1314
BIOCHEMISTRY:
ARNON
ET
AL.
PROC.
N.
A.
S.
he modynamic
o ces
owa d
he
mos
p obable
o m,
na i e
ibonuclease.
Some
o
he
less
likely
possibili ies
men ioned
abo e
can
only
be
igo ously
excluded
upon
comple ion
o
cu en
expe imen s
on
he
na u e
o
he
pai ing
o
hal -cys ine
esidues
du ing
he
lag
phase.
The
au ho s
wish
o
hank
M s.
Juani a
Cooke
o
he
expe
assis ance
in
many
o
hese
expe imen s.
*
P esen
add ess:
Massachuse s
Gene al
Hospi al,
Bos on
14,
Massachuse s.
On
lea e
o
absence
om
The
Weizmann
Ins i u e
o
Science,
Reho o h,
Is ael.
1
Sela,
M.,
F.
H.
Whi e,
J .,
and
C.
B.
An insen,
Biochim.
e
Biophys.
Ac a,
31,
417
(1959).
2
Whi e,
F.
H.,
J .,
J.
Biol.
Chem.,
236,
1353
(1961).
3An insen, C.
B.,
and
E.
Habe ,
ibid.,
236,
1361
(1961).
4Aq is ,
S.
E.
G.,
and
C. B.
An insen,
ibid.,
234,
1112
(1959).
6
Pe e son,
E.
A.,
and
H.
A.
Sobe ,
J.
Am.
Chem.
Soc.,
78,
751
(1956)-.
6
Hi s,
C.
H.
W.,
S.
Moo e,
and
W.
H.
S ein,
J.
Biol.
Chem.,
200,
493
(1953).
7
Spackman,
D.
H.,
W.
H.
S ein,
and
S.
Moo e,
Anal.
Chem.,
30,
1190
(1958).
8
An insen,
C.
B.,
R. R.
Red ield,
W.
L.
Choa e,
J.
Page,
and
W.
R.
Ca oll,
J.
Biol.
Chem.,
207,
201
(1954).
9
C es ield,
A.
M.,
Smi h,
K.
C.,
and
F.
W.
Allen,
ibid.,
216,
185
(1956).
10
Richa ds,
F.
M.,
Comp .
end.
a .
lIb.
Ca lsbe g,
Se .
Chim.,
29,
315
(1955).
11
Boye ,
P.
D.,
J.
Am.
Chem.
Soc.,
76,
4331
(1954).
12
B ay,
G.
A.,
Anal.
Biochem.,
1,
279
(1960).
13
Sela,
M.,
and
C.
B.
An insen,
Biochim.
e
Biophys.
Ac a,
24,
229
(1957).
14
Sela,
M.,
C.
B.
An insen,
and
W.
F.
Ha ing on,
ibid.,
26,
502
(1957).
-
Habe ,
E.,
M.
Sela,
and
C.
B.
An insen,
Fede a ion
P oc.,
20,
Pa
I,
217
(1961).
16
Expe imen al
wo k
is
now
in
p og ess
o
de e mine
he
na u e
o
he
pai ing
o
hal -cys ine
esidues
a
a ious
imes
du ing
he
ea ly
s ages
o
eoxida ion.
P elimina y
esul s
indica e
ha
pai ing
is
qui e
andom
and
ha
"inco ec ly"
o med
bonds
a e
p esen .
17
Ha ing on,
W.
F.,
and
J.
A.
Schellman,
Comp .
end.
a .
lib.
Ca lsbe g,
Se .
Chim.,
30,
21
(1956).
18
Sluy e man,
L.
A.
A.,
Biochim.
e
Biophys.
Ac a,
48,
429
(1961).
19
Habe ,
E.,
and
C.
B.
An insen,
unpublished
da a.
PHOTOSYNTHETIC
PHOSPHORYLATION
AND
MOLECULAR
OXYGEN*
BY
DANIEL
I.
ARNON
M.
LOSADA,
F.
R.
WHATLEY,
H.
Y.
TsuJIMOTO,
D.
0.
HALL,
AND
A. A.
HORTON
DEPARTMENT
OF
CELL
PHYSIOLOGY,
UNIVERSITY
OF
CALIFORNIA,
BERKELEY
Communica ed
July
3,
1961
Oxygen
and
pho osyn hesis
we e
i s
linked
abou
200
yea s
ago
when
bo h
we e
disco e ed
almos
simul aneously.
The
ea lies
concep
o
pho osyn hesis
was
ha
o
plane a y
en ila ion
in
which
illumina ed
plan s
exchanged
CO2
o
"bad
ai "
o
02
o
" i al
ai "
(see
his o ical
e iew1).
A
mechanism
o
his
gas
exchange
was
p oposed
in
1796
by
Ingenhousz.2
G een
plan s,
he
sugges ed,
abso b
om
"ca bonic
acid
in
he
sunshine,
he
ca bon,
h owing
ou
a
ha
ime
he
oxygen
alone,
and
keeping
he
ca bon
o
i sel
as
nou ishmen ."
2
Fo
o e
a
hund ed
yea s
a e wa d,
he
iew
ha
CO2
assimila ion
always
in-
ol ed
a
libe a ion
o
oxygen
gas
was
so
i mly
en enched
ha
i
was
e en
ex-
ended
o
he
da k
CO2
assimila ion
by
chemosyn he ic
bac e ia.3-5
The
idea
VOL.
47,
1961
BIOCHEMISTRY:
ARNON
ET
AL.
1315
o
pho osyn hesis
wi hou
oxygen
e olu ion
seemed
a
con adic ion
o
e ms
un il
he
conclusi e
wo k
o
an
Niel6'
7wi h
pho osyn he ic
bac e ia
i mly
es ablished
ha ,
basically,
he
assimila ion
o
CO2
a
he
expense
o
ligh
ene gy
does
no
de-
pend
on
he
libe a ion
o
oxygen
o
on
he
p esence
o
oxygen.
Bac e ial
pho o-
syn hesis
occu s
unde
s ic ly
anae obic
condi ions
and
includes
o ganisms
ha
a e
obliga e
anae obes
( o
example,
Chlo obium
and
Ch oma ium6,
7).
Al hough
molecula
oxygen
was
shown
o
be
unnecessa y
and
o en
e en
de i-
men al
o
bac e ial
pho osyn hesis,
i s
ole
in
he
ene gy
ans o ma ions
ha
occu
du ing
pho osyn hesis
in
g een
plan s
has
gained
mo e
a en ion
wi h
ime.
In
1939,
Hill
pos ula ed8
ha
he
consump ion
o
molecula
oxygen
is
necessa y
o
pho osyn hesis
o
g een
plan s
in
o de
o
supply
addi ional
ene gy
o
CO2
assimi-
la ion.
He
en isaged
" i s
a
educ ion
o
a
subs ance
by
chlo oplas s
gi ing
oxygen
and
hen
a
eoxida ion
o
he
educed
subs ances
by
pa
o
he
oxygen
oge he
wi h
a
simul aneous
educ ion
o
he
necessa y
equi alen
o
ca bon
di-
oxide."
9
In
1951
Bu k
and
Wa bu g'0
epo ed
he
sepa a ion
o
pho osyn hesis
in o
a
ligh
eac ion
and
a
da k
eac ion
on
he
basis
o
manome ic
measu emen s
wi h
in ac
Chlo ella
cells.
In
he
wo ds
o
Wa bu g,
"in
he
ligh
eac ion,
one
molecule
o
02
will
de elop
pe
molecule
o
chlo ophyll
...
whe eas
du ing
he
da k
pe iod
ollowing
he
end
o
illumina ion
i
can
be
obse ed
manome ically
...
ha
wo- hi ds
o
he
oxygen
gas
de eloped
du ing
he
ligh
pe iod
unde goes
a
back
eac ion."
'1
This
da k
consump ion
o
oxygen
is,
in
Wa bu g
and
Bu k's
iew,
a
pho osyn he ically
induced
espi a ion
ha
supplies
wo- hi ds
o
he
needed
ene gy
and
is,
he e o e,
indispensable
o
CO2
assimila ion
in
pho osyn hesis.
As
summed
up
ecen ly
by
Wa bu g,
"Keine
(CO2)
Fixie ung
ohne
A mung."
12
A
biochemical
model
o
oxygen
consump ion
in
pho osyn hesis
in
acco dance
wi h
he
iews
o
Hill
and
Wa bu g
was
p oposed
by
Vishniac
and
Ochoa.13
They
sugges ed
ha
he
ATP
used
o
CO2
assimila ion
in
pho osyn hesis
is
o med
by
a
collabo a ion
be ween
chlo oplas s
and
mi ochond ia.
In
hei
model,
chlo o-
plas s
educed
py idine
nucleo ide
in
ligh
and
ATP
was
o med
when
mi ochond ia
eoxidized
he
educed
py idine
nucleo ide
wi h
molecula
oxygen,
by
he
da k
p ocess
o
oxida i e
phospho yla ion.
A
simila
model
o
ATP
o ma ion
has
been
used
in
se e al
gene al
schemes
o
pho osyn hesis,
as,
o
example,
in
he
one
p oposed
in
1951
by
Holze l4
and
in
1954
by
Cal in's
g oup
( e .
1a,
Fig.
7).
In
all
hese
schemes,
oxida i e
phospho yla ion
by
mi ochond ia,
and
hence
oxygen
consump ion,
was
an
essen ial
ea u e
o
he
mechanism
o
pho osyn hesis.
