Electron Transfer between Plastocyanin and P700 in Highly - J-Stage

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Jun 9, 1983 - MgCl2 as compared to the case of. P700-chlorophyll a protein complex. .... further extensively purified. The supernatant solution of crude. Triton .... heat treatment of the sample. (20). The concentration of protein was deter-.
J. Biochem.

Electron

Transfer

Photosystem

between

Plastocyanin

I Reaction

and Subunit

Peptide

Teruhiro and

Center

and P700

Complex.

94, 1901-1911

in Highly -Purified

Effects

of pH

, Cations,

Composition

TAKABE,1,*

Shigeru

Hiroshi

ISHIKAWA

,* Satsuki

NIWA,*

ITOH**

*Department

of Chemistry , Faculty of Science and Technology, Meijo University, Tenpaku-ku, Nagoya , Aichi 468, and **National Institute for Basic Biology, Okazaki, Aichi 444 Received

for publication,

Treatment

of

repeated

which

At

values

the

photooxidized lower

at

trivalent of from

which

consists

this

directly

to of

protein

pH

transfer

largest

the pH

complex.

largest and

peptide

cations a

complex

protein seemed

to

of

and

the

and only

no

effects case

a

minor

of of

,

complex

The role

in

the

trivalent

At

pH

pH

with

monovalent the

invol

electron

trans

center

protein,

centers, to

pH

was

the

and

case

metal

of ions

P700-chlorophyll donates and

between

plastocyanin.

of

compared

plastocyanin

interaction

as

iron-sulfur

similar the

well

reaction

as

in

or

indicating rate

the

a protein

to

as

The

that

P700-chlorophyll

have

P700

plastocyanin

decreasing

pH,

MgCl2

as

suggest

complex

low

in

subunit

observed

due

free

per

complex.

with

regions.

Essentially

mainly

virtually

spinach

salts

at

P700

strongly of

are

pH

presence

were

results

is

monovalent

protein

Divalent

peptide

reaction

not

increased

rate

those

the

by

DEAE-

chlorophylls

reduced a

drastically

photooxidized

in

but from

reduction in

7.0

These

P700-chlorophyll

the

the

at

complex.

effects of

only

cations, transfer

cations.

P700

and

protein

complex 30

P700-chlorophyll

without

followed

S-300

X.

divalent

the

X-100

P700-chlorophyll ƒ¿ protein

approximately

and

rate

the

a protein

electron

protein

4.3

to

50%

P700-chlorophyll on

in

Triton Sephacryl

purified The

electron

interaction

of

only

6,

the

electrostatic

B,

reaction

pH

highly

with

the

decreased

plastocyanin

reduced

than

the

about

salts

vement fer

6,

a

A,

P700

than

maximum

or

centers

accelerated

with and

polypeptides. b>10)

higher

efficiently

values

five

(Chl ƒ¿/Chl

fragments

centrifugations

yielded of

iron-sulfur

pH

cations,

a

b

contains

to

thylakoid

gradient

consists

chlorophyll

and

spinach

density

chromatographies

complex of

isolated

sucrose

Sephacel

June 9, 1983

the four

the

observed

largest

small

a electrons

peptide subunits

reaction

with

oxidized

P700;

in

plasto-

cyanin.

1 To

whom

correspondence

Abbreviations:

P700,

SDS,

sodium

et hanesulfonate;

Vol. 94, No.

dodecyl Tricine,

6, 1983

should reaction sulfate;

be center

HEPES,

addressed chlorophyll

. of

photosystem

I;

P700+,

N-2-hydroxyethylpiperazine-N•Œ-2-ethanesulfonate;

N-tris(hydroxymethyl)-metbylglycine;

kobs,

1901

pseudo-first-order

MES, rate

Chl,

chlorophyll;

2-(N-morpholino)-

constant.

