Bioscience Reports 2, 743-749 (1982)
Printed in Great Britain
743
L o c a l i z e d e n e r g y c o u p l i n g during p h o t o p h o s p h o r y l a t i o n by
chromatophores of Rhodopseudomonas capsulata N22
G. Duncan HITCHENS and Douglas B. KELL*
Department of Botany and Microbiology, University
College of Wales, Aberystwyth, Dyfed SY23 3DA, U.K.
(Received 31 July 1982)
The principle of the dual inhibitor titration method for
testing models of electron-transport phosphorylation is
outlined, and the method is applied to the study of
photophosphorylation in bacterial chromatophores. It is
concluded that energy coupling is strictly localized in
nature in this system, in the sense that free energy
released by a particular electron-transport chain may be
used only by a particular H+-ATP synthase.
Dual
inhibitor titrations using the uncoupler SF 68t~7 and the
H+-ATP s y n t h a s e i n h i b i t o r oligomycin indicate that
u n c o u p l e r s a c t by s h u t t l i n g r a p i d l y b e t w e e n the
localized energy-coupling sites.
An important current problem in bioenergetics may be summarized
by the q u e s t i o n "is the 'energized intermediate' coupling electron
transport and ATP synthesis detocalized at the level of the intact
v e s i c l e , as in m a c r o s c o p i c versions of the chemiosmotic coupling
hypothesis (see e.g. Mitchell, 1979; Nichols, 1982), or do there exist
more localized and direct free-energy-transferring interactions between
t h e redox and ATP synthase proton-pumping complexes in energy~
coupling membranes (e.g. Ernster, 1977; Williams, 1978; Kell, 1979;
Baccarini-Melandri et al., 1981; Conover & Azzone, 1 9 g l ) ? "
We (Hitchens & Kell, 1982), and others (Melandri et al., 1981;
Venturoli & Melandri~ 1982) have recently addressed ourselves to this
question by using the double inhibitor titration method (Baum et al.,
1971; Kell et al., 1979) with bacterial chromatophores, and have
concluded that the free energy transfer between electron transport and
ATP synthase complexes in chromatophores is fully localized in nature,
in the sense t h a t the ' h i g h - e n e r g y intermediate' generated by a
particular electron-transport chain may be used only by a particular
H+-ATP synthase. This conclusion was arrived at from the results of
experiments in which the e f f e c t on the photophosphorylation rate of
t h e c o v a l e n t H+-ATP s y n t h a s e inhibitor dicyclohexyl carbodiimide
(DCCD) was studied at different rates of electron transport (Melandri
et al., 1981; Hitchens & Ketl, 1982; Venturoli & Melandri, 1992).
However, to strengthen t h i s conclusion further, it would be desirable
to test other H+-ATP synthase inhibitors, and it has been suggested
(Westerhoff et al., 1982a,b) that the double inhibitor titration method
*To whom correspondence should be addressed.
01982
The Biochemical Society
7/#4
HITCHENS & KELL
is only completely reliable when the e f f e c t of an electron-transport
i n h i b i t o r on the titration curve of an ATP synthase inhibitor for
inhibiting phosphorylation is tested in this type of experiment.
It is
t h e purpose of the present article to report the results of such
experiments in bacterial chromatophores, using the electron-transport
inhibitor Antimycin A and the H+-ATP synthase inhibitor oligomycin,
and it is concluded that the energy coupling in electron-transport
phosphorylation is indeed 'localized' in na:ture in those membranes.
F u r t h e r , it is shown t h a t the p o t e n t uncoupler 3,5-di-t-butyl-#hydroxybenzylidene malononitrile (SF 68#7) acts 'substoichiometrically'
by s h u t t l i n g rapidly between the localized energy-coupling sites in
these membranes.
M a t e r i a l s and Methods
The growth and maintenance of Rhodopseudomonas capsulata N22,
the preparation of chromatophores therefrom, t h e assay of bacteriochlorophyll content and photophosphorylation rate, and the sources of
chemicals and biochemicals have been described elsewhere (Hitchens &
Kell, 1982), except that oligomycin was obtained from Sigma (Cat.
No. 0 #g76).
Results
The principle o f the double inhibitor t i t r a t i o n method (see 5aum et
at.9 1971; Kell et al., 1979; Hitchens & Kell, 1992) may briefly be
s u m m a r i z e d as follows.
If a reaction sequence A E~ B .+_ C is
e n v i s a g e d , in which, implicitly, A represents electron transport, B
s t a n d s for a ' h i g h - e n e r g y ' i n t e r m e d i a t e , and C constitutes phosp h o r y l a t i o n , inhibition of the net rate of phosphorylation by the
addition of an H+-ATP synthase (E 2) inhibitor will, if the 'high-energy'
intermediate B is fully delocalized, make the electron-transport-driven
formation of B less rate-limiting, i.e. the addition of an electrontransport (E 1) inhibitor such as antimycin A will have relatively less
e f f e c t on the rate of phosphorylation in the presence of an ATPase
inhibitor than in its absence. If, however, B is fully localized, in the
sense that the 'high-energy' intermediate generated by a particular
electron-transport chain may be used only by a particular H+-ATP
synthase, then the electron-transport inhibitor will be equally effective
in each case.