A
mechanism
o
pho osyn hesis
ha
includes
a
consump ion
o
molecula
oxygen
canno
apply
o
he
s ic ly
anae obic
bac e ial
pho osyn hesis
and
leads,
he e-
o e,
o
he
in e ence
ha
he
mechanism
o
ene gy
con e sion
in
bac e ial
pho o-
syn hesis
is
basically
di e en
om
ha
in
plan
pho osyn hesis.
Howe e ,
he
disco e y
o
pho osyn he ic
phospho yla ion
in
isola ed
spinach
chlo oplas s16
and,
sho ly
he ea e ,
in
cell- ee
p epa a ions
o
Rhodospi illum
ub um,
17
has
poin ed
o
mechanisms
o
ATP
o ma ion
a
he
expense
o
ligh
ene gy
ha
a e
common
o
g een
plan s
and
pho osyn he ic
bac e ia.
A
uni ied
concep
o
pho osyn hesis
in
plan s
and
bac e ia,
based
on
pho osyn he ic
phospho yla ion
as
he
common
denomina o
is
now
possible
and
has
been
p esen ed
elsewhe e.
18-20
A
key
p emise
in
his
concep
is
ha ,
in
g een
plan s
as
in
baq e ia,
pho osyn he ic
phospho yla ion
is
undamen ally
independen
o
molecula
oxygen.
Since
his
1316
BIOCHEMISTRY:
ARNON
ET
AL.
PROC.
N.
A.
S.
has
ecen ly
been
ques ioned12'
21
we
ha e
e-examined
he
ela ion
o
oxygen
o
pho osyn he ic
phospho yla ion.
The
pu pose
o
his
a icle
is
o
b ing
oge he
and
add
o
he
e idence
which
con i ms
and
ex ends
ou
ea lie
conclusions
ha
pho osyn he ic
phospho yla ion,
bo h
in
g een
plan s
and
pho osyn he ic
bac e ia,
is
basically
independen
o
molecula
oxygen
and
hence
o
espi a ion.
Cyclic
and
Noncyclic
Pho ophospho yla ion.-Pho osyn he ic
phospho yla ion
(pho ophospho yla ion)
is
a
e m
coined
in
1954
o
desc ibe
a
ligh -induced
ATP
o ma ion
by
isola ed
chlo oplas s,
wi hou
he
aid
o
mi ochond ia
and
wi hou
he
consump ion
o
oxygen.16
The
o e -all
eac ion
o
pho osyn he ic
phospho yla-
ion
is
ep esen ed
by
equa ion
(1):
ligh
ADP
+
P
ATP
(1)
Reac ion
1,
in
which
he
sole
p oduc
is
ATP,
was
subsequen ly
designa ed
cyclic
pho ophospho yla ion,
o
dis inguish
i
om
a
second
pho ophospho yla ion
eac ion
by
isola ed
chlo oplas s
(equa ion
(2))
which
was
ound
a
ew
yea s
la e 22
and
named
noncyclic
pho ophospho yla ion.18
ligh
TPN
+
2H+
+
ADP
+
P
+
20H-
)
TPNH2
+
ATP
+
H20
+
'/202
(2)
The
e ms
"cyclic"
and
"noncyclic"
e e
o
he
elec on
low
mechanisms
which
ha e
been
p oposed'8'
19
o
hese
wo
eac ions
ha
now
join ly
cons i u e
he
p ocess
o
pho osyn he ic
phospho yla ion.
In
cyclic
pho ophospho yla ion
all
o
he
biochemically
e ec i e
ligh
ene gy
is
used
o
ATP
o ma ion.
In
non-
cyclic
pho ophospho yla ion
only
a
po ion
o
he
biochemically
e ec i e
ligh
ene gy
is
used
o
he
o ma ion
o
ATP;
he
emainde
is
used
o
he
o ma ion
o
a
educ an ,
TPNH2,
and
he
exc e ion
(e olu ion)
o
oxygen.
A
nonphysiologi-
cal
a ian
o
noncyclic
pho ophospho yla ion
( eac ion
2)
is
eac ion
3,
in
which
TPN
is
eplaced
by
e icyanide22'
23
( ep esen ed
he e
by
Fe3+).
ligh
2Fe3+
+
ADP
+
P
+
20H-
-
2Fe2+
+
ATP
+
H20
+
1/202
(3)
The
signi icance
o
cyclic
and
noncyclic
pho ophospho yla ion
o
he
o e -all
mechanism
o
pho osyn hesis
in
g een
plan s
is
ha
hey
supply-aside
om
oxygen
as
he
exc e ed
by-p oduc - he
wo
p oduc s,
ATP
and
TPNH2,
which
alone
su ice
o
con e ing
C02
in o
ca bohyd a es
in
he
da k.24
25
Bo h
ATP
and
TPNH2
(also
oxygen)
a e
o med
by
noncyclic
pho ophospho yla ion
(equa ion
(2))
bu
cyclic
pho ophospho yla ion
(equa ion
(1)),
which
supplies
only
ATP,
is
also
needed
o
C02
assimila ion
because
he
ATP
o med
in
noncyclic
pho ophos-
pho yla ion
is
insu icien
o
con e
CO2
o
ca bohyd a e.26
Chlo oplas
agmen s
("g ana")
do
no
espi e27'
28
and
hence,
acco ding
o
Wa bu g,
would
no
be
expec ed
o
ix
CO2
("G ana
a men
nich
und
ixie ien
nich "
12).
Howe e ,
by
ac iona ing
isola ed
chlo oplas s
we
ha e
shown
ha
g ana
ail
o
assimila e
C02
no
because
hey
a e
unable
o
espi e
bu
because
hey
lack
he
equisi e
chlo oplas
enzymes.
These
a e
wa e -soluble
and
a e
eadily
leached
ou
in
he
p epa a ion
o
"g ana."29
The
soluble
chlo oplas
enzymes,
ei he
in
si u
o
when
ex ac ed
om
chlo oplas s,
assimila e
CO2
o
he
le el
o
ca bohyd a es
and
do
so
solely24
a
he
expense
o
ATP
and
TPNH2-
VOL.
47,
1961
BIOCHEMISTRY:
ARNON
ET
AL.
1317
he
wo
compounds
o med
in
chlo oplas s
by
cyclic
and
noncyclic
pho ophos-
pho yla ion
(equa ions
(1)
and
(2))
and
no
by
espi a ion.
CO2
assimila ion
p ope
in
pho osyn hesis,
whe he
in
whole
cells30
o
in
isola ed
chlo oplas s,24'
25
consis s
o
he
same
exclusi ely
da k,
enzyma ic
eac ions3'
ha
a e
now
known
o
ope a e
in
nonpho osyn he ic
cells.32-34
The e
is,
so
a
as
we
know,
no
alid
expe imen al
basis,
o
pos ula ing
in
pho osyn hesis
a
peculia
pho ochemically
p oduced
" educing
powe "
ha
is
capable
o
di ec
educ ion
o
CO2
o
he
le el
o
ca bohyd a e
by
some
special
mechanism
unknown
elsewhe e
in
biochemis y
(see,
o
example,
e .
35
and
36).
I
we
abandon
he
no ion
o
an
unspeci ied,
hypo he ical
" educing
powe "
and
in e p e
pho osyn hesis
in
e ms
o
known
biochemical
mechanisms,
hen
he
unique
ea u e
o
pho osyn hesis
as
a
biological
p ocess
is
no
CO2
assimila ion-a
da k
p ocess
d i en,
in
bo h
pho osyn he ic
and
nonpho osyn he ic
cells,
by
ATP
and
educed
py idine
nucleo ide-bu
he
o ma ion
o
hese
wo
compounds
by
cyclic
and
noncyclic
pho ophospho yla ion.
The
disco e y
o
pho osyn he ic
phospho yla ion
was
pa
o
a
coinciden ,
b oade
inding'6'
27
ha
isola ed
chlo oplas s
a e
capable
o
ca ying
ou
a
com-
ple e
ex acellula
pho osyn hesis,
i.e.,
a
con e sion
o
CO
o
s a ch
and
suga ,
a
physiological
empe a u es
and
wi h
no
ene gy
supply
excep
isible
ligh .
The
iew
ha
chlo oplas s
a e
he
si es
o
comple e
pho osyn hesis
was
once
widely
held
wi hou
he
suppo
o
c i ical
expe imen al
e idence
and
was
la e
abandoned
because
o
e idence
o
he
con a y,
only
o
be
o mula ed
anew
on
he
basis
o
imp o ed
me hods
o
isola ing
unc ional
chlo oplas s
om
lea es
(see
e iew').
The
disco e y
o
cyclic
and
noncyclic
pho ophospho yla ion
by
isola ed
chlo o-
plas s
(also
o
CO2
assimila ion,
see
e iew')
was
con i med
and
ex ended
in
o he
labo a o ies,
no ably
hose
o
Jagendo ,37-40
Wessels,41
42
Vennesland,43
44
and
Hill.
Mos
o
he
wo k
has
been
done
wi h
spinach
chlo oplas s
bu
ecen ly
Wha ley
e
al.46
ha e
also
demons a ed
cyclic
and
noncyclic
pho ophospho yla ion
(and
CO2
assimila ion)
in
chlo oplas s
isola ed
om
se e al
o he
species
o
plan s.47
O
he
wo
pho ophospho yla ion
eac ions
(equa ions
(1)
and
(2)),
he
cyclic
ype
(equa ion
(1))
appea ed
he
mo e
basic
om
he
s andpoin
o
a
gene al
mechanism
o
ene gy
ans o ma ion
in
pho osyn hesis
since
his
eac ion
was
ound
in
ep esen a i es
o
all
he
di e en
g oups
o
pho osyn he ic
o ganisms.