(1983)

1902

Plastocyanin is a blue copper (type I) protein which acts as an electron carrier in photosynthetic electron transport between cytochrome f and pho tosystem I reaction center (P700) (1-3). Recent studies have shown that cations regulate the rate of electron transport from reduced plastocyanin to oxidized P700 (P700+) (2-16). However, the ef fects of cations are complex and are still not fully understood (3, 14-16). In sonicated chloroplasts and photosystem I-enriched digitonin- or Tritonparticles, low concentrations of monovalent ( 14 times at the same concentration) (9). The results suggest some specific roles of divalent cations in the reaction between plastocyanin and P700-Chl a protein complex. However, the poly peptide compositions of P700-Chl a protein com plex have not been characterized at all in those preparations. It is desirable to study the effect of cations in relation to the molecular structure of P700-Chl a protein complex. Another interesting point to be studied is the effect of medium pH on the electron transfer from plastocyanin to P700+. If the screening of the negative charges on both the plastocyanin and the P700-Chl a protein complex is essential in the salt effects, a drastic stimulation would be anticipated at low pH due to the decrease of the negative charges on both the plastocyanin and the P700-Chl a protein complex. In addition, under illumina tion, the internal side of the thylakoid membrane, in which most of the plastocyanin is located, is expected to be at low pH. Hitherto, drastic ac celeration at low pH has not been reported. In this paper, we report the preparation of a

T. TAKABE, highly

H. ISHIKAWA,

purified

consists

P700-Chl ƒ¿

of

sulfur

five

on

A,

the

electron

and or

in

concluded

a

to

largest

the

change

tide

of

in

of

metal

ions

that

the

protein it

of

are

protein

the

was

electrons

di

P700-Chl of

cations

mainly

between

P700-Chl ƒ¿

a

effects

study

interaction

on

protein,

subunit

this

detailed

spinach

P700-Chl

center donates

and

observed

a

reduced

the

peptide

complex

pH

in

plastocyanin

the

protein

and

and

iron

From

between

reaction

which

contains

X.

pH

P700+

that

rectly

and of

the

complex

and

reactions

plastocyanin complex

B,

influence

transfer

and S. ITOH

protein

polypeptides

centers,

study

S. NIWA,

the

due

to

largest

complex

pep

and

plasto

cyanin.

MATERIALS The

P700-Chl ƒ¿

from

spinach

that

of

protein

Lien

and

Triton was

density

gradient rich

buffer

mm

and

cm

supernatant

x

17

gradient

NaCl

rich

more

by

eluted

sucrose

Then

density

phacryl

S-300

3 cm2),

and

0.05

Triton column

the

same

liters

of

0

were

buffer. a

and

linear

500

mm.

examined

gradient

once

centrifugation chromatography.

complexes

were

examined

centrifugation,

chromatography

finally

%

0.8

gradient

column

frac against

with

column a protein

sucrose

sucrose

dialyzed and

content

density

P700-Chl

by

of

protein

stepped

between

DEAE-Sephacel

to

further

solution

a

8.0

with

P700

was

DEAE-Sephacel

solution

in

it

centrifugation,

equilibrated

was

Fractions

of

pH a

similar

P700-Chl ƒ¿

were

Tris-HC1

cm2)

of

top

content

to

but

of

After

applied

protein

and

The

on

prepared

method

(9),

(9). P700

were

a

Pietro

extract

layered

in

(10

X-100)

The

San

X-100

complex

(20

by

purified.

crude

METHODS

complexes

chloroplasts

extensively

tions

AND

sucrose

Se (110

density

cm

gradient

x

cen

trifugation. The only

P700

the

centers

(A,

The

for

loaded

on

sucrose, pH

lected, HCl

a

0.1%

the

a

at

centrifugation rich

dialyzed and

0.05

The

particles

and

Triton

(15

10

then

(0.1-1-O mm

220,000 content

buffer

dodecyl were

gradient

P700

follows.

lithium

at

against %

as

1 %

in

has

iron-sulfur

complexes

with

acid,

After

8.0

prepared

sucrose

cholic

which the

protein

4•Ž.

linear

lacks

was

fractions and

pH

X)

incubated

1 h

8.0). h,

and

protein

and

P700-Chl were

sulfate

center

peptide

B,

purified

ƒÊM P700)

13

reaction

largest

x were

(10

m

Tris-HCl

mm

g

for col Tris-

X-100).

J. Biochem.

ELECTRON The

TRANSFER

photooxidation

reduction

of

tinuous a

using

and

nm

750

R-69

and

697

the

the

used

a heat-absorbing

lated

from

the

measured

at

equipped

700

nm

or

filter

a

signal

the (Corning

(2 ƒÊs

lated

by

of

Ke

protein

was

used

the

red

actinic

R-67

according

ascorbate,

and

various

to

wise

mentioned.

