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Mössbauer effect in Scenedesmus and spinach ferredoxins. The mechanism of electron transfer in plant-type iron–sulphur proteins

Authors:
  • Transgene Biotek Ltd

Abstract

1. The Mössbauer spectra of Scenedesmus ferredoxin enriched in (57)Fe were measured and found to be identical with those of two other plant-type ferredoxins (from spinach and Euglena) that had been previously measured. Better resolved Mössbauer spectra of spinach ferredoxin are also reported from protein enriched in (57)Fe. All these iron-sulphur proteins are known to contain two iron atoms in a molecule that takes up one electron on reduction. 2. The Mössbauer spectra at 195 degrees K have electric hyperfine structure only and show that on reduction the electron goes to one of the iron atoms, the other appearing to remain unchanged. 3. In the oxidized state, both iron atoms are in a similar chemical state, which appears from the chemical shift and quadrupole splitting to be high-spin Fe(3+), but they are in slightly different environments. In the reduced state the iron atoms are different and the molecule appears to contain one high-spin Fe(2+) and one high-spin Fe(3+) atom. 4. At lower temperatures (77 and 4.2 degrees K) the spectra of both iron atoms in the reduced proteins show magnetic hyperfine structure which suggests that the iron in the oxidized state also has unpaired electrons. This provides experimental evidence for earlier suggestions that in the oxidized state there is antiferromagnetic exchange coupling, which would result in a low value for the magnetic susceptibility. 5. In a small magnetic field the spectrum of the reduced ferredoxin shows a Zeeman splitting with hyperfine field (H(n)) of 180kG at the nuclei. On application of a strong magnetic field H the spectrum splits into two spectra with effective fields H(n)+/-H, thus confirming the presence of the two antiferromagnetically coupled iron atoms. 6. These results are in agreement with the model proposed by Gibson, Hall, Thornley & Whatley (1966); in the oxidized state there are two Fe(3+) atoms (high spin) antiferromagnetically coupled and on reduction of the ferredoxin by one electron one of the ferric atoms becomes Fe(2+) (high spin).
Biochem.
J.
(1971)
122,
257-265
257
Printed
in
Great
Britain
Mossbauer
Effect
in
Scenedesmus
and
Spinach
Ferredoxins
THE
MECHANISM
OF
ELECTRON
TRANSFER
IN
PLANT-TYPE
IRON-SULPHUR
PROTEINS
BY
K. K.
RAO,
R.
CAMMACK,
D.
0.
HALL
AND
C.
E.
JOHNSON*
Department
of
Botany,
University
of
London
King's
College,
68
Half
Moon
Lane,
LondonS.E.24,
and
Atomic
Energy
Research
Establishment,
Harwell,
Berk8.,
U.K.
(Received
9
November
1970)
1.
The
Mossbauer
spectra
of
Scenedesmus
ferredoxin
enriched
in
57Fe
were
measured
and
found
to
be
identical
with
those
of
two
other
plant-type
ferre-
doxins
(from
spinach
and
Euglena)
that
had
been
previously
measured.
Better
resolved
M6ssbauer
spectra
of
spinach
ferredoxin
are
also
reported
from
protein
enriched
in
57Fe.
All
these
iron-sulphur
proteins
are
known
to
contain
two
iron
atoms
in
a
molecule
that
takes
up
one
electron
on
reduction.
2.
The
Moss-
bauer
spectra
at
195°K
have
electric
hyperfine
structure
only
and
show
that
on
reduction
the
electron
goes
to
one
of
the
iron
atoms,
the
other
appearing
to
remain
unchanged.
3.
In
the
oxidized
state,
both
iron
atoms
are
in
a
similar
chemical
state,
which
appears
from
the
chemical
shift
and
quadrupole
splitting
to
be
high-spin
Fe3+,
but
they
are
in
slightly
different
environments.
In
the
reduced
state
the
iron
atoms
are
different
and
the
molecule
appears
to
contain
one
high-spin
Fe2+
and
one
high-spin
Fe3+
atom.
4.