Williamsw
ound
pho osyn he ic
phospho yla ion
in
he
obliga ely
anae obic
pho osyn he ic
bac e ia,
Ch oma ium
and
Chlo obium.
Cyclic
pho ophospho yla-
ion
in
algal
p epa a ions
was
ound
by
Thomas
and
Haans6'
and
Pe ack
and
Lipmann.52
(Fo
a
mo e
comple e
e iew
o
li e a u e
see
e .
53.)
Special
Fea u es
o
Cyclic
and
Noncyclic
Pho ophospho yla ion.-The
unique
ea u es
o
cyclic
pho ophospho yla ion
a e
ha
ATP
is
o med
wi h
no
added
elec on
dono
and
no
added
elec on
accep o .
Cyclic
pho ophospho yla ion
con-
sumes
nei he
espi a o y
subs a e
no
molecula
oxygen.
Bo h
a e
eplaced
by
ligh 53
which
p o ides
he
ee
ene gy
equi ed
o
he
syn hesis
o
py ophospha e
bonds
o
ATP.
This
seemed
a
i s
no
o
be
he
case
in
pho osyn he ic
bac e ia.
F enkel's
cell- ee
p epa a ions
o
R.
ub um
became
subs a e-dependen
a e
washing;
he
a e
o
phospho yla ion
was
doubled
on
adding
a-ke oglu a a e.17
Bu
in
subsequen expe imen s
F enkel54
uled
ou
he
dependence
on
an
added
chemical
subs a e
and
once
his
undamen al
poin
was
cla i ied,
he
equi alence
1318
BIOCHEMISTRY:
ARNON
ET
AL.
PROC.
N.
A.
S.
o
cyclic
pho ophospho yla ion
in
chlo oplas s
and
bac e ial
pa icles
seemed
p obable.55
The
independence
o
pho osyn he ic
phospho yla ion
om
a
espi a o y
sub-
s a e
is
con i med
by
he
independence
o
his
p ocess
om
CO2
assimila ion.
Pho osyn he ic
phospho yla ion
occu s
when
he
enzymes
o
he
educ i e
ca bon
cycle
a e
emo ed
by
washing
he
chlo oplas s,
CO2
is
no
supplied
o
he
eac ion
mix u e,
and
he
eac ion
essels
con ain
KOH
in
he
cen e
well.
This
would
no
exclude
a
possible
ca aly ic
pa icipa ion
o
CO2
in
pho ophospho yla ion
(c .
Wa bu g
e
al.12
and
Vennesland
e
al.56).
Wha
can
be
de ini ely
excluded
is
ha
subs a e
amoun s
o
ca bon
compound(s)
a e
i s
syn hesized
in
ligh
om
CO2
and
a e
hen
used
as
elec on
dono s
o
he
o ma ion
o
ATP.
While
cyclic
pho ophospho yla ion
was
ecognized
as
a
common
denomina o
o
all
pho osyn heses,
noncyclic
pho ophospho yla ion
as
ep esen ed
by
equa ion
(2)-a
eac ion
ha
libe a es
oxygen-seemed
a
i s
des ined
o
emain
a
special
ea u e
o
plan
pho osyn hesis,
since
oxygen
is
no
e ol ed
in
bac e ial
pho o-
syn hesis.
Howe e ,
Losada
e
al.57
(c .
also
Duysens
e
al.58)
ha e
ecen ly
sepa-
a ed
eac ion
2
in o
wo
dis inc
pho ochemical
eac ions
(a)
a
pho ooxida ion
o
hyd oxyl
ions
ha
yields
oxygen
(equa ion
(4))
and
(b)
a
pho ophospho yla ion
eac ion
p ope
in
which
he
pho o educ ion
o
iphosphopy idine
nucleo ide
is
coupled
wi h
he
o ma ion
o
adenosine
iphospha e
(equa ion
5).
ligh
2A
+
20H-
>
2A-
+
H2O
+
1/202
(4)
ligh
TPN
+
2H+
+
ADP
+
P
+
2A-
>-TPNH2+
ATP
+
2A
(5)
ligh
Sum:
TPN+2H++ADP+P+2OH---
TPNH2
+
ATP
+
H20
+
1/202
(2)
Only
he
pho ooxida ion
o
hyd oxyl
ions
(equa ion
(4))
is
peculia
o
g een
plan s
since
only
g een
plan s
seem
capable
o
using
OH-
as
an
elec on
dono
wi h
he
esul an
exc e ion
o
molecula
oxygen.
Reac ion
5,
he
noncyclic
pho ophospho yla ion
eac ion
p ope ,
occu s
also
in
pho osyn he ic
bac e ia.59
Since
hey
use
ino ganic
o
o ganic
elec on
dono s,
such
as
hiosul a e
o
succina e
ins ead
o
OH-,
oxygen
e olu ion
does
no
occu
in
he
eac ion.
Figu e
1
depic s
he
o e -all
scheme
o
noncyclic
pho ophospho yla ion
in
g een
plan s
ha
we
now
en isage.
The
in e media e
A
in
Figu e
1
is
bo h
he
elec on
accep o
o
he
i s
ligh
eac ion
and
he
elec on
dono
o
he
second
ligh
eac-
ion
(c .
equa ions
(4)
and
(5)).
We
ha e
used
indophenol
dyes
as
an
expe imen al
de ice
o
sepa a e
he
wo
pho ochemical
eac ions.
The
na u al
in e media es
wi h
which
he
dyes
ha e
in e ac ed
ha e
no
been
iden i ied.
They
may
include
cy och ome
componen s60
o
a
quinone.61,
62
The
e minal
physiological
elec on
accep o
in
noncyclic
pho ophospho yla ion
(B
in
Fig.
1)
is
py idine
nucleo ide.
Ou
o iginal
scheme
o
noncyclic
pho ophospho yla ion
( e .
22,
Fig.
3)
en-
isaged
he
phospho yla ion
s ep
as
occu ing
be ween
he
unknown
educ an
(H),
o med
by
he
pho olysis
o
wa e
and
TPN,
whe eas
ou
p esen
scheme,
based
on
he
elec on
low
heo y,18'
53
links
he
phospho yla ion
s ep
wi h
he
oxida ion
o
cy och ome
by
pho oac i a ed
chlo ophyll.

VOL.
47,
1961
BIOCHEMISTRY:
ARNON
ET
AL.
1319
(?e,--
B
-
e
Chi
~+)
Cy
-
A
I
e !
LIGHT
pigmen
J(4,
OH
O
-
H20
[OH]
LIGHT
4°2
FIG.
1.-Scheme
o
noncyclic
pho ophospho yla ion
o
he
g een
plan
ype.
In
he
i s
ligh
eac ion,
he
pigmen
molecule
(see
ex )
becomes
exci ed
by
he
abso p ion
o
a
quan um
o
ligh .
The
exci ed
pigmen
dona es
i s
high-ene gy
elec on
(e
-)
o
an
in e media e
elec on
accep o
(A)
and
accep s
an
elec on
om
an
hyd oxyl
ion.
The
oxida ion
p oduc
(OH)
o
he
hyd oxyl
ion
is
he
p ecu so
o
molecula
oxygen.
In
he
second
ligh
eac ion,
he
chlo ophyll
molecule
(Chl),
exci ed
by
he
abso p ion
o
a
quan um
o
ligh ,
dona es
i s
high-ene gy
elec on
(e-)
o
he
e minal
elec on
accep o
(B)
and
accep s,
ia
he
cy och ome
sys em
(Cy ),
an
elec on
om
he
educed
in e media e
(A-)
o med
in
he
i s
ligh
eac ion.
The
phospho yla ion
s ep
is
linked
wi h
he
ans e
o
he
elec on
om
cy och ome
o
chlo ophyll.
(.e-,.
B
[g
cz~ e-
e-
A
P.
LIGHT
FIG.
2.
-Scheme
o
noncyclic
pho ophospho yla ion
o
he
bac e ial
ype.
The
chlo ophyll
molecule
(Chl)
becomes
exci ed
by
he
abso p ion
o
a
quan um
o
ligh .
The
exci ed
chlo-
ophyll
dona es
i s
high
ene gy
elec on
(e-)
o
he
elec on
accep o
(B)
and
accep s,
ia
he
cy och ome
sys em
(Cy ),
an
elec on
om
an
ex e nal
elec on
dono
(A
-).
The
phospho yla ion
s ep
is
linked
wi h
he
ans e
o
he
elec on
om
cy och ome
o
chlo o-
phyll.
TPN
educ ion
in
noncyclic
pho ophospho yla ion
( eac ion
2),
al hough
no -
mally
coupled
wi h,
may
also
be
uncoupled
om,
ATP
o ma ion.
T ebs
e
al.
( e .
63,
Table
1)
ha e
shown
ha
ammonia64
supp esses
almos
comple ely
he
o ma ion
o
ATP
in
eac ion
2
in
ligh
wi hou
inhibi ing
he
o ma ion
o
TPNH2.
These
esul s
speak
agains
he
sugges ion
o
Chance
and
Olson65
ha
he
pho o-
educ ion
o
TPN
may
be
d i en
by
ATP,
o med
pe haps
by
cyclic
pho ophos-
pho yla ion
(c .
also
Ma 6
and
Fo i66).