100,

which

was

blue

protect

the

xenon

flash

MF-270)

iso

The

P700

of

3ƒÊM

than

purified P700-Chl a protein complex. The P700Chl a protein complex contains five polypeptides, including one major diffuse band at 60-65K daltons and four other bands of 23, 20, 18, and 12K daltons as determined by SDS-polyacrylamide gel electrophoresis (Fig. 1A). This P700-

and

Arnon

P700-Chl

a

P700,

2.5

unless of

%

other-

Triton

P700-Chl

0.001

Isolation of P700-Chl a Protein Complex and P700 Reaction Center Protein-Treatment of iso lated spinach thylakoid fragments with Triton X-100 followed by repeated sucrose density gradient centrifugations and Sephacryl S-300 and DEAE-Sephacel chromatographies yielded a highly

plastocyanin,

salt,

concentration the

for kinetic measurements.

were

0.25 ƒÊM

of

of solutions

coeffi

Hiyama

method

buffer,

lower

a

extinction

to

accompanied

complexes,

to

contained

concentrations The

TMC

nm,

by

the

equivalent 10 mm

authors)

concentrations

mixture

complexes

beam

filter. the

chlorophyll

reaction

mm

glass using

were

a split the

430

• cm -1 determined

The

The

by

ferrihexacyanide

RESULTS

P700

(Kawasaki

4-96)

calculated

determined (18).

using

Sugawara

Hoya

64 mM-1

(17).

P700+.

at

half-duration,

was

cient

of of

preparation

A

time

averager

from

a

content

nm

measurement

photomultiplier light

430

layer). calcu

changes

with

filter

was

(manufactured

with

glass

(6 cm

oxidation

from

4-96

(kobs)

half-reduction

spectrophotometer

For

filter

1903

photo-

isolated

Corning

constant

absorbance

the

light a

water rate

Flash-induced

200).

by

beam A Hoya

protect

actinic

lamp

pseudo-first-order

monitoring

P700 PROTEIN

(24) and 32 mm-1 •cm-1 at 554.5 nm after reduction with dithiothreitol (25), respectively. Reagent-grade chemicals were used throughout, and deionized distilled water was used in the

mentioned,

beam.

to

con

spectropho

the

PURIFIED

1 at 597 nm after

dark

under

reference

blue

tungsten

as

WITH

subsequent

otherwise

light

as was

from W

the measured,

dual-wavelength

nm

filter

multiplier 200

557

light

glass

and

unless

Hitachi

tometer

PLASTOCYANIN

were

illumination

with

a

P700+

OF

a

in

the

X-

protein reaction

mixture. Sodium gel

dodecyl

sulfate

electrophoresis

apparatus ing

was

using

gel

and

running

a

gel

sample

a

6 % (w/v)

7.5-15

(19).

(20).

The

mined

by

spectra

%

The

treatment

of Laemmli

(SDS)-polyacrylamide

performed

gradient

were

out

of Lowry

obtained

by

treatment

concentration a modified

slab

of

procedure a

and the

protein

with

gel

stack

buffer

carried

heat

a

polyacrylamide

electrode

were

without

in

polyacrylamide

the

method

the

sample

was

deter-

(21).

ESR

Brucker

ER-200D Fig.

spectrometer

equipped

with

an

Oxford

of

ESR

9 liquid

helium

Plastocyanin from as

spinach previously

using

and

and

extinction

Vol. 94, No.

6, 1983

cryostat. f

were

komatsuna, (22,

23).

SDS-polyacrylamide

P700-Chl

center

cytochrome

Brassica

described

of plastocyanin by

flow and

1.

gel

electrophoretic

pattern

Instruments

purified

respectively, Concentrations

the

proteins

daltons), bumin

f were

determined

soybean

coefficients

of

mm-1

(14.4K

• cm-

of

proteins;

serum

daltons),

trypsin daltons).

Numbers

marker

bovine (43K

complexes

(B).

positions

cytochrome

4.9

a protein

carbonic

inhibitor 0,

albumin

origin

(20.1K of the

(A) on

and

the

P700

left

side

phosphorylase (67K anhydrase

reaction indicate b (94K

daltons), (30K

oval daltons),

daltons), ƒ¿-lactalbumin running

gel;

P, pigment.