At
lower
temperatures
(77
and
4.2°K)
the
spectra
of
both
iron
atoms
in
the
reduced
proteins
show
magnetic
hyperfine
structure
which
suggests
that
the
iron
in
the
oxidized
state
also
has
unpaired
electrons.
This
provides
experimental
evidence
for
earlier
suggestions
that
in
the
oxidized
state
there
is
antiferromagnetic
exchange
coupling,
which
would
result
in
a
low
value
for
the
magnetic
susceptibility.
5.
In
a
small
magnetic
field
the
spectrum
of
the
reduced
ferredoxin
shows
a
Zeeman
splitting
with
hyperfine
field
(H.)
of
180kG
at
the
nuclei.
On
application
of
a
strong
magnetic
field
H
the
spectrum
splits
into
two
spectra
with
effective
fields
Hn
+H,
thus
confirming
the
presence
of
the
two
antiferro-
magnetically
coupled
iron
atoms.
6.
These
results
are
in
agreement
with
the
model
proposed
by
Gibson,
Hall,
Thornley
&
Whatley
(1966);
in
the
oxidized
state
there
are
two
Fe3+
atoms
(high
spin)
antiferromagnetically
coupled
and
on
reduction
of
the
ferredoxin
by
one
electron
one
of
the
ferric
atoms
becomes
Fe2+
(high
spin).
Plant
ferredoxins
are
among
the
simpler
iron-
sulphur
proteins,
since
their
molecules
contain
only
two
iron
atoms
and
on
reduction
they
take
on
only
one
electron
(see
review
by
Hall
&
Evans,
1969).
Mossbauer
spectra
of
57Fe
have
been
measured
in
ferredoxins
from
spinach
(Johnson
&
Hall,
1968;
Johnson,
Bray,
Cammack
&
Hall,
1969)
and
from
Euglena
enriched
by
growth
on
s7Fe
(Johnson
et
al.
1968).
The
spectra
were
similar
to
those
ob-
tained
from
the
more
complex
metalloflavoprotein,
xanthine
oxidase
(Johnson,
Knowles
&
Bray,
1967;
Johnson
et
al.
1969).
We
present
here
results
on
another
plant-type
ferredoxin
from
the
green
alga
Scenedesmus,
to-
gether
with
improved
results
on
spinach
ferredoxin.
The
purified
proteins
were
enriched
in
57Fe
by
exchange,
by
using
a
method
derived
from
that
of
*
Present
address:
Oliver
Lodge
Laboratory,
Univer-
sity
of
Liverpool,
Liverpool
L69
3BX,
U.K.
9
Lovenberg,
Buchanan
&
Rabinowitz
(1963).
This
procedure
greatly
increases
the
sensitivity
of
the
Mossbauer
method,
since
naturally
occurring
iron
contains
only
2.2%
of
57Fe.
However,
the
method
is
capable
in
some
circumstances
of
producing
artifacts
that
have
a
similar
absorption
spectrum
to
the
plant-type
ferredoxins
(see
Yang
&
Huennekens,
1970);
moreover
the
product
may
be
contaminated
with
excess
of
"7Fe,
which
may
be
bound
very
tightly
to
the
ferredoxin
apoprotein
(Keresztes-
Nagy
&
Margoliash,
1966).
This
excess
of
iron
would
also
contribute
to
the
M6ssbauer
spectrum,
producing
two
lines
in
the
centre
of
the
spectrum.
These
lines
are
similar
to
the
spectrum
of
the
oxi-
dized
ferredoxin,
and
are
thus
detectable
only
in
the
reduced
state.
Some
previously
published
M6ss-
bauer
spectra
of
reduced
spinach
ferredoxin
(Moss,
Bearden,
Bartsch,
Cusanovitch
&
San
Pietro,
1968;
Johnson
&
Hall,
1968)
showed
these
two
lines
to
a
Bioch.
1971,
122
K.
K.
RAO,
R.
CAMMACK,
D.
0.
HALL
AND
C.
E.
JOHNSON
greater
or
lesser
extent
and
as
a
result
were
incorrectly
interpreted.