The
con a y
is,
in
ac ,
he
case.
Elec-
1320
BIOCHEMISTRY:
ARNON
ET
AL.
PROC.
N.
A.
S.
/00
02
p oduced
80
~~~~~~~~/00
02
p oduced
%
60
80
Xi
40
-
/-
60
_a
TPN//2
~~~ o med
I
'a,~~~~~~~~~~~~~~~~~~q
z,
,00
40
0_A_
oxygene olu onand
TPNH
educ io med
o
20
4~~~~~~0
Oxygen
was
measu ed
manome iATP
o med
0630644
66/
677
699
FIG.
3.-E ec i eness
o
monoch oma ic
66
6
6
6
ligh ,
in
he
ed
egion
o
he
spec um,
on
oxygen
e olu ion
and
TPN
educ ion,
occu -
(m
ing
as
sepa a e
chlo oplas
eac ions.57
Oxygen
was
measu ed
manome ically
when
FIg.
e olu ionwss
o
dsc ibed
he
pho ochemical
eac ion
was
limi ed
o
Fig.
4ig
e
ec aegin
o
mo
nc h oma o
he
pho ooxida ion
o
OH-
(equa ion
(4))
ligh n
m h
ed
egio
o hespe um
do
in
a
eac ion
mix u e
which
included,
in
a
oxgn
ou ion
andlume
cylc
oophosp
ho la-
inal
olume
o
3
ml,
chlo oplas
agmen s
ioyen,
u igassep n s
mcls op
as
eaciens.
(Cae)
con aining
1
mg.
o
chlo ophyll
and
Oxgn
e olu i
on
meason
as
dylesc
p ibed
he
ollowing
in
Mmoles:
is/ace a e
bu -
ih
Fu e,3.
AP
o ma ion
by;
cyclic
mpho o
e ,
pH
8,
40;
po assium
e icyanide,
15;
phospho yla
(eque ean in
()
was
measu ed
and
2,
6
-
dichlo ophenol
indophenol
0i
ap llel
e
imn
inal
ea io
mi
(DCPIP),
0.2.
TPN
educ ion
(measu ed
wi
inclue
ina
ial
oum
ae
o
m
spec opho ome ically
a
340
mM)
was
chlo oplas
agmen s
(C18)
con aining
1
mg
ca ied
ou
in
a
pa allel
expe imen
in
which
chlo ophyll
and
he
ollowing
in
Mmoles:
is!
he
pho ooxida ion
o
wa e
was
blocked
by
HCl
bu e ,
pH
8.3,
80;
MgSO4,
5;
ADP,
10;
he
omission
o
chlo ide
and
he
addi ion
K5)).
10;i
an d ian
K0
(mnaedione)
o e
2ecin
X
e
un
Mo
CMU
The
ele o
dono
0.3.s
qiaen
o010Ml
P e
sys em
euc
cons
do
100
on
he
o dina e
scale
is
equi alen
o
moles
asco ba e
and
0.05
moles
o
2,3',6-
a oms
oxygen
e ol ed
pe
ichlo ophenol
indophenol
(equa ion
(5)).
quan a
o
ligh
abso bed.
60
on
he
o dina e
The
eac ions
we e
un
o
15
mm
a
15'C.
scale
is
equi alen
o
0.10
,umole
ATP
o med
Gas
phase,
ni ogen.
pe
/mole
quan a
o
ligh
abso bed.
100
on
he
o dina e
scale
is
equi alen
o
0.15
MAa oms
oxygen
e ol ed
pe
M~mole
quan a
o
ligh
abso bed.
58
on
he
o dina e
scale
is
equi alen
o
0.09
M~moles
TPNH2
o med
pe
/Amole
quan a
o
ligh
abso bed.
on
anspo ,
esul ing
in
TPN
educ ion,
may
p oceed
wi hou
ATP
o ma ion,
bu
no
pho ophospho yla ion
can
occu
unless
i
is
coupled
wi h
elec on
anspo .
Expe imen ally,
i
is
possible
o
con e
noncyclic
pho ophospho yla ion
in
chlo oplas s
(equa ion
(2)
and
Fig.
1)
o
a
"bac e ial"
ype
(equa ion
(5)
and
Fig.
VOL.
47,
1961
BIOCHEMISTRY:
ARNON
ET
AL.
1321
2)
by
eplacing
OH-
wi h
an
exogenous
elec on
dono
a
he
le el
o
A-.
Reduc-
ion
o
py idine
nucleo ide
is
hen
coupled
wi h
he
oxida ion
o
he
exogenous
elec-
on
dono
and
a
simul aneous
ATP
o ma ion
bu
wi hou
oxygen
e olu ion.57
We
ha e
no
de ini ely
iden i ied
he
"pigmen "
shown
in
Figu e
1
ha
is
in-
ol ed
in
he
pho ooxida ion
o
hyd oxyl
ions.
Recen
expe imen s
on
he
e -
ec i eness
o
monoch oma ic
ligh
on
he
pho ochemical
eac ions
o
chlo oplas s
sugges
ha
his
may
be
a
special
pho osyn he ic
pigmen
such
as
chlo ophyll
b
o
ano he
accesso y
pigmen
ha
is
ound
only
in
oxygen-e ol ing
o ganisms,
i.e.
highe
plan s
and
algae,
a he
han
chlo ophyll
a
(o
he
closely
ela ed
bac e io-
chlo ophyll)
ound
in
all
pho osyn he ic
cells.
(See
e iew
o
ela ed
li e a u e
in
e .
57.)
In
he
ed
egion
o
he
spec um
he
pho ooxida ion
o
OH-
(equa ion
(4))
was
mos
e ec i e
a ound
644
miu,
which
co esponds
o
he
maximum
abso p-
ion
peak67
in
he
ed
o
chlo ophyll
b
(Figs.
3
and
4).
The
e ec i eness
o
mono-
ch oma ic
ligh
in
he
ed
egion
on
pho o educ ion
o
TPN
(equa ion
(5))
and
cyclic
pho ophospho yla ion
(equa ion
(1))
was
dis inc ly
di e en
om
ha
on
oxygen
e olu ion
(equa ion
(4)
and
Figs.
3
and
4),
hus
suppo ing
he
iew
ha
hese
a e
di e en
ligh
eac ions.
The
expe imen s
wi h
monoch oma ic
ligh
a o
he
conclusion
ha
he
pho ochemical
eac ion
which
p oduces
oxygen
is
di e en
om
he
o he
pho ochemical
eac ions
which
p oduce
ATP
and
educed
py idine
nucleo ide.
The
ela ion
o
oxygen
p oduced
o
ATP
o ma ion
in
noncyclic
pho o-
phospho yla ion
will
now
be
examined
in
mo e
de ail.
Oxygen
as
an
Exc e ed
By-P oduc
o
Noncyclic
Pho ophospho yla ion.-The
es-
sence
o
noncyclic
pho ophospho yla ion,
bo h
in
plan s
and
in
pho osyn he ic
bac e ia,
appea s
o
be
he
o ma ion
o
ATP
which
is
coupled
wi h
an
elec on
anspo
om
an
ex e nal elec on
dono
o
py idine
nucleo ide
ac ing
as
he
elec on
accep o .
As
al eady
men ioned,
in
plan s,
as
in
pho osyn he ic
bac e ia,
oxygen
is
no
consumed
in
hese
essen ial
aspec s
o
noncyclic
pho ophospho yla ion.
Oxygen
may
be
libe a ed
in
his
p ocess
bu
only
in
he
speci ic
ins ance,
ha
is
cha ac e is ic
o
plan
bu
no
o
bac e ial
pho osyn hesis,
when
OH-
is
he
ex-
e nal
elec on
dono .
This
concep
is
suppo ed
by
he
expe imen al
indings
ha
ATP
o ma ion
g ea ly
inc eases
he
a e
o
oxygen
e olu ion
and
he
concomi an
educ ion
o
he
elec on
accep o ,
whe he
i is
e icyanide
o
TPN22
23,
39,
68,
69
(equa ions
(2)
and
(3)).
These
esul s
un
coun e
o
Hill's
o iginal
hypo hesis
ha
in
chlo oplas s
" he
educed
p oduc
o med
in
he
ligh
migh
be
eoxidized
by
molecula
oxygen
o
gi e
a
phospho yla ing
sys em
simila
o
ha
in
chemosyn he ic
bac e ia."
45
As
Hill
and
Bonne
ha e
ecen ly
poin ed
ou ,
"In
such
a
hypo hesis
one
would
expec
ha
addi ion
o
ino ganic
phospha e
and
ADP
o
he
sys em
would
esul
in
dec eased
a es
o
(H)
accep o
educ ion
and
o
oxygen
p oduc ion"
60
(i alics
ou s).
Hill
has
accep ed
he
new
expe imen al
indings
and
concluded
ha
" he
abo e
...
hypo hesis
has
o
be
abandoned."
60
Wa bu g,'2
howe e ,
has
e ained
he
hy-
po hesis
o
oxygen
consump ion
in
ene gy
ans o ma ions
in
pho osyn hesis
and
ques ioned
he
new
expe imen al
ac s.
Speci ically,
Wa bu g
e
al.12
ound
ha
wi h
naph hoquinone
sul ona e
as
he
hyd ogen
accep o ,
he
s oichiome y
o
oxygen
e olu ion
by
chlo oplas s
is
no
a ec ed
by
phospho yla ion.