Fig. l shows the data from such an experiment with chromatophores from Rhodopseudomonas capsulata N22, where it may be s e e n
that, far from making the electron-transport-driven formation of the
' h i g h - e n e r g y ' i n t e r m e d i a t e less rate-limiting, partial restriction of
photophosphorylation with oligomycin actually decreases the concentration
of a n t i m y c i n r e q u i r e d to inhibit p h o t o p h o s p h o r y l a t i o n
completely.
The symmetrical experiment, in which the photophosp h o r y l a t i o n r a t e is t i t r a t e d with oligomycin in the presence and
a b s e n c e of a partially inhibitory concentration of Antimycin A, is
displayed in Fig. 2, wherein it may be observed that data entirely
c o m p a r a b l e to those of Fig. 1 are obtained, as predicted by the
'thumb rule' (see Westerhoff et al., 19g2a,b) analysis of this type of
experiment when energy coupling is fully localized in nature.
CHROMATOPHORE PHOTOPHOSPHORYLATION
7#5
0.6
_
~ 0"5
e-
,
~ o.~
E
0.3
~J
C
=o 0.2
0
e-
r-
0~
~
~
- ~
[ Antimycin A ]
06
0~
( pN )
Fig. 1.
Effect of antimycin A and oligomycin on
photophosphorylation by chromatophores of Rps.
capsu2ata N22. Photophosphorylation was measured as
described in 'Materials and Methods' in a 6-ml
reaction mixture containing, at 25~
and pH 7.8,
3 mM KH2P04,
I0 mM m a g n e s i u m acetate, 30 mM
potassium acetate, 0.2 mM sodium succinate, 1.5 mM
sodium ADP, 5 pM Pi,p5-bis-(5'-adenosyl) pentaphosphate,
800 ~g of carbonic anhydrase, and
chromatophores
corresponding
to a bacteriochlorophyll concentration of 20 pM. Antimycin A was
added to the concentrations indicated (O, 9
and
where
indicated ( 9
the chromatophores were
preincubated for 20 min with oligomycin (0.1 ~g/ml).
The uncoupler SF 6947 is apparently the most potent uncoupler
known ( T e r a d a , 1 9 g l ) , and causes complete uncoupling of photop h o s p h o r y l a t i o n in our c h r o m a t o p h o r e system at a concentration
corresponding to 0.3 molecules per e l e c t r o n - t r a n s p o r t chain (Hitchens
& Kell, 1992). The question arose (Hitchens & Kell, 1992) as to how
one might reconcile the localized energy coupling observed (Hitchens
& Kell, 1992; and see above) with the 'substoichiometric' uncoupling
catalysed by SF 68#7.
We countenanced two possibilities (Hitchens &
Kell, 1982): e i t h e r (i) SF 6g#7 s h u t t l e d b e t w e e n the localized
energy-coupling sites at a rate sufficiently rapid to uncouple at least 3
sites during one turnover Of the photophosphorylation apparatus, or
(ii) th e simple binding of a given SF 6g#7 molecule could lead to the
cooperative uncoupling of (here) 3 or more 'coupling sites'. T h e
7146
HITCHENS
"
t
i
~
1
& KELL
1
1"2'
.-.=
-5
E
:3,..
~04~
r
.g
c.6
...-
I
0"4
.s
"6
~
0"2
0,1
0"2
C8
O~
[ Otigomycin ] (,ug/mt)
=
0'5
Fig. 2.
Effect of oligomycin and antimycin A on
photophosphorylation
by chromatophores of Rps.
capsu2ata N22.
Photophosphorylation was assayed as
described in the legend to Fig. i, except that
chromatophores were preincubated with the concentrations of oligomycin indicated for periods ranging
from 20 to 45 min, prior to initiating photophosphorylation by illuminating the reaction vessel.
Where indicated ( 9
antimycin A (0.2 ~M) was also
present.
following experiment was performed to distinguish these possibilities.
A titration curve for the inhibition of photophosphorylation by SF 6847
was established.
Then photophosphorylation was partially inhibited by
p r e i n c u b a t i n g the c h r o m a t o p h o r e s with an a p p r o p r i a t e t i t r e of
oligomycin. For a 'shuttling' model it is to be expected that SF 6847
will uncouple even more effectively when the rate of phosphorylation
in the absence of uncoupler is partially decreased by oligomycin, whilst
the 'binding' model would predict that a similar titre of the uncoupler
would be required for full inhibition of photophosphorylation in both
the presence and absence of a partially inhibitory concentration of
oligomycin. The experimental data are shown in Fig. 3; it is evident
that they strongly support a model in which the uncoupler molecules
can shuttle rapidly between the (localized) energy-coupling sites.
Discussion
It is important, in experiments of the type described herein, that
the chromatophores do not contain a significant native energy 'leak',
and the evidence against this possibility, and also against that of the
CHROMATOPHORE PHoTOPHOSPHORYLATION
7#7
A(
E
::3..
s
._o
S
c-,
o
,r"
n
[ SF66.4.7 ] (nM)
Fig. 3.
Effect of SF 6847 and oligomycin on
p h