Kinetics of reactions involving C-550 and cytochrome b559 in chloroplasts at low temperature. Evidence for two photoreactions

Vol.

56, No.

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

AND CYTOCHROMB b559 IN CHLOROPLASTS AT LOW TEMPERATURE. EVIDENCE FOR TWO PHOTOREACTIONS.

KINETICS

OP REACTIONS INVOLVING

Paul Service

BIOCHEMICAL

3, 1974

MATHIS,

Michel

de Biophysique, de Saclay,

Received

November

C-550

and Andrd

MICHEL-VILLA2

VERMBGLIO

DLpartement de Biologie. Centre d’Etudes B.P. n* 2 -91190GIF-sur-Wl?TTE.France.

Nuclcaires

28,1973

Summary : The kinetics of reduction of C-550 and of oxidation of cytochrome b55g are studied with spinach chloroplasts, at -170*, under light-limited conditions at different light intensities. The rate of reduction of C-550 of b55g is is proportional to the light intensity I; the rate of oxidation 2-3 times slower and not proportional to I. We propose that two light reactions occur at the reaction center of Photosystem(RC-II) at low temperature. The photoreduction been observed arguments

in photosynthetic

allow

the

of Photosystemdark

of C-550

reaction

(5i.8).

of C-550

and cyt

affected

by the In this

materials

identification (6,7).

of

work,

the

the

the

that

the

oxydation

reactions

reduction

with

under

Several

the

electron

acceptor

results is

(9,lO) of

to be a secondary for

the

presented

are (1 I).

and of oxidation

of

at different

by Butler

to I.

leads us to propose 11 at low temperature.

location

photoreaction

conditions,

proportional

the

by Kok --et al

of C-550

b55g

in Photosystem

assumed

as defined

light-limited

to the

primary

properties

of reduction

b55g have

( 1-5 ).

b55g is

center,

of cyt

temperature

arguments

: several reaction

of C-550

of cytochrome

occur

low

of cyt

different

kinetics

at -170’, cyt b559 are studied Iight intensities. In contrast we find

of C-550

at RC-II

state

photooxidation

at

The oxidation

We provided

b55g

and the

that

--et al (12), The behaviour of at least

two photo-

MATERIAL AND METHODS. Chloroplasts the

grinding

buffer

, prepared ( 0.4

5% dimethylsulfoxide. in liquid

from

spinach

M sucrose;

The suspension

0.02

leaves M Trls,

(9), pH

was distributed

were 7.8;

resuspended 0.01

M NaCl > plus

in 0.5 ml tubes

N 2’ Differential

spectrophotometer Abbreviation

absorption

spectra

were

559 : cytochrome

b 559

(9). : cyt

b

recorded

with

in

a Perkin-Elmer

and kept

Vol. 56, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL

of kinetics

of absorbance

For measurements were

thawed

and transferred

inserted

in a copper

then the transferred

cell

cedure

to a cuvette

frame.

-170’(+

The

actinic

the

plane

the

the

dark.

was dark-adapted

A thermocouple,

The suspension beams,

beam,

cuvette.

windows

( 2 min at

20.);

be referred

in the

euvette,

in a microcrystallized to each

by a 625-w

other,

state.

were

On a parallel

path

filter,

a Schott

of

tungrtene-iodine

the

beam were

a Compur

RG 610-3

at 45’

as I = 100;

smaller

values

of

lamp,

inserted

electronic

mm filter,

of

was focu-

: a 2-cm water

shutter

( opening

time

a MT0 (630 nm ) interference beam at the position of the

The maximum intensity of the actinic -2 ( measured with a calibrated was 25 mW.cm

cuvette

Lucite

inserted

was frozen

perpendicular

provided

(Balzers)

: 8 ms ),

filter.

chloroplasts

cuvette.

a Calflex

1O-90

in

5’).

The actinic sed on the

the

( e = I mm ) made of

The suspension

and measuring of

filter,

changes

uas dipped into a liquid N2 container for 60 s. The cell was then to a partly unsilvered Dewar flask, containing liquid N2. This pro-

was accomplished

indicated

RESEARCH COMMUNICATIONS

Eppley

I were

thermopile.

obtained

with

It

Schott

will

neutral

filters. The measuring a Bausch

light

was provided

sample

and was received

Schott

BG 18-3 mm and two Wratten

500 lQ;a

variable

required

electrical

V, output, signal

change; signals

with

in order

it

0.5

a Didac

The PM output

(Intertechnique)

had no detectable

to improve

the

( load

amplifier analyser. of the

In most

cuvettes

were

: whose

multichannel

effect.

successive

resistor

The amplified

measurement

the

the minimum

voltage.

photochemical

to several

on

by a

to have

W differential

as comparison

beam was .gpened I min before

corresponding

lamp mounted

The beam crossed

I uF was inserted

a Tektronix

was used

mm ).