Wa bu g
e
al.12
sugges ed
ha
he
inc ease
in
pho op oduc ion
o
oxygen
as
a
co sequence
o
1322
BIOCHEMISTRY:
ARNON
ET
AL.
PROC.
N.
A.
S.
pho ophospho yla ion- i s
obse ed
by
o he
in es iga o s22'
68
wi h
e icyanide
(equa ion
(3))-was
he
esul
o
an
uncon olled
change
in
acidi y.
Since
he
inc eased
p oduc ion
o
oxygen
ha
accompanied
pho ophospho yla ion
in
he
e icyanide
sys em
was
ound
in
solu ions
ha
we e
s ongly bu e ed
wi h
is
(hyd oxyme hyl)
aminome hane
bu e ,23
i
seemed
unlikely
ha
he
ob-
se ed
e ec
was
he
esul
o
uncon olled
pH
changes
as
sugges ed
by
Wa bu g
e
al.'2
Mo eo e ,
Da enpo 69
has
ecen ly
ound
ha
he
a e
o
pho o educ ion
o
TPN,
he
physiological
hyd ogen
accep o
in
noncyclic
pho ophospho yla ion
(equa ion
(2)),
was
also
g ea ly
inc eased
a
wo
di e en
pH's
when
a
phospha e-
accep ing
sys em
was
p esen .
We
ha e
now
ein es iga ed
he
e ec
o
pho ophospho yla ion
on
oxygen
e olu ion
by
isola ed
chlo oplas s
a
di e en
hyd ogen
ion
concen a ions,
using
ei he
TPN
o
e icyanide
as
elec on
accep o s.
The
pH
alue
o
each
eac ion
mix u e
was
checked
a
he
end
o
he
expe imen .
The
esul s
a e
shown
in
Figu es
5
and
6.
Wi h
ei he
TPN
o
e icyanide
as
he
elec on
accep o ,
we
obse ed
a
signi i-
TPN
#
(ADP+
P)
qj
TPN
I
7.6
7.8
80
8.2
84
8.6
pH
FIG.
5.-E ec
o
pho ophospho yla ion
a
a ious
pH
alues
on
oxygen
e olu ion
accom-
panying
TPN
educ ion.
The
eac ion
was
ca ied
ou
in
Wa bu g
manome e
essels
a
150C.
Illumina ion,
25,000
Lux
o
6
min.;
gas
phase,
ni ogen.
In
all
essels
he
eac-
ion
mix u e
con ained,
in
a
inal
olume
o
3
ml,
200
Mimoles
is-HCl
bu e ,
4
,moles
TPN,
pu i ied
TPN- educ ase
om
spinach,
and
b oken
chlo oplas s
(Pi,)
con aining
0.2
mg
chlo ophyll.
To
he
phospho yla ing
se ies
[TPN
plus
(ADP
plus
P)]
we e
added
10
Mmoles
MgSO4,
10 Mmoles
po assium
phospha e
and
10
/Amoles
ADP.
T is
bu e ,
po as-
sium
phospha e,
and
ADP
we e
each
adjus ed
o
he
selec ed
pH;
he
TPN
was
adjus ed
o
pH
7.5.
The
pH
shi ,
as
de e mined
a
he
end
o
he
eac ion,
was
ound
no
o
ex-
ceed
0.1
uni .
VOL.
47,
1961
BIOCHEMISTRY:
ARNON
ET
AL.
1329
o
a
ela i ely
high
concen a ion
o
chlo oplas
ma e ial
sugges s
ha
he
anae obic
FMN
and
i amin
K
sys ems
equi e
mo e
chlo oplas
ac o (s)
han
he
pseudo-
cyclic
oxygen-dependen
sys em.
No
simila
equi emen s
we e
obse ed
o
he
anae obic
phenazine
me hosul a e
sys em.
Despi e
he
simila i y
o
he
o e -all
eac ions
(equa ions
(1)
and
(8)),
he
anae o-
bic,
cyclic
pho ophospho yla ions
ca alyzed
by
i amin
K
and
FMN
can
be
dis in-
guished
expe imen ally
om
hei
oxygen-dependen ,
pseudocyclic
analogues.
The
la e
depend
on
he
pho op oduc ion
o
oxygen
(equa ions
(6),
(7),
and
8))
which
e-
qui es
chlo ide",
80
and
a e
sensi i e
o
wo
well-known
inhibi o s
o
oxygen
e olu ion
in
pho osyn hesis,
CMU8'
(p-chlo ophenyldime hyl
u ea)
and
o-phenan h oline.80
Table
1
shows
ha ,
unde
expe imen al
condi ions
a o able
o
each
sys em,
TABLE
1
EFFECT
OF
CHLORIDE
ON
PHOTOPHOSPHORYLATION
IN
NITROGEN
OR
AIR
pmoles
ATP
Fo med-
T ea men
Minus
chlo ide
Plus
chlo ide
Ni ogen,
FMN
5.1
5.7
Ni ogen,
i amin
K,
9.7
9.9
Ai ,
FMN
0.5
6.1
Ai ,
i amin
K3
0.4
5.5
In
he
ni ogen
se ies
he
illumina ion
was
2,000
Lux
o
30
min,
and
he
eac ion
mix u e
included,
in
a
inal
olume
o
3
ml,
chlo oplas
agmen s
(Ci.)
con aining
2.5
mg
chlo ophyll
and
0.3
moles
o
FMN
o
i amin
Ks.
In
he
ai
se ies,
he
illumina ion
was
50,000
Lux
o
30
min
and
he
eac ion
mix u e
included,
in
a
inal
olume
o
3
ml,
chlo oplas
agmen s
(Ci,)
con aining
0.2
mg
chlo ophyll
and
0.003
pumoles
o
FMN
o
i amin
Ka.
O he
componen s
o
he
eac ion
mix u e
we e,
in
pmoles:
is
bu e ,
pH
8.3,
80;
K H32PO4,
15;
ADP,
15;
and
MgSO4,
5.
Chlo oplas s
we e
p epa ed
in
0.5
M
suc ose
and
chlo ide- ee
eagen s
we e
used.
In
he
plus
chlo ide
se ies
10
jsmoles
NaCl
we e
added.
Ni ogen
gas
was
pu i-
ied
by
passing
h ough
a
zinc-me hylene
blue
solu ion
(A.
T.
Jagendo ,
pe sonal
com-
munica ion).
he
omission
o
chlo ide
had
sca cely
an
e ec
on
he
anae obic
cyclic
pho ophos-
pho yla ion
wi h
FMN
o
i amin
K,
bu
se e ely
inhibi ed
hei
oxygen-de-
penden ,
pseudocyclic
coun e pa s.
Likewise,
CMU
(also
o-phenan h oline53)
inhibi ed
he
oxygen-dependen ,
pseudocyclic
pho ophospho yla ion
bu
had
ela i ely
li le
e ec
on
hei
anae obic,
cyclic
coun e pa s
(Table
2).
TABLE
2
EFFECT
OF
CMU
ON
PHOTOPHOSPHORYLATION
IN
NITROGEN
AND
AIR
WITH
HIGH
CHLOROPHYLL
AT
Low
LIGHT
INTENSITY
pmoles
ATP
Fo med
T eamn
-
Ni ogen
Ai -
T ea men
Con ol
CMU
Con ol
CMU
FMN
7.0
6.6
4.2
1.7
Vi amin
K3
8.1
7.6
3.3
1.3
PMS
2.7 2.4
2.9
2.8
The
eac ion
mix u e
included,
in
a
inal
olume
o
3.0 ml,
chlo oplas
agmen s
(Ci.)
con aining
2.5
mg
chlo ophyll
and,
in
moles:
is
bu e ,
pH
8.3,
80;
MgSO4,
10;
K2H32PO4,
15;
ADP,
15;
and,
whe e
indica ed,
FMN,
0.3;
i amin
Ks,
0.3,
o ,
phenazine
me hosul a e
(PMS),
0.1.
The
inal
concen a ion
o
CQMU
was
2
X
10-6
M.
The
eac ion
was
un
o
30
min
a
an
illumina ion
o
2,000
Lux.
The
ni ogen
gas
was
pu i ied
as
desc ibed
in
Table
1.
When
some
expe imen al
condi ions
a e
un a o able
o
anae obic,
cyclic
pho o-
phospho yla ion,
he
high
a ini y
o
educed
FMN
o
i amin
K,
o
molecula
oxygen
may
s ill
gi e
ise
o
a
pseudocyclic,
oxygen-dependen
pho ophospho yla-
ion.
Thus,
we
ha e
obse ed
in
he
p esence
o
10-4
M
FMN
o
i amin
K3
a
a
high
ligh
in ensi y,
and
wi h
a
ela i ely
low
concen a ion
o
chlo oplas
ma e ial
(0.25
mg
chlo ophyll/3
ml)
ha
pho ophospho yla ion
became
oxygen-
dependen ,
e en
when
he
eac ion
was
s a ed
in
pu i ied
ni ogen
gas
ha
was

1330
BIOCHEMISTRY:
ARNON
ET
AL.
PROC.
N.
A.
S.
