascorbate

rate

b56 1 re-

dependent

by the dianion

b56l

c (midpoint

corbate L . moi

are much

constant

for reduction

potential

= +0.26

potential

However,

the

ascorbate

(+0.

rate

. 1

ofoxidation

is also

for

6.0 is 35 L

L#{149} mol

310

rate

14 V), should

constant

at pH

and

corbate

mol

.

the

at

be reduced

reduction .

‘

faster

than

b56 surface

surface b561

expected

when

(k1).

compared

a rate

potential mol

.

has a midpoint

of +0.42 .

reduction

be reduced more slowly. (k’) of 2 X 106 L . action

between

occurs

too

transfer. The

anomalous

transfer.

of

quickly

permits

ascorbate

b56 1 and

be

attributed

transfer

reduction

also

facilitate

outer-sphere

between

involving monoanion.

will

of 6.2 which

V (Table

1) should

be

concerted

the

rationalized

in

proton-electron

of cytochrome This

electron

ascorbate-semide-

b56 1 can

reaction

faster than a reaction requiring the ascorbate the rate will be much less pH dependent. electron

(kr)

semidehydnoascorbate to

of reaction

cytochrome

a mechanism

constant

of +0.076

the

Instead, we measured a rate constant . s’. Again, this suggests that the re-

rt

rates

with

(Table 2). a midpoint

Semidehydroascorbate,

potential

to

and

This

6.0.

cytochrome

hydroascorbate terms

V, with

at pH

1

1

by semidehydroas-

reduction L

slowly.

at the internal

external

ofcytochrome

more

by ferncyanide which has

l0

V) by as-

of cytochrome

rate of oxidation ofcytochrome b56l Cytochrome b56l reduces ferricyanide,

x

faster of cy-

at pH 6.0 has been measured as 2.5 (23) or 0.83 (12) ‘ . ‘ . Cytochrome b56l, which has a lower midpoint

reduction

(ks’)

) are

2, cytochnome

with

(12).

be expected.

tochrome

by

is consistent dianion

for reduction

rates

abundant

outer-sphere

by ascorbate 23). Moreover,

by a factor

duction

The con-

to

by using the of cytochrome The

con-

measured

(22). ascorbate

the

ascorbate

cytochrome.

be calculated and

to

of steady-state

(22).

these

rate

), the

may be monitored directly (Fig 4, C). The reduction

monitored

rate

directly,

redox

(k)

of the

the

of external semidehydroascorbate electron transfer from external

chrome bS6l flow method

k

(k

measured

from

(k).

to

oxidase to the suspension from internal ascorbate

be measured

rate constant

redox

ascorbate transfer

be used

transfer

Although

cannot

the internal

electron

semidehydroascorbate

of ascorbate and initiates electron

centration,

for

(Fig 4, B) may

This

c by the

be expected

(20, 21).

A similar

pH unit.

every

ascorbate

reductase;

b56 therefore

dianion. Concerted

oxidation

1 by may Moreover, proton-

of cytochrome

the be

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FIG 3. Mechanism ofascorbic acid regeneration b56l; 3, dopamine 5-monooxygenase (reprinted

1181S

TRANSFER

1 182S

NJUS

ET

AL TABLE 2 Sum mary of rate constants5 k4

pH

k

k’

35 55 9.4

±

12

±

12

3.6

±

(6.2 (6.7

±

(5.1

± 5.1)

SD. Values

S ;

±

2.7) x l0 3.4) X l0

for k’ and

s’

(2.0 (1.2 (1.7

l0

X

.

k were

x 106

0.7) 0.5) 0.2)

± ± ±

X

106

X

l0

determined

310 ± 10 450 ± 190 570 ± 190

using

the reaction

scheme shown in Figure 4, A (21). Values for k and k1 were determined using the reaction schemes shown in Figures 4, B and C, respectively (22).

b56 1 by semidehydroascorbate becomes

the

ascorbate

unfavorable We

ascorbate

have

chosen

to emphasize

the

because monoanion

semidehydroascorbate

instead

of the

energetically

dianion. the

term

distinct

concerted

roles

proton-electron

of H

and

e

and

transfer not

to convey

any mechanistic connotations. In terms ofmechanism, one could imagine two possibilities: first, when ascorbate passes an electron to the heme, the proton could be physically transferred to a

B

protonatable

AH#{149}

7t:t.

group

H

cytochrome.

Alternatively,

the

Ionic

dianion

form.

bonding

between

the

ascorbate

mono-

anion and a cationic group in the cytochrome could cause the pK for ascorbate bound to the cytochrome to be considerably lower than pK2 for free ascorbate. This would substantially indissociation complex

of the second proton and therefore facilitate

ofconcerted

ben of significant

from ascorbate electron transfer.

proton-electron

implications.

