Vol.

179,

No.

September

BIOCHEMICAL

2, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

16, 1991

N,N-DIETHYLHYDROXYLAMINE:A

S-C.

T.

Sabat,

ELECTRON

NEW

Sudhakar

School of Life Jawaharlal Nehru New Delhi - 110 Received

August

DONOR TO PHOTOSYSTEM

and Prasanna

Babu

1127-1133

II

Hohanty

Sciences, University,

067,

India

7, 1991

Diethylhydroxylamine, when added to beet spinach thylakoid membranes in the reaction mixture enhanced both photosystem II mediated dichlorophenolindophenol photoreduction and whole chain electron transport supported by methyl viologen. dichlorophenolindophenol Diethylhydroxylamine supports photoreduction when oxygen evolving complex is inactivated by hydroxylamine washings. All the electron transport assays were found to be highly sensitive to diuron, indicating that diethylhydroxylamine donates electrons to the photosystem II the herbicide binding site. The stimulation of before the photochemical activity by diethylhydroxylamine is not solely due to its action as an uncoupler. It was also observed tnat the action of diethylhydroxylamine was not altered by preincubations of thylakoids in light in the presence of diethylhydroxylamlne. Also, thylakoid membranes did not lose their benzoquinone Hill activity by the pre-incubations wltn diethylhydroxylamine either In light or in dark. Thus, unlike the photosystem II electron donor, hydroxylamine, diethylhydroxylamine was found to donate electrons without the inactivations of oxygen evolving complex. It is suggested that dlethylhydcoxylamine 1s a useful electron donor to the photosystem II. 191991Acadrmic Press, Inc.

During chemicals

have

study

of

these

donors

and (3,4),

the

last been

photosystem are

semrcarbaslde hydroxylamlne

three

decades,

described IL

as

(2s

II)

hydroquLnanes, (l),

electron

exogenous

donors

photochemistry.

I

(L), 33corbate

(1)

All

the of

benzldlne

dLpht?nyLsarbazLde

Abbreviations: DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethyl2,6-dichlorophenolindophenol; urea: DCPIP, diethylhydroxylamine; MV, methylviologen; evolving complex: pBQ, para-benzoquinone.

1127

for Some

phenylenediamina;,

cysteine (5th)

several

a:ld

c.ltecnol

DEHA, OEC ,

Copwighr e I9YI rights of reproduction

N,Noxygen

0006-291X/91 $1.50 0~ Academic Press. Im.. irr an! form reserwd.

Vol.

179, No. 2, 1991

electrons

only

Hydroxylamine, inhibitor

apart

an electron

the

to

physiological

of

the

other

D1

this

hand,

evolution

activity

(DEHA),

(10,

13).

donor

PS

has

used

in animal

(15)

and

However, donor

the

as

to our knowledge, for

photosynthetic

communication,

but

for

II.

we present

and

it

has (11).

very

close residue

(12). loss

The

of

NH20H irreversibly

O2

damages

N,N-diethylhydroxylamine acts

as an

efficient

DEHA has been

shown

to

reduction

of quinones

systems

as

an agent

as

to

DEHA has

results,

NH20H or DPC, DEHA is

unlike

NHZOH, DEHA does not

a

delay

not

electron

like

MATERIAL

The

tyrosyl

causes

that

as an

(10).

centres

of hydroxylamine

to

acts

feed

the

reaction

We show

reagent

scavenger

donor

(4).

2+ to Mn ions

close

Z (5),

light,

feeds

inactivated

uncertain

hydroxylamine

potential been

feeds

donor

and in

a derivative

electron

DPC is

in PS II by

DP2

low concentrations

of

donor

is

NH20H has been shown to

protein

donation

PS II

at

electron

electron

that

being

donation

been assumed that

the

from

hydroxyiamine

dowever,

capacity

electron

On

used.

when O2 evolving

of o2 evolution of

RESEARCH COMMUNICATIONS

(DPC) and

known and extensively

best

site

AND BIOPHYSICAL

diphenylcarbazide

Of these,

(R19).

are

BIOCHEMICAL

(14). free

an efficient inactivate

radical

been studied

clearly

a

DEHA

ageing

transport. which

be

In

(16). as

a

this

demonstrate

donor

to PS II,

OEC in light.

AND METHODS

chloroplasts (thylakoid membranes) Broken from beet spinach ( Beta vulgaris palanga) leaves were prepared following the procedure desribed earlier (17). Electron transport rates were monitored polarographically using either 0.5 mM MV or 0.5 mM pBQ as electron acceptors. DCPIP photoreduction was monitored spectrophotometrically as per the procedure mentioned in ref (18). All electron transport measurements -yere done under rate saturating light intensity (-43d W m ). The electron reaction mixture in 1 ml contained 100 mM sucrose, 10 mM NaCl, 5 mM MgC12, 1 mM 1128

Vol.

179,

No.