Therefore
in
the
present
work
the
57Fe-reconstituted
ferredoxins
were
care-
fully
purified
by
chromatography
on
DEAE-
cellulose
under
anaerobic
conditions
and
the
pro-
ducts
were
examined
by
microchemical
analysis,
by
biological
activity
and
by
a
number
of
spectral
methods
to
check
that
they
were
chemically
iden-
tical
with
the
native
proteins.
The
results
on
57Fe-enriched
spinach
ferredoxin
agreed
very
well
as
far
as
could
be
seen
with
those
on
the
unenriched
protein,
though
of
course
there
was
far
more
detail
observable
in
the
present
results.
At
the
same
temperature
and
magnetic
field
the
spectra
were
essentially
identical
for
Scendesmus,
spinach
and
Euglena
ferredoxins.
Gibson,
Hall,
Thornley
&
Whatley
(1966)
pro-
posed
a
model
for
the
active
centre
of
spinach
ferre-
doxin
mainly
from
e.p.r.*
evidence.
In
this
model
the
oxidized
ferredoxin
molecule
contains
two
high-
spin
ferric
atoms
and
the
reduced
molecule
contains
one
high-spin
ferric
atom
and
one
high-spin
ferrous
atom.
The
low
magnetic
susceptibilities
that
have
been
reported
(Thornley,
Gibson,
Whatley
&
Hall,
1966;
Moss,
Petering
&
Palmer,
1969)
are
explained
by
antiferromagnetic
exchange
coupling
between
the
iron
atoms.
The
present
work
was
undertaken
to
examine
carefully
the
model
by
using
improved
Mossbauer
techniques.
The
substance
of
this
paper
was
presented
at
the
4th
International
Conference
on
Magnetic
Reson-
ance
in
Biological
Systems,
Oxford,
August
1970
(C.
E.
Johnson,
K.
K.
Rao,
R.
Cammack,
M.
C.
W.
Evans
&
D.
0.
Hall).
EXPERIMENTAL
Materials
Scenede8mus
cells
similar
to
those
used
by
Matsubara
(1968)
were
kindly
supplied
by
Dr
H.
K.
Gee,
Richmond
Field
Station,
University
of
California,
U.S.A.
Spinach
was
obtained
from
Covent
Garden
Market,
London
W.C.2,
U.K.
Iron
enriched
with
87.8%
of
57Fe
was
obtained
as
the
element
from
the
Isotope
Separation
Group
of
the
Atomic
Energy
Research
Establishment,
Harwell,
Berks.,
U.K.;
the
sample
contained
0.03%
of
copper
and
less
than
0.015%
of
other
heavy
metals.
It
was
dissolved
in
3M-
H2SO4
and
neutralized
with
NaOH
before
use.
DEAE-
cellulose
(Whatman
DE23
and
DE52)
was
obtained
from
W.
and
R.
Balston
Ltd.,
Maidstone,
Kent,
U.K.
NADP
was
from
the
Boehringer
Corp.
(London)
Ltd.,
London
W.5,
U.K.
All
other
chemicals
were
the
purest
products
of
B.
D.
H.
Chemicals
Ltd.,
Poole,
Dorset,
U.K.
Methods
M6ssbauer
spectra
were
measured
in
the
apparatus
previously
described
(Cranshaw,
1964).
A
strong
*
Abbreviation:
e.p.r.,
electron
paramagnetic
resonance.
narrow
line
(0.21
min/s)
source
of
57Co
in
palladium
foil
(0.00025in
thick)
was
obtained
from
the
Radiochemical
Centre,
Amersham,
Bucks.,
U.K.
E.p.r.
spectra
were
recorded
on
a
Varian
E4
spectrometer
(Varian
Associates
Ltd.,
Walton-on-Thames,
Surrey,
U.K.)
by
using
a
liquid-
N2
insert
Dewar.
Circular-dichroism
spectra
were
recorded
in
a
Spectropol
I
spectropolarimeter
(SoFICA,
St
Denis,
France).
