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
(