BIOCHEMICAL

2, 1991

azide and 50 sodium Whenever necessary, different maintain (inactivated OEC) procedure mentioned

AND

mM Hepes buffered appropriate buffer values of pH. thylakoids were in reference (8).

RESULTS Whole H20

--j

addition

MV exhibited of

DEHA

stimulation addition Addition

of

of

whole

an enhancement

in

reaction

chain

to

suggest

that

the

PS

II

(Fig.

electron

mM) than

mixture transport

at about

2.0

(Fig.

2.5

The

by

mM of

the DEHA.

activity

of activity

by

DEHA

curve).

These

donor

and

are much higher

when H20 was used as an electron

6OOr

1).

rates

to

the

by

the

as an electron rates

rates

as

inhibited

1; dotted

transport

measured

the

electron

stimulation

DEHA acts

to pH 7.5 with KO!;. systems were used to Hydroxylamine washed the prepared after

activities

the

COMMUNICATIONS

DISCUSSION

10 uM DCMU completely that

limited

2.5

to

RESEARCH

transport

of DEHA saturated

suggesting

donated

AND

electron

chain

BIOPHYSICAL

is

results the

DEHA

(4 times

at

donor.

As

in

I

“0 YE

01

u.J zl-oo-~-~---~---~---2~---295 0.5 DEHA,mM

Figure 1. Whole chain (-), the presence

Effect of varying electron transport control thylakoids: of 10 PM DCMU.

figure 2. Effect of varying photoreduction. (-), NH20H washed thylakoids: presence of 10 PM DCMU.

0

o-5

1.0

1.5

2.0

DEHA,mM

concentrations of DEHA activities (Hz0 --j MV). (C-----O), thylakoids

on in

concentrations of DEHA on DCPIP control thylakoids; (S), (-1 thylakoids in the

1129

2.5

Vol.

179,

No.

2, 1991

the

case

DCPIP

BIOCHEMICAL

of

whole

photoreduction

concentrations in

chain

the

of

when

aslo

donate

electrons

active.

before

the

As DEHA

like

light

assayed

show

that

light

exhibited

mM DEHA. the

dark

and

DEHA and

then PS II

remained incubation

does

not

washed

with

mediated

the

not

(Fig.

4,

25 min

OEC,

it 1130

or

in

3 (A &

the

B)

electron

presence

were in

light

remove

--3

pBQ)

that

light

did

not

Thus,

the

DEHA

unlike electrons

and

to

pBQ (H20

of

of

incubated

buffer

capacity. that

donates

light

indicating

02 evolving

but

in

pre-incubations

both

with

amount

3 and 4 suggest,

II

were

the

dark

suspension

saturating the

inhibit

membranes

A Z 8)

that

PS

in

by

O2 evolution

to

possible

Figure

stimulation

mM DEHA for

Figures inactivate

of

of

2.5

is

either

and

thylakoid

impair of

results

light

Simiiarly,

with

irreversibly

both

found

thylakoids

MV activity.

as

as

electrons

inctivates

H20 ---)

extent

unaltered

it

did

also

well

2).

possibility,

pre-incubation

presence

NH2OH,

25 min

for

similar

Fig.

to under

as

DEHA donates

mM DEHA for

DEHA,

by

this

seems side

was

(see

washed DCPIP

DEHA

inactive

irreversibly

To test 2.5

and

site

may also

NH20H

oxidizing

that

DCMU.

of

Thus,

the

of

to

transport:

In

photoreduction

a derivative

13).

dark

at

indicating

photoreduction

electron

2).

of

increasing

susceptible

either

binding

is

with

donation

DCPIP

activity

rates

(Fig.

PS II

COMMUNICATIONS

the

DCPIP

DEHA was

OEC is

DCMU,

NH20H

(10,

incubated

in

the

herbicide

DEHA

the

NH20H washing.

observed

DEHA mediated to

with

appreciable

when

sensitive

the

DEHA mediates by

to

conditions

2.5

that

was

transport,

2) and of

membranes,

photoreduction

RESEARCH

stimulated

absence

inactivated

thylakoid

be

also (Fig.

observed

OEC is

the

DEHA

and

BIOPHYSICAL

electron

was

presence

We have

AND

NH2OH, t3

DEHA the

PS

Vol.

179,

No.

BIOCHEMICAL

2, 1991

AND

BIOPHYSICAL

OFF

04

3 3 Fiaure 3. Whole chain electron NV) of thylako&ds pre -incubated in dark (at 4 C) or in light was assayed as in Fig. l.-yalufs pm01 O2 consumed (mg.Zhi) .h A. Thylakoids ), ( ),

( Figure

II

4,

in

pre-incubated mM DEHA.

in

.ight.

2.5

of

thylakobds

of washing

dark.

parentheses

indicatepmol

A. Thylakoids ), (

pre-incubated mM DEHA.

in

dark.

2.5

8. (

pre-incubated mM DEHA.

in

light.