TABLE
3
EFFECT
OF
CMU
ON
PHOTOPHOSPHORYLATION
IN
NITROGEN
AND
AIR
WITH
Low
CHLOROPHYLL
AT
HIGH
LIGHT
INTENSITY
-
yjmoles
ATP
Fo med
Ni ogen
Ai
T ea men
Con ol
CMU
Con ol
C
1[U
FMN
5.6
1.5
5.4
1.1
Vi amin
K3
8.6
4.7 5.3
1.0
PMS
9.3
9.9
8.9
8.6
Expe imen al
condi ions
as
in
Table
2
excep
ha
less
chlo oplas
ma e ial
(Ci.)
was
used
(0.25
mg
chlo ophyll)
and
he
illumina ion
was
inc eased
o
20,000
Lux.
Reac ion
ime,
15
min.
ea ed
o
emo e
oxygen
impu i ies
(Table
3).
Phospho yla ion
became
oxygen-
dependen
when
he
ca aly ic
amoun s
o
FMN
o
i amin
K?
we e
pho ochemically
educed
by
chlo oplas s.
An
equi alen
amoun
o
oxygen
was
hen
libe a ed
which
was
su icien
o
sus ain
an
oxygen-dependen
pho ophospho yla ion
(c .
T ebs
and
Eck75).
The
oxygen
dependence
o
he
sys em
was
de e mined
by
blocking
he
oxygen
e olu ion
eac ion
wi h
CMU.
ATP
o ma ion
hen
became
s ongly
inhibi ed.
(I
seems
likely
ha
a
simila
dependence
on
pho ochemically
gene a ed
oxygen
accoun s
o
he
esul s
epo ed
by
Vennesland
e
al.
in
e .
56,
Table
2.)
Pho ophospho yla ions
ca alyzed
by
phenazine
me hosul a e
we e
esis an
o
inhibi ion
by
CMU
and
o-phenan h oline,53
ega dless
whe he
he
eac ion
was
ca ied
ou
unde
condi ions
a o ing
he
anae obic
cyclic,
o
he
oxygen-dependen
pseudocyclic
pa hway
(Tables
2
and
3).
Concluding
Rema ks.-F om
he
s andpoin
o
cellula
physiology,
he
ole
o
oxygen
in
ATP
o ma ion
in
pho osyn hesis
p o ides
an
in e es ing
con as
o
he
ole
o
oxygen
in
ATP
o ma ion
in
espi a ion.
The
high
he modynamic
e iciency
o
espi a ion,
and
i s
supe io i y
o e
e men a ion
in
using
he
ee
ene gy
o
he
deg ada ion
o
oods u s
o
syn hesis
o
he
py ophospha e
bonds
o
ATP,
is
made
possible
by
he
key
ole
ha
molecula
oxygen
plays
as
he
e -
minal
elec on
accep o
in
oxida i e
phospho yla ion
by
mi ochond ia.
Only
wi h
oxygen
can
he
oxida ion
o
oods u s
be
comple e
( o
CO
a
ld
wa e )
and
hei
ee
ene gy
eleased
in
ull
o
me abolic
pu poses.
In
he
las
se en
yea s
biochelnical
in es iga ions
o
pho osyn hesis
in
cell- ee
sys ems
e ealed
no
such
ole
o
oxygen
in
ATP
o ma ion
by
chlo oplas s
and
bac e ial
pa icles.
He e,
ATP
o ma ion
occu s
in
cyclic
pho ophospho yla ion
unde
s ic ly
anae obic
condi ions
whe e
he
pa icipa ion
o
oxygen
is
excluded.
I
i
noncyclic
pho o-
phospho yla ion,
oxygen
may
eplace
TPN
as
a
e minal
elec on
accep o ,
bu ,
as
discussed
la e ,
his
esul s
in
a
loss
ins ead
o
a
gain
in
he
yield
o
physiologically
use ul
ene gy.
The
s ic ly
anae obic
cha ac e
o
cyclic
pho ophospho yla ion
in
bac e ial
p epa a ions
needs
no
elabo a ion
since
pho osyn hesis
in.
bac e ia
is
i mly
es-
ablished
as
an
anae obic
p ocess.7
In
isola ed
chlo oplas s,
anae obic
cyclic
pho ophospho yla ion
has
been
demons a ed
wi h
phenazine
me hosul a e
as
a
ca alys ,
and
also
wi h
i amin
K3
and
FMN.
Wi h
he
la e ,
a
a
mo e
igid
con ol
o
expe imen al
condi ions
was
equi ed
han
wi h
phenazine
me hosul a e.
Special
expe imen al
sa egua ds
a e
needed
o
demons a ing
in
chlo oplas s
an
anae obic
cyclic
pho ophospho yla ion
ca alyzed
by
i amin
K3
and
FMN,
because
hese
subs ances,
when
educed'
pho ochemically,
eadily
eac
wi h
small
concen a ions
o
molecula
oxygen.
Phenazine
me hosul a e
also
eac s
eadily
VOL.
47,
1961
BIOCHEMISTRY:
ARNON
ET
AL.
1331
wi h
oxygen,
bu
i s
a ini y
o
oxidized
cy och omes82
seems
o
be
g ea e
han
o
oxygen;
i
is
hus
able
o
ca alyze
a
cyclic
elec on
low
(Fig.
8)
e en
in
he
p esence
o
molecula
oxygen.
Noncyclic
pho ophospho yla ion
consis s
o
ATP
o ma ion
ha
is
coupled
wi h
he
educ ion
o
py idine
nucleo ide
and
he
oxida ion
o
an
exogenous
elec-
on
dono .
The
p ocess,
which
was
o iginally
ound
in
isola ed
chlo oplas s,22
has
now
also
been
demons a ed
in
bac e ial
pa icles.59
The
di e ence
be ween
noncyclic
pho ophospho yla ion
in
he
wo
cases
cen e s
on
he
elec on
dono .
Fo
isola ed
chlo oplas s
he
elec on
dono s
a e
hyd oxyl
ions
which
on
oxida ion
o m
oxygen;
his
accoun s
o
oxygen
e olu ion
in
plan
pho osyn hesis.
Pho o-
syn he ic
bac e ia
canno
use
hyd oxyl
ions
bu
only
such
elec on
dono s
as
o ganic
acids
o
educed
sul u
compounds6'
7;
his
accoun s
o
he
absence
o
oxygen
e olu ion
in
bac e ial
pho osyn hesis.
Ins ead
o
oxygen,
oxidized
bac-
e ial
elec on
dono s
a e
o med,
which
may
be
u he
me abolized
o
may
ac-
cumula e
and
yield,
o
example,
elemen al
sul u .6
The
ole
o
oxygen
in
pho osyn hesis
is,
he e o e,
only
inciden al
and
no
i al
o
he
ene gy
ans o ma ion
eac ions,
as
i
is
in
espi a ion.
Oxygen
is
e ol ed
because
i
is
an
exc e ed
p oduc
o
he
oxida ion
o
hyd oxyl
ions- he
elec on
dono
in
noncyclic
pho ophospho yla ion
by
chlo oplas s.
Expe imen ally,
when
ano he
elec on
dono
is
subs i u ed
o
hyd oxyl
ions,
noncyclic
pho o-
phospho yla ion
in
chlo oplas s
becomes
con e ed
o
he
bac e ial
ype:
ATP
o ma ion
is
coupled
wi h
py idine
nucleo ide
educ ion
bu
no
oxygen
is
e ol ed.57
In
a
cell- ee
sys em,
oxygen
e olu ion
by
chlo oplas s
will
be
obse ed
mano-
me ically
only
unde
ce ain
condi ions.
Fi s ,
he
accep o
o
he
elec ons
"expelled"
om
chlo ophyll
in
he
p ima y
pho ochemical
eac ion
mus
no
eac
p e e en ially
wi h
he
componen s
o
he
pho osyn he ic
elec on
anspo
chain
(c .
T ebs
and
Eck83).
I
i
does
eac ,
as
in
he
case
o
phenazine
me hosul a e,
hen
cyclic
pho ophospho yla ion,
in
which
oxygen
is
no
e ol ed,
will
esul .
Second,
he
elec on
accep o
mus
no
be
easily
oxidized
by
molecula
oxygen.
This
is
o en,
bu
no
always,
ela ed
o
edox
po en ial.
Thus,
TPN
wi h
a
edox
po en ial,
a
pH
7,
o
E'o
=
-324
mV
sha es
his
p ope y
wi h
e icyanide
(E'o
=
360
mV)
and
p-benzoquinone
(E'o
=
293
mV).
In
he
in ac
plan ,
oxygen
is
no mally
e ol ed
du ing
pho osyn hesis
because
he
TPNH2
o med
in
non-
cyclic
pho ophospho yla ion
is
no
eoxidized
by
he
oxygen
o med,
bu
is
used
up
in
ca bon
assimila ion.
A
special
case
o
noncyclic
pho ophospho yla ion
by
isola ed
chlo oplas s
occu s
when
oxygen
ac s
as
he
e minal
elec on
accep o
and
becomes
educed
o
hyd o-
gen
pe oxide.
The
pho o educ ion
o
oxygen
by
isola ed
chlo oplas s,
disco e ed
by
Mehle ,72
has
been
in es iga ed
by
B own
and
Good84
and
Good
and
Hill,76
who
sugges ed
ha
i
is
"an
a i ac "
84
and
ha
" he
di ec
educ ion
o
oxygen
does
no
o dina ily
occu
o
a
signi ican
ex en
in
i o."
76
In
a
cell- ee
sys em
con aining
mic oca aly ic
amoun s
o
FMN
o
i amin
K3,
oxygen
may
eplace
TPN
as
he
elec on
accep o
in
noncyclic
pho ophospho yla-
ion
by
chlo oplas s
and
gi e
ise
o
an
oxygen-dependen ,
pseudocyclic
pho o-
phospho yla ion.