1904

T. TAKABE,

Chl of

a

protein

imately were

b

30

at

1.94,

ironsulfur

and

The

P700-Chi ƒ¿ and five of

of

70K,

25K,

Recently,

have

been

hardii

(27)

particles

protein

and

daltons)

and

daltons).

three

Although been

a

reported

for

protein

sp.

(28,

than spinach

four

by con

daltons

These

(8-19K small

P700-Chl ƒ¿

absence B,

2B,

peptides pro

X)

the

In

P700

reaction

the

P700

be

30,

which

is

complex.

been

sp.

(28).

mg)

in

lithium

fact,

the

no

reported The the

reaction 7.7,

as

P700-Chl ƒ¿ center

sulfate-treated

in

Figs.

and

present protein

suggests

was

case

protein

B).

The

per

P700

found

the The

P700

and small

to

P700-Chl ƒ¿

that

chlorophyll.

spec 1B

lacks

(A

of

SDS-

ESR

protein

that

protein

of

and

center

the

The centers

basis

the

centers

of

to

isolated

on

molecules

for ratios

was

iron-sulfur

shown

as

similar

dodecyl

center

same

the

sulfate.

electrophoresis

This have

protein

reported

iron-sulfur chlorophyll

and

dodecyl and

reaction

of

in

subunits

the

of

essentially

29).

center

subunits

in

and S. ITOH composition

is (26)

(28,

lithium

gel

and

and

chard sp.

been

(31).

number

5.3

Swiss

reaction

has

P700

complex

small

and

subunits

has

polypeptide

with of

tein

(60-70K

polypeptides

P700

troscopy

2C.

the

of

treatment

polyacrylamide

rein 29).

by

was

complexes

polypeptides

small

Fig.

that

S. NIWA,

protein

Synechococcus

particles

molecular 16K

Chlamydomonas

large

more

and

as of

(A,

micro

with

Synechococcus two

1.75

chard

a

The

isolated Swiss

18K,

at

reduced

in

(30),

P700-Chl

The mm

signals

power

polypeptides

from

contain

200

g=

complex

20K,

presence

shown

from

P700-Chl isolated

at

high as

the and

the

complex

our

that

observed.

X

of

coworkers

classes

(26).

of

temperature,

his

to

were

tein

same

spectra complexes

ESR

due

B

conditions

lower

2A,

1.89

and

spectrum

under

wave

have

ESR

in

Fig.

and A

ESR

obtained

weights

1.92,

centers

high-field

in

approx

protein

illumination

shown

free

with

P700.

P700-Chl ƒ¿

during

As

g=2.05,

tains

10)

dichlorophenolindophenol

ascorbate.

Nelson

is virtually b>

per

for

77K

1 mm

preparation

(Chl ƒ¿/Chl

chlorophylls

recorded

frozen of

complex

chlorophyll

H. ISHIKAWA,

of to

four

small

same

result

Synechococcus protein

(nmol/

complex were

pro

and found

the to

be

respectively.

Fig. 2. ESR spectra of P700-Chl a protein complexes (A and C) and P700 reaction center proteins (B). Instrument settings for ESR spectra A and B were: microwave power, 20 mW; frequency, 9.27 GHz; modulation ampli tude, 2 mT; temperature, 18K. Those for C were: microwave power, 100 mW; frequency, 9.22 GHz; modulation amplitude, 4 mT; temperature, 11K. The concentrations of P700 in A, B, and C were 7.7, 5.8, and 7.7 /LM, respectively. Other experimental conditions are described in " MATERI ALS AND METHODS."

J. Biochem.

ELECTRON

TRANSFER

Rate

Law

Reduced

for

a

reverse

30

ms

in

of

those (32).

P700+

was

for the

reduced

the

that

the

of

efflux

acceptors influx

electrons

rapid

P700+

This the

complex

from

very

par

tion

to

the

not

side

rate

plastocyanin.

of

This

in

poor

in

Fig.

MgCl2.