2.5

while

the

OEC is

be

stimulating

DEHA may seems

the

quite

relatively

although,

the

also

extent

Less

the

case of

DEHA treated

the

reason

The

pH dependence

suggesting rates effictive

at

that low electron

(-------),

than

Hill

COntcOl;

pre-incubated

t-------j,

with

of

the

to

activity

the

maximal

Thus, PS II 1131

electron

low PH.

in

(Table

1),

of

DEHA

stimulation be

due

in to

uncoupling.

was quite

DEHA can be at

uncoupler

presence

partial

MV activity

that

NH4Cl

membranes could

induces

DEHA supports

donor

in

an

uncoupler,

The lesser

thylakoid

5).

as

activity

the control.

pH (Fig.

control;

The possibility

stimulated

Hz0 --j

control:

(------),

addition

DEHA possibly of

control;

(-------),

of stimulation

is somewhat

that

J

intact.

the The

mixture

O.Lmln

O2

marginal.

reaction

COMMUNICATIONS

transport activity (H20 --min withD2.5 mM DEHA for 25 consumption (at 25 C). The 0 i n the parent ?Ieses indicate .

pre-incubated mM DEHA.

Effect

Thylakoids ),

dN

2.5

8. Thylakoids

RESEARCH

broad, transport

used

as

an

Vol.

179, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table 1. Effect of uncoupler NH4Cl (5 mM) on the whole chain electron transport rate of beet spinach thylakoids in the presence and absence of 2.5 mM DEHA. For whole chain electron transport assay, the reaction mixture contained 0.5 mM MV, 1 mM sodium aside, 100 mM sucrose, 10 mM NaCl, 5 mM MgC12 in 50 mM Hepes buffer adjusted to pH 7.5. Chloroplasts equivalent to 10 pg of Chl were used. Electron

Transport

Rate

Addition

Donor

Acceptor

-NH4Cl

-NH4Cl

Ratio (+/-)

Control (No DEHA)

H20

MV

105*14

184_+07

1.75

+DEHA (2.5 mM)

H2O/DEHA

MV

445+11

548~14

1.23

In that

in

this

diethylhydroxylamine

donor in

conclusion,

to light.

the

PS II.

Unlike

(DEHA) DEHA

NH20H,

500

communication

the

o-4

-6.0

does

is

not

effect

we

an seem

of

demonstrate

efficient to

electron

inactivate

DEHA

on

the

PS thylakoid

H20--MV DEHA-MV

6.5

7.0

7.5

8.0

8.5

9.0

PH

Fiaure thylakoids (M), III the

5. The pH depandence of MV supported in absence and the presence of presence

control of 2.5

thylakoids: m,M DEHA.

1132

(M),

02 uptake 2.5 mM DEHA. thylakoids

of

II

Vol.

179,

No.

BIOCHEMICAL

2, 1991

membranes

is

activity.

DEHA seems

to

suggest

that

intact.

We

electron

donor

reversible

to

by DEHA however,

AND

BIOPHYSICAL

without donate

remains

electrons

to

exact

COMMUNICATIONS

loss

significant

DEHA can The

PS II.

RESEARCH

when

OEC is

be used

as

site

electron

of

of

an

any still

effective donation

be ascertained.

Acknowledgment The

work

was

supported

by the

project

FG-IN-679,

IN ARS

402.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. LO. 11. 12. 13. 14. 15. 16. 17. ia.

Yamashita, T., and Butler, W.L. (1969) Plant Physiol. 44, 1342-1346. Katoh, S., and San Pietro, A. (1967) Arch. Biochem. 122, 144-152. Biophys. Vernon, L.P., and Shaw, E.R. (1969a) Biochem. Biophys. Res. Commun. 36, 878-884. Vernon, L-P., and Shaw, E.R. (1969b) Plant Physiol. 44, 1645-1649. anA Joliot, A. (1969) Biochim. Biophys. Bennoun, P., hcta 189, s5-94. Mohanty, P., Mar. T., and Govindjee (1971) Biochim. Biophys. Acta 253, 213-221. Bohme, H., and Trebst, A. (1969) Biochim. Biophys. Acta 180, 137-148. Ort, D.R., and Izawa, S. (1973) Plant Physiol. 52, 59560). Wavare, R-A., Prusti, R.K., and Mohanty, P. (1989) Ind. J. Biochem. Biophys. 26, 19-23. Cheniae, G-M., and Martin, 1-F. (1971) Plant Physiol. 47, 568-575. Hsl~, B-D., Lee, J-Y., and Pan, R.L. (1987) Biochim. Biophys. Acta 890, 89-93. Takahashi, Y., Takahashi, M., and Satoh, K. (1986) FEBS 208, 347-351. Lett. Katoh, S., Ikegami, I., and Takamiya, A. (1970) Arch. Biochem. Biophys. 141, 207-218. FLIJ ita, S. , and Sane, K. (1375) Tetrahedron Lett. 21, 1695-1696. RPddY, c-c., Hsicklen, J., Scholz, R-W., Ho, C-Y., Uurgess,