Iron
was
determined,
after
digestion
of
the
protein
with
1M-HCl
at
80°C
for
15min,
by
using
bathophenanthroline
(4,7-diphenyl-1,10-phenanthroline)
as
described
by
Diehl
&
Smith
(1965).
Labile
sulphur
was
determined
by
the
method
of
Fogo
&
Popowsky
(1949),
as
modified
by
Lovenberg
et
al.
(1963).
Protein
was
determined
by
Kjeldahl
nitrogen
determination,
the
nitrogen
content
being
taken
to
be
16%.
Purification
of
spinach
ferredoxin
The
method
used
was
developed
from
the
methods
of
Keresztes-Nagy
&
Margoliash
(1966)
and
Matsubara
(1968),
and
was
designed
for
the
handling
of
large
quantities
of
plant
material.
Ferredoxin
was
recovered
from
the
plant
homogenate
by
adding
DEAE-cellulose
and
recovering
it
by
centrifugation;
this
procedure
proved
to
be
much
more
rapid
than
passing
the
homogenate
directly
through
a
column
of
DEAE-cellulose
as
in pre-
viously
used
methods,
and
has
been
found
to
be
applicable
to
the
preparation
of
a
wide
variety
of
iron-sulphur
pro-
teins.
A
20mM-potassium
phosphate
buffer,
pH7.5,
was
used
throughout
the
procedure
instead
of
tris-HCl
because
of
the
affinity
of
tris
for
adventitious
iron.
Samples
of
ferredoxin
prepared
with
phosphate
did
not
show
the
e.p.r.
signal
at
g
=
4.27
in
the
oxidized
state
due
to
contaminant
iron
that
was
seen
in
some
samples
pre-
pared
in
tris
(Hall,
Gibson
&
Whatley,
1966).
To
minimize
denaturation
of
the
protein,
all
steps
were
carried
out
at
0-40C,
and
buffers
for
chromatography
and
dialysis
were
bubbled
with
N2
to
remove
02.
Homogenization.
Washed
spinach
leaves
(20kg)
were
homogenized
in
a
1-gallon
Waring
Blendor
in
800
g
batches,
each
with
1.5
litres
of
cold
buffer,
at
maximum
speed
for
2
min.
The
homogenate
was
allowed
to
settle
in
a
large
aspirator
and
the
copious
froth
was
removed.
The
liquid
was
run
'from
the
bottom
and
centrifuged
at
2500rev./min
in
the
6
x
1-litre
rotor
of
an
MSE
Mistral
refrigerated
centrifuge
for
15min.
The
dark-
green
supernatant,
about 40
litres
in
all,
was
poured
through
muslin.
Batchwise
DEAE-cellulose
treatment.
Solid
NaCl
(6g/l)
and
Whatman
DE
23
DEAE-cellulose
equilibrated
with
buffer
(20
ml
packed
wet
volume/i)
were
added
with
stirring
and
the
suspension
was
centrifuged
at
5OOg
for
1
min.
The
supernatant
was
discarded
and
the
precipitate
washed
three
times
by
resuspension
in
6
litres
of
buffer
and
centrifugation.
Finally
the
DEAE-cellulose
was
transferred
to
a
column
(30
cm
x
8
8cm)
and
washed
with
2
litres
of
0.2
m-NaCl
in
buffer.
The
ferredoxin
was
eluted
with
0.5m-NaCl
in
about
600ml
of
dark-red
solution.
Ammonium
sulphate
treatment.
(NH4)2SO4
(50%,
w/v)
was
added
to
the
solution
with
stirring.
After
15min
the
solution
was
centrifuged
at
20000g
for
30min.
The
preci-
pitate
of
greenish
protein
material
was
discarded
and
the
258
1971
MOSSBAUER
EFFECT
IN
PLANT
FERREDOXINS
supernatant
dialysed
overnight
against
30
vol.
of
buffer.
DEAE-cell?Ulose
chromatography.
The
ferredoxin
was
concentrated
by
passing
the
solution
through
a
column
(15cmx2.5cm)
of
Whatman
DE23
DEAE-cellulose
and
eluting
with
0.8M-NaCl
in
buffer.
It
was
then
diluted