However,

a

stimulation

electron

3,

con-

drastic Traces

B

absolute the

as

those

of

transfer

by

from

purified

(3, P700+

cally

9,

3,

Trace

pH

is

reduction

an

important

was

sults

of

and

the

cytochrome

flow in

f

vitro

also

from vivo

Dependence

plastocyanin

for

reaction

mixture

corbate,

10 mm

P700+

MM

temperature

the

7.0 at

(25•Ž);

P700,

same

buffer same

as

The 2.5

mm

concentrations

(HEPES C,

of

plastocyanin.

various

8•Ž;

D,

by

concentration

0.25 ƒÊM and

pH

MgCl2

on

reduction

buffer, A,

10

kobs

contained

plastocyanin. A plus

of

A

as

at

25•Ž);

B except

except

for

for the

as

Linearity confirmed

flash

reported

cyanin P700+ are

buffer

at

pH

concentration by

indicates

ascorbate.

described

Vol. 94, No.

in

4.8).

Other

" MATERIALS

6, 1983

The

point the

at reduction

experimental AND

zero

conditions METHODS."

These

transfer

re

between

34)

obtained

of

electron

via

plastocyanin

by

plastocyanin

be

to

at

pH

7.0

conditions

of

studied

50 ƒÊM

with

10

by

(Fig.

plastocyanin

MM

MgCl2

measurements

not in

Therefore,

terms

P700+

of

and

mechanisms or

a

bimolec

plastocyanin. have

chloroplasts

and using

shown).

explained

reaction broken

concentration

least

between

in

P700+

in

also

been

photosystem

pH

plasto rate

photooxidized

P700+

the

(data

can

reaction

I-enriched (acetate

at

illumination

Bimolecular

P700-Chl

be

(33,

of

all

up

results

B,

koma

our

studied.

increasing

methylviologen

ular

(Fig. that

Brassica for

occurrence to

with

under

was

100 ƒÊM

f

4.8

reaction.

not

the

rates

linearly

of

the

plastocyanin

drasti

pH

35).

3).

the

and

re that

indicates

electron

support

plastocyanin

3.

high

reduction

increased

Fig.

very

cytochrome

(2,

The

could

highly been

was

from

conditions

Al-

further

at

donor

and the

found

this

purified

electron

complex, any

in has

clearly for

same

electron

P700+

MgCl2

the

9).

the

plastocyanin

factor

poor

(3,

for

we

result

f

a

ƒ¿ protein under

15), by

This

(Fig.

the

al.

cations

without

Cytochrome tsuna

in

et

to

13,

added,

plastocyanin

complex

12,

D).

by

essentially

of

accelerated

was

also

and

are

plastocyanin

7,

without

observed

Pietro

P700-Chl ƒ¿ protein

ported

7.0

MgCl2 was

P700+

San

requirement

shown).

characteristics

MgCl2

reported a

mm rate

of

and

not

is

pH

a

rate

small

plastocyanin at

10 the

rate

presence

though

A,

These

reduction

in

the

C).

reduction

(data

P700+

of

is addi

was

cases

when

and

the

Trace for

electron

was

3,

reductant

ascorbate

effect

most

and

Although

accelerated

neglected

shown

a

3,

P700+.

its

pH

plastocyanin

reaction.

Fig.

ascorbate,

flashes

of

by

reverse

for

of

continuous

effects

P700+

from

slightly

by

be

As

suggests

the

Mg2+

could

reducing

to

of

the

seen

reductant

P700+

the of

of

be

series

under

study

influence

poor

of

can

reduction

can

a

Therefore,

we

little

1905

using

shown).

the

As

same

did

P700+.

compared

with

first-order

measurements

on

(data

illumination, with

by

Mg2+

the

P700 PROTEIN

not

illumination,

about

I-enriched

from

protein is

to

of

(data

of

methylviologen

rate

of

P700-Chl ƒ¿

essentially

plastocyanin

reduction

firmed

flash

probably

phase

continuous

by

Addition

change

are

ms

PURIFIED

from

Methyl

30

photosystem

After

kinetics.

this

results

reported

ticles

rapidly, half-time

methylviologen.

eliminated

These

the

reduced a

WITH

Reaction

P700+-After

with

absence

(100 ƒÊM)

shown).

of

was

reaction,

the

viologen

PLASTOCYANIN Transfer

to

P700+

by

as

Electron

Plastocyanin

illumination,

not

OF

particles

Effects

of

(5, Salts

on

11, the

14,

16,

36,

Reduction

37). of

P700+

by

of Plastocyanin-In or

in

thylakoid

photosystem membranes,

I-enriched low

concentrations

particles of

1906

T. TAKABE,

monovalent

(