The
subs i u ion
o
oxygen
o
TPN
is
analogous
o
he
sub-
s i u ion
o
e icyanide
o
TPN.
ATP
is
s ill
o med
in
each
case,
bu
he
e-
duc an
p oduced
is
no
longe
TPNH2
bu
ei he e ocyanide,
o ,
when
oxygen
1332
BIOCHEMISTRY:
ARNON
ET
AL.
PROC.
N.
A.
S.
is
he
elec on
accep o ,
H202
o
wa e .
Thus,
he
in e en ion
o
oxygen
in
noncyclic
pho ophospho yla ion
esul s
in
a
loss
o
biochemically
use ul
ene gy
which
would
ha e
no mally
been
used
o
o ming
a
s ong
educ an ,
i.e.
TPNH2.
When
oxygen
is
he
elec on
accep o
and
wa e
is
he
inal
p oduc
o
oxygen
educ ion
(equa ions
(6)
and
(7))
he
ene gy
loss
is
equi alen
o
a
edox
po en ial
o
1139
mV
(di e ence
be ween.
Eo'
-815
mV
o
he
wa e /oxygen
sys em
an(d
Eo'
=
-324
mV
o
he
TPN/TPNH2
sys em).
I
hyd ogen
pe oxide
is
allowed
o
accumula e
as
he
inal
p oduc
o
he
oxygen
educ ion,
he
compu ed
ene gy
loss
would
be
equi alen
o
a
edox
po en ial
o
1006
mV.
*
The
ollowing
abb e ia ions
a e
used:
ATP,
adenosine
iphospha e;
ADP,
adenosine
di-
phospha e;
P,
o hophospha e;
TPN,
TPNH2,
oxidized
and
educed
o ms
o
iiphosphopy idine
nucleo ide;
FMN,
ibo la in
phospha e
( la in
mononucleo ide);
PMS,
phenazine
me hosul a e.
Aided
by
g an s
om
he
Na ional
Ins i u es
o
Heal h
and
he
O ice
o
Na al
Resea ch.
1
A non,
D.
I.,
Bull.
To ey
Bo an.
Club,
88
(1961).
2
Ingenhousz,
J.,
Essay
on
he
ood
o
plan s
and
he
eno a ion
o
soils,
London
(1796).
3Lebede ,
A.,
Biochem.
Z.,
7,
1
(1907).
4Lebede ,
A.,
Be .
deu .
bo an.
Ges.,
27,
598
(1909).
6
Lebede ,
A.
F.,
Iz es .
Donsko o
Gosuda s .
Uni .,
3,
25
(1921),
ansla ed
in
Am.
Re .
So ie
Med.,
5,
15
(1948).
6Van
Niel,
C.
B.,
A ch.
Mik obiol.,
3,
1
(1931).
7
Van
Niel,
C.
B.,
in
Pho osyn hesis
in
Plan s,
ed.
J.
F anck
and
W.
E.
Loomis
(Ames,
Iowa:
Iowa
S a e
College
P ess,
1949),
p.
437.
8
Hill,
R.,
P oc.
Roy.
Soc.
(London),
B
127,
209
(1939).
"
Hill,
R.,
Symposia
Soc.
Exp l.
Biol.,
5,
221
(1951).
10
Bu k,
D.,
and
0.
Wa bu g,
Z.
Na u o sch.,
6b,
12
(1951).
"1
Wa bu g,
O.,
Science,
128,
68
(1958).
12
Wa bu g,
O.,
G.
K ippahl,
H.
S.
Gewi z,
and
W.
Z.
Volke ,
Z.
Na u o sch.,
14b,
712
(1959).
13
Vishniac,
W.,
and
S.
Ochoa,
J.
Biol.
Chem.,
198,
501
(1952).
14
Holze ,
H.,
Z.
Na u o sch.,
6b,
424
(1951).
15
Bassham,
J.
A.,
A.
A.
Benson,
L.
D.
Kay,
A.
Z.
Ha is,
A.
T.
Wilson,
and
M.
Cal in,
J.
Am.
Chem.
Soc.,
76,
1760
(1954).
16
A non,
D.
I.,
M.
B.
Allen,
and
F.
R.
Wha ley,
Na u e,
174,
394
(1954).
17
F enkel,
A.
W.,
J.
Am.
Chem.
Soc.,
76,
5568
(1954).
18
A non,
D.
I.,
Na u e,
184,
10
(1959).
19
A non,
D.
I.,
M.
Losada,
M.
Nozaki,
and
K.
Tagawa,
ibid.,
190,
601
(1961).
20
S anie ,
R.
Y.,
Bac e iol.
Re .,
25,
1
(1961).
21
K ogmann,
D.
W.,
and
B.
Vennesland,
J.
Biol.
Chem.,
234,
2205
(1959).
22
A non,
D.
I.,
F.
R.
Wha ley,
and
M.
B.
Allen,
Science,
127,
1026
(1958).
23
A non,
D.
I.,
F.
R.
Wha ley,
and
M.
B.
Allen,
Biochim.
e
Biophys.
Ac a,
32,
47
(1959).
24
T ebs ,
A.
V.,
H.
Y.
Tsujimo o,
and
D.
I.
A non,
Na u e,
182,
351
(1958).
25
Losada,
M.,
A.
V.
T ebs ,
and
D.
I.
A non,
J.
Biol.
Chem.,
235,
832
(1960).
28
T ebs ,
A.
V.,
M.
Losada,
and
D.
I.
A non,
ibid.,
234,
3055
(1959).
27
A non,
D.
I.,
in
Enzymes:
Uni s
o
Biological
S uc u e
and
Func ion,
ed.
0.
H.
Gaeble
(New
Yo k:
Academic
P ess,
Inc.,
1956),
p.
279.
28
A non,
D.
I.,
M.
B.
Allen,
and
F.
R.
Wha ley,
Biochim.
e
Biophys.
Ac a,
20,
449
(1956).
29
Wha ley,
F.
R.,
M.
B.
Allen,
L.
L.
Rosenbe g,
J.
B.
Capindale,
and
D.
I.
A non,
ibid.,
20,
462
(1956).
30
Cal in,
M.,
P oc.
In e n.
Cong .
Biochem.,
3 d
Cong .,
B ussels,
1955
(New
Yo k:
Academic
P ess,
Inc.,
1956),
p.
211.
31
Vishniac,
W.,
B.
L.
Ho ecke ,
and
S.
Ochoa,
Ad ances
in
Enzymol.,
19,
1
(1957).
32
T udinge ,
P.
A.,
Biochem.
J.,
64,
274
(1956).
33
Aube ,
J.
P.,
G.
Milhaud,
and
J.
Mille ,
Ann.
ins .
Pas eu ,
92,
515
(1957).
34
McFadden,
B.
A.,
and
D.
E.
A kinson,
A ch.
Biochem.
Biophys.,
66,
16
(1957);
McFadden,
B.
A.,
J.
Bac e iol.,
77,
339
(1959).
VOL.
47,
1961
BIOCHEMISTRY:
ARNON
ET
AL.
1333
'5
S ehle ,
B.
L.,
A ch.
Biochem.
Biophys.,
43,
67
(1953).
36
Ga on,
H.,
in
Au o ophic
Mic oo ganisms,
ed.
B.
A.
F y
and
J.
L.
Peel
(Camb idge,
Eng-
land:
Camb idge
Uni e si y
P ess,
1954),
p.
163.
37
A on,
M.,
and
A.
T.
Jagendo ,
Na u e,
179,
428
(1957).
38
A on,
M.,
A.
T.
Jagendo ,
and
M.
E ans,
Biochim.
e
Biophys.
Ac a,
26,
262
(1957).
39
Jagendo ,
A.
T.,
in
The
Pho ochemical
Appa a us,
B ookha en
Symposia
in
Biology,
No.
11
(1958).
40
Jagendo ,
A.
T.,
and
M.
A on,
J.
Biol.
Chem.,
231,
277
(1958).
41
Wessels,
J. S.
C.,
Biochim.
e
Biophys.
Ac a,
25,
97
(1957).
42
Ibid.,
29,
113
(1958).
43
Chow,
C.
T.,
and
B.
Vennesland,
Plan
Physiol.,
32
(Supp.),
i
(1957).
44
Nakamo o,
T.,
D.
W.
K ogmann,
and
B.
Vennesland,
J.
Biol.
Chem.,
234,
2783
(1959).
46
Hill,
R.,
and
D.
A.
Walke ,
Plan
Physiol.,
34,
240
(1959).
46
Wha ley,
F.
R.,
M.
B.
Allen,
A.
V.
T ebs ,
and
D.
I.
A non,
ibid.,
35,
188
(1960).
47
O he accoun s
o
he
disco e y
o
pho osyn he ic
phospho yla ion
and
CO2
assimila ion
by
isola ed
chlo oplas s
a e
some imes
ound
in
he
li e a u e.
Thus
in
1956,
J.
A.
Bassham
and
M.
Cal in,
in
Cu en s
in
Biochemical
Resea ch,
ed.
D.
B.
G een
(New
Yo k:
In e science,
1956),
asc ibed
he
disco e y
o
CO2
assimila ion
by
isola ed
chlo oplas s
o
Boychenko
and
Ba ano .48
In
1959,
Cal in
( e .
49,
p.