(EM1 9558 B ) protected

64 filters. 10 nF to

) fed

at 2 volts,

The measuring the

of

bandwith

fixed

( slits

by a photomultiplier

capacitor

was measured

bance

by a 108-W tungsten

and Lomb 500 mm monochromator

absor-

experiments

stored

and added

S/N ratio.

RESULTS.

Comparison The

of

the

kinetics

effect

of

at

illumination

la,

as

a differential

to

the

oxidation

of

cyt

to

the

reduction

of

C-550.

The

mere

studied

of 542

and

b559

reduction 556

light of

absorption

kinetics at

different

chloroplasts

spectrum. and

the

of

intensities. at The

S-shaped

C-550

nm respectively,

-

negative signal

and

of in

170’

is

peak (+

at

542

oxidation the

range

reported at

of of

in

556 nm,

nm -

cytochrome excitation

at

Fig.

is

due 546

b55S inten-

nm)

BIOCHEMICAL

Vol. 56, No. 3, 1974

AND BIOPHYSICAL

I

AA.O.M)l

al u-1 530

:

t

556

I 1

SLO

550

RESEARCH COMMLJ,\(ICATIONS

560

I

530

I

1

!xo

I

1

550

I

L

560

hnm Figure

1.

sities

from

absorbance presented

Difference spectra recorded after illumination of the sample cuvette at -196’ a - Chloroplest suspension used for the results of Fig. 2-4. Illumination by white light (Is).Chlorophyll concentration : 202 ng.ml-1 bIllumination during 15 min by monochromatic light (611 run) of very low intensity ( top curve ) and then during IS bylwhite light (bottom). Chlorophyll concentration : 250 ug ml .

25 mW.cm -2 (I = 100) changes in Fig.

were 2; they

The reciprocal At 542 nm (reduction seen by a straight not

shown,

were

that

the

reduction

the

of

to 0.098

same at all

mW.cm-*

are practically the

t ,,2

for

at 546 nm).

of C-550

is

Typical

of first reaction

of C-550), the rate (tl,2-I) line with a slope of I in the

obtained

(I = 0.39).

intensities.

This

a primary

is

the

saturation, are

order

at both

wavelengths.

is plotted

versus

I in Fig.3.

is proportional log

plot

in agreement

photoreaction.

684

At

kinetics

to I,

(similar with

as

results, the

assumption

An important

departure

Vol. 56, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I = 55

I = 3.75

5L2 nm AA

I

t

hv

hu

556 nm AA

Figure

from At

2.

Time course intensities. of averaged

linearity, 556

reported

nm,

tendency

the to

Evolution

rate

larger of

once

the is

t

112

the

absorbance

ratio

at

is

the

not

of

progressive as

must

dark

in

lb

is

slower,

in

our

with

a

experiments clear

reaction. the

illumination

achieved,

the

proportion

oxidized

cyt

b559.

difference

spectra after the

also

observed

times

when

minutes

(see

not

a limited

of

when

Fig.

2-3

kinetics,

phototransformation, seen

is

after

C-550

a few

was

intensities.

proportion

observed

(12)

highest

described

the

al

change

the

reduction than

et

the

in

spectrophotometer,

shape,

are

the

2 in

of

recorded

9).

such with

So

has some

a

a Perkin-

: at

spectrum ref.

stopped reduced

However

illumination

difference

Fig.

is

any the

dark

part

of

same reaction

occur. Fig.

the

dark

the

cyt

dark

& represents after

reaction however

A time

gap

due

slow

to

an

attempt

photoreduction

b559.

We see

(about

of

Butler

of

to

greater

difference Elmer

by

absorbance

According

C-550

of absorbance changes at 542 and 556 nm, at two light The arrows indicate the opening of the shutter. Number experiments : 6 for I = 55, 3 for I I 3.76 .

No

evolution

is

a reverse that

evolution

between

evolutions

of due

to

60%

the the several

follow of

the

observed,

oxidation

any

remains

1 minute,

of is

to

so of

has abilities baseline) manual

the C-550 that

C-550 to

evolution and we

or

slow

of

the

kinetic

and

of

685

the

of

decide

a dark

very

absorbance

oxidation

cannot

be

operations

of

oxidation (t ,,2

greater

apparatus

Perkin-Elmer following

in

25-30% whether of

of the

cyt than

b559V 5s).

(about spectrophotometer

the

illumination).

10

s,

Vol.

56,

No.

BIOCHEMICAL

3, 1974

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

/

I

,' I'

ix_ 01

/' ,I

D , ,,.' ,' I ,. ,' I 1

IO

III

L I(

Figure

3.

A logarithmic plot the reciprocal of the half-time of absorbance changes at 542 nm (reduction of C-550) and at 556 nm (oxidation Line has a slope of cyt b559) versus light intensity I. The dashed of 1. The continuons line is a best fit for cyt b559. In that plot the straight line with a slope different of 1 has no well-defined signification.