First,

because

duction electron

in many of these cases transfer. Second, concerted

account Because

for the unique biological properties outer-sphere oxidation ofascorbate

by the exceedingly

small

fraction

has a num-

ascorbate

functions

effective

unreactive

reductant. until

of ascorbic acid. is kinetically limited

in the dianion

Consequently,

it encounters

an enzyme

form,

ascorbate

proton-electron use ascorbate

ascorbic

acid

designed

unique combination, relative stability in the high reactivity in specific metabolic reactions, acid especially attractive as a reducing agent

AH

occur at transfer that re-

occurs via concerted protonproton-electron transfer may

is a fairly stable reductant. Because concerted transfer occurs quickly, specific enzymes can very

in

transfer

as a reducing agent in many biological reactions that neutral or slightly acidic pH and outer-sphere electron is inefficient under these conditions, it is quite likely

C

H

the

The concept +

the

not to the protein but to water. The protein’s site could induce ascorbate to deprotonate to

crease the the reaction A-

on

could be transferred ascorbate-binding

as a

is relatively to use it. This

environment and makes ascorbic in biological sys-

tems.

a

References lyanagi I, Yamazaki I, Anan KF. One-electron oxidation-reduction properties of ascorbic acid. Biochim Biophys Acts l985;806:25561. 2. Steenken 5, Nets P. Electron transfer rates and equilibria between 1.

FIG 4. Reactions

schemes

for rate-constant

measurements.

substituted phenoxide ions and phenoxyl radicals. i Phys Chem 1979;83:l 134-7. 3. Everling VFB, Weis W, Staudingen H. Determination ofthe standard reduction potential (pH 7.0) of L-(+)-asconbate/semidehydno-L(+)ascorbate by nonenzymatic reaction of L(+)-ascorbate/semidehydro-

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6.0 7.0 8.0

mo/’

.

L

AH

k1

PROTON-ELECTRON

4.

6.

7.

8.

9. 10. 1 1. 12. 13.

in biological membranes. Reading, MA: 1981:365-74. J, Cook C, Kelley PM. Electron transfer across the chromaffin granule membrane. i Biol Chem l983;258:27-30. 15. Njus D, Kelley PM, Hannadek Gi. Bioenergetics ofsecnetory vesicles. Biochim Biophys Acts l986;853:237-65. 16. Njus D, Ozkan ED, Kelley PM. Bioenergetics and the adrenal medulla. In: Dulbecco R, ed. Encyclopedia of human biology. Vol 1. San Diego: Academic Press, 1991:641-53. 17. Apps DK, Boisclair MD, Gavine ES, Pettignew GW. Unusual redox behaviour of cytochnome b-56 1 from bovine chnomaffin granule membranes. Biochim Biophys Acts l984;764:8-l6. 18. Degli Eposti M, Kamensky YA, Arutjunjan AM, Konstantinov AA. A model for the molecular organization of cytochrome b-56l in chromaffin granule membranes. FEBS Lett l989;254:74-8. 19. Perin MS, Fried VA, Slaughter CA, SUdhof IC. The structure of cytochnome b561 , a secretory vesicle-specific electron transfer proosmotic

proton

circuits

Addison-Wesley, 14. Njus D, Knoth

tein.

20.

EMBO i 1988;7:2697-703.

Kelley PM, Njus D. A kinetic analysis of electron transport acnoss chromaffin vesicle membranes. i Biol Chem l988;263:3799-804. 21. Jalukan V, Kelley PM, Njus D. Reaction of ascorbic acid with cytochnome b56l: concerted electron and proton transfer. i Biol Chem 199 1;266:6878-82. 22. Kelley PM, ialukar V, Njus D. Rate of electron transfer between cytochnome b56 1 and extravesiculan ascorbic acid. J Biol Chem 1990;265: 19409-13. 23. Yamazaki I. The reduction of cytochnome c by enzyme-generated ascorbic free radical. J Biol Chem l962;237:224-9.

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L(+)-asconbate with cytochnome b5(Fe2/cytochnome b5(Fe3). Hoppe Seylers Z Physiol Chem l969;350:886-8. Pelizzetti E, Mentasti E, Pramauro E. Kinetics and mechanism of the oxidation ofascorbic acid by Tris(l,lO-phenanthroline)inon (III) and its derivatives in aqueous acidic perchlorate media. Inorg Chem 1976; 15:2898-900. Pelizzetti E, Mentasti E, Pramauro E. Outer-sphere oxidation of ascorbic acid. Inong Chem 1978;l7:l 18 1-6. Iaqui Khan MM, Martell AE. Kinetics of metal ion and metal chelate catalyzed oxidation ofascorbic acid. IV. Uranyl ion catalyzed oxidation. i Am Chem Soc l969;9 1:4668-72. LaroffGP, Fessenden RW, Schuler RH. The electron spin resonance spectra of radical intermediates in the oxidation of ascorbic acid and related substances. i Am Chem Soc l972;94:9062-73. Ilan YA, Czapski G, Meisel D. The one-electron transfer redox potentials of free radicals. I. The oxygen-superoxide system. Biochim Biophys Acts l976;430:209-24. Williams NH, Yandell JK. Outer-sphere electron-transfer reactions ofascorbate anions. Aust J Chem 1982;35:l 133-44. Creutz C. The complexities ofasconbate as a reducing agent. Inong Chem 198 l;20:4449-52. Njus D, Kelley PM. Vitamins C and E donate single hydrogen atoms in vivo. FEBS Lett l99l;284:l47-51. Al-Ayash Al, Wilson MI. The mechanism of reduction of singlesite nedox proteins by ascorbic acid. Biochem J 1979;l77:64l-8. Njus D, Zallakian M, Knoth i. The chnomaffin granule: protoncycling in the slow lane. In: Skulachev VP, Hinkle PC, eds. Chemi-

ll83S

TRANSFER