152)
asc ibed
he
disco e y
o
bo h
CO2
assimila ion
and
ATP
syn-
hesis
by
isola ed
chlo oplas s
o
his
own
labo a o y.
48
Boychenko,
E.
A.,
and
V.
I.
Ba ano ,
Doklady
Akad.
Nauk
S.S.S.R.,
95,
1025
(1954);
Chem.
Abs .,
48,
8881
(1954).
49
Cal in,
M.,
Re .
Mode n
Phys.,
31,
147
(1959);
also
in
Biophysical
Science-A
S udy
P o-
g am,
ed.
J.
C.
Oncley
(New
Yo k:
John
Wiley
and
Sons,
1959),
p.
152.
50
Williams,
A.
M.,
Biochim.
e
Biophys.
Ac a,
19,
570
(1956).
51
Thomas,
J.
B.,
and
A.
M.
Haans,
ibid.,
18,
286
(1955).
52
Pe ack,
B.,
and
F.
Lipmann,
in
Ligh
and
Li e,
ed.
W.
D.
McEl oy
and
B.
Glass
(Bal imo e:
Johns
Hopkins
P ess,
1961),
p.
621.
6
A non,
D.
I.,
ibid.,
p.
489.
64"F enkel,
A.
W.,
J.
Biol.
Chem.,
222,
823
(1956).
56
Gelle ,
D.
M.,
"Pho ophospho yla ion
by
Rhodospi illum
ub um
p epa a ions,"
Doc o al
Disse a ion,
Di .
Med.
Sci.,
Ha a d
Uni e si y
(1957).
66
Vennesland,
B.,
T.
Nakamo o,
and
B.
S e n,
in
Ligh
and
Li e,
ed.
W.
D.
McEl oy
and
B.
Glass
(Bal imo e:
Johns
Hopkins
Uni e si y
P ess,
1961),
p.
609.
67
Losada,
M.,
F.
R.
Wha ley,
and
D.
I.
A non,
Na u e,
190,
606
(1961.)
58
Duysens,
L.
N.
M.,
J.
Amesz,
and
B.
M.
Kamp,
ibid.,
190,
510
(1961).
69
Nozaki,
M.,
K.
Tagawa,
and
D.
I.
A non,
hese
PROCEEDINGS,
47,
1334
(1961).
60
Hill,
R.,
and
W.
D.
Bonne ,
in
Ligh
and
Li e,
ed.
W.
D.
McEl oy
and
B.
Glass
(Bal imo e:
Johns
Hopkins
Uni e si y
P ess,
1961),
p.
424.
61
C ane,
F.
L.,
Plan
Physiol.,
34,
128
(1959);
Bishop,
N.
I.,
hese
PROCEEDINGS,
45,
1696
(1959).
62
K ogmann,
D..
W.,
Biochem.
Biophys.,
Resea ch
Commun.,
4,
275
(1961).
63T ebs ,
A.
V.,
M.
Losada,
and
D.
I.
A non,
J.
Biol.
Chem.,
235,
840
(1960).
64
K ogmann,
D.
W.,
A.
T.
Jagendo ,
and
M.
A on,
Plan
Physiol.,
34,
272
(1959).
65
Chance,
B.,
and
J.
M.
Olson,
A ch.
Biochem.
Biophys.,
88,
54
(1960).
"
Ma b,
E.,
and
E.
Fo i,
Science,
126,
976
(1957).
67
F ench,
C.
S.,
in
Handbuch
de
P anzenphysiolqgie,
ed.
W.
Ruhland
(Heidelbe g:
Sp inge ,
1960),
ol.
5,
pa
1,
p.
252.
68
A on,
M.,
D.
W.
K ogmann,
and
A.
T.
Jagendo ,
Biochim.
e
Biophys.
Ac a,
30,
144
(1958).
69
Da enpo ,
H.
E.,
Biochem.
J.,
77,
471
(1960).
70
A non,
D.
I.,
pape
p esen ed
a
he
Cell
Symposium,
Ame .
Assn.
o
he
Ad ancemen
o
Sci.,
Be keley
Mee ing
(1954);
Science,
122,
9
(1955).
71
A non,
D.
I.,
M.
B.
Allen,
and
F.
R.
Wha ley,
Biochim.
e
Biophys.
Ac a,
20,
449
(1956).
72
Mehle ,
A.
H.,
A ch.
Biochem.
Biophys.,
33,
65
(1951).
73
Ibid.,
34,
339
(1951).
74
Mehle ,
A.
H.,
and
A.
H.
B own,
ibid.,
38,
365
(1952).
75
T ebs ,
A.,
and
H.
Eck,
Z.
Na u o sch.
(in
p ess).
1334
BIOCHEMISTRY:
NOZAKI
ET
AL.
PROC.
N.
A.
S.
76
Good,
N.,
and
R.
Hill,
A ch.
Biochem.
Biophys.,
57,
355
(1955).
77
K ogmann,
D.
W.,
J.
Biol.
Chem.,
235,
3630
(1960).
78
A non,
D.
I.,
in
Handbuch
de
P lanzenphysiologie,
ed.
W.
Ruhland
(Heidelbe g:
Sp inge ,
1960),
ol.
5,
pa
1,
p.
773.
79
Bal sche sky,
H.,
S ensk
Kemisk
Tidsk i ,
72,
4
(1960).
'I
Wa bu g,
O.,
in
Hea y
Me al
P os he ic
G oups
and
Enzyme
Ac ion
(Ox o d,
England:
Cla en-
don
P ess,
1949),
p.
213.
81
Wessels,
J.
S.
C.,
Biochim.
e
Biophys.
Ac a,
19,
548
(1956).
82
Massey,
V.,
ibid.,
34,
255
(1959).
83T ebs ,
A.,
and
H.
Eck,
Z.
Na u o sch.,
16b,
44
(1961).
84
B own,
A.
H.,
and
N.
Good,
A ch.
Biochem.
Biophys.,
57,
340
(1955).
NONCYCLIC
PHOTOPHOSPHORYLATION
IN
PHOTOSYNTHETIC
BACTERIA
BY
M.
NOZAKI,
K.
TAGAWA,
AND
DANIEL
I.
ARNON*
LABORATORY
OF
CELL
PHYSIOLOGY,
UNIVERSITY
OF
CALIFORNIA,
BERKELEY
Communica ed
Augus
2,
1961
Noncyclic
pho ophospho yla ion
is
he
o ma ion
o
adenosine
iphospha e
in
a
ligh -induced
elec on
low
om
an
ex e nal
elec on
dono
o
an
elec on
accep o
such
as
py idine
nucleo ide.
In
pho osyn hesis
o
g een
plan s
he
elec on
dono s
a e
he
hyd oxyl
ions
o
wa e
and
hence
he
o e -all
eac ion
also
p oduces,
aside
om
ATP
and
PNH2,l
molecula
oxygen,
which
is
libe a ed
when
OH-
is
pho o-
oxidized
(see
Fig.
1
in
p eceding
a icle2).
Pho osyn he ic
bac e ia
canno
use
OH-
as
an
elec on
dono
o
pho osyn hesis
bu
use
a
a ie y
o
educ an s
s onge
han
OH-,
among
hem
hyd ogen
gas,
succina e,
o
hiosul a e.
Wi h
hyd ogen
gas
no
inpu
o
ligh
ene gy
is
equi ed
o
he
educ ion
o
py idine
nucleo ide,
since
hyd ogen
gas,
wi h
he
aid
o
bac-
e ial
hyd ogenase,
can
educe
py idine
nucleo ide
in
he
da k.3
The
con ibu ion
o
ligh
o
bac e ial
pho osyn hesis
is
hen
limi ed
o
he
o ma ion
o
ATP
by
cyclic
pho ophospho yla ion.4
I
Wi h
o he
elec on
dono s
such
as,
o
example,
succina e,
he
addi ional
inpu
o
ene gy
ha
is
necessa y
o
educe
py idine
nucleo-
ide
is
p o ided
by
ligh .
Thus,
in
bac e ial
pho osyn hesis
wi h
succina e
as
he
elec on
dono ,
ligh
ene gy
se es
a
dual
pu pose:
i
supplies
ATP
by
cyclic
pho ophospho yla ion
and,
by
pho ooxidizing
he
elec on
dono ,
i
also
p o ides
elec ons
o
educing
py idine
nucleo ides
by
a
noncyclic
elec on
low
mechanism.
Pho o educ ion
o
py idine
nucleo ide
(DPN)
by
cell- ee
p epa a ions
o
Rhodo-
spi illum
ub um6
I
and
Ch oma ium3
has
been
obse ed
bu
a emp s
o
ind
a
simul aneous
ATP
o ma ion,
analogous
o
he
noncyclic
pho ophospho yla ion
in
chlo oplas s,
led
o
nega i e
esul s.7'
8
As
poin ed
ou
by
Ve non
and
Ash,8
a
demons a ion
o
ATP
o ma ion
by
a
noncyclic
elec on
low
mechanism
is
mo e
di icul
in
ch oma opho es
han
in
chlo oplas s
because
in
ch oma opho es
i
canno
be
expe imen ally
dis inguished
om
a
simul aneous
ATP
o ma ion
by
cyclic
pho ophospho yla ion.
In ch oma opho es,
a
igo ous
cyclic
pho ophos-
pho yla ion
occu s
unde
anae obic
condi ions
and
wi hou
added
co ac o s,3
whe eas
in
chlo oplas s
cyclic
pho ophospho yla ion
is
always
unde
expe imen al