Figure

4.

Time course of absorbance changes at 542 and 556 nm, with and a shutter opening of l/8 s (negative square signal). the vertical step corresponds 8 experiments were averaged; AA = 1.1 x 10-3. At 542 nm, 6 experiments were averaged; vertical step corresponds to AA = 1.8 x 10m3.

I At

= 16.25 556 nm, to

the

DISCUSSION. Our at

-17d

accounted

results its for

confirm rate

of

by

the

that

C-550

reduction

is

currently

used

behaves

as

proportional model

686

a primary to

for

the

I.

electron However

low-temperature

acceptor they

cannot reactions

: be

Vol. 56, No. 3, 1974

BIOCHEMICAL

of Photosystem-

(8,Y) hv -

c550

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

: C - - Chl+ 550

- b55y -

kb

C

- Chl 550

+ - b55y

r where

is

%

a ratio

a dark

reaction.

of 2-3 between in our

b559 reaction,

a : c550

range

Indeed

the

of

rates

according

- Chl

- b55y

We are

to one of a1

r

no value

of reduction

intensities.

e.g.

for

the

of kb this

of C-550

led

to

the

- b55y

Predict

--!% c

of cyt

of a second

models

kr

can

and of oxidation

proposal

two following

C 550 - Chl+

model

light

:

C - - Chl 550

- b;5y

kb

a1 - (Chl

c550

- D)

7

C - - (Chl 550

b:

- D)+

b,

kr

!sE -

(A - Chl)

donor

- A : electron

(A - Chl) - bssy (D:

electron

Model

a/ can readily

with

I and the

time

after

identical

a partial

xidation

of cyt

model

correctly

a definite

predict

the

assumptiorsare

dation

of cyt

b55y

products Model

of b/

donor-acceptor

in the

photoreactions

presents

at RC-II

of

variation

C-550

of the

and of oxidized

However

it

predicts,

we were

not

able

the

reaction

absorbance

cyt for

b55y the

to detect. changes;

kinetics

The second however

is

the

addiof oxibetween

and b2. with

the

on the possible

photooxidation

S2 and S3 states

some

photoo-

proposal

by Joliot

and Joliot

of

electron

temperature. of cyt donor

occurence of these The second reaction b55y : very

and fluorescence low temperatures

two reactions could explain induction (12,14),

-55'

(10,15).

Knaff, D.B. and Arnon D.I.(1969)Proc. Nat. Acad. Sci. Knaff D.B. and Arnon D.I. (1969)Proc. Nat. Acad. Sci. Boardman,N.K., Anderson, J.M. and Hiller, R.G. (1971) Acta 234, 126-136

687

U.S. 63, U.S. 63, Biochim.

in the

in ca-

REFERENCES.

:: 3.

two

(13).

between b55y

that

course

similarities

no information

cyt

lag

b,

at room

ses where

of reduced

time

photosynthesis,

parallelism

observed

)

needed to account for the non-linearity in the rate ( versus I ) and some interaction has to be postulated

pairs

We have normal

the

amount

b2

acceptor

phototransformation.

b55y,

tional the

explain

+ - b559

963-969 956-962 Biophys.

Vol.

56,

No.

3, 1974

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

4. 5.

Bendall, D. S. and Sofrova, D. (1971) Biochim. Floyd,R.A., Chance, B. and Devault, D. (1971)

6.

Erixon, K.and Butler, W.L. (1971) Biochim. Biophys. Acta 234, 381-389 Vermeglio,A. and Mathis, P. (1973) Bioelectrochemistry and Bioenergetics I, in the press. Butler, W.L. (1973) Act. Chem. Res. 6, 177-184 Vermeglio, A. and Mathis P. (1973) Biochim. Biophys. Acta 292, 763-771 Vermeglio, A. and Mathis, P. (1973) Biochim. Biophys. Acta 314, 57-65 Kok, B.,Forbusch, B. and MC Gloin, M. (1970) Photochem. Photobiol. II,

103-I 7.

8. 9. 10. II.

Biophys. Biochim.

COMMUNICATIONS

Acta 234, 371-380 Biophys. Acta 226

12

457-475

12. 13.

14. 15.

Butler, W.L., Visser, J.W.M. and Simons, H.L. (1973) Biochim. Biophys.Acta 292, 140-151 Joliot, P. and Joliot,A. (1972) in Proc. 2nd Int. Congr. Photosynth. (Forti G ., Avron, M. and Melandri, A., eds) Vol. I, pp.26-38, Dr. Junk N.V. Publishers, The Hague. Okayama, S. and Butler, W.L. (1972) Biochim. Biophys. Acta, 267, 523-529 Joliot, P. and Joliot, A. (1973) Biochim. Biophys. Acta 305, 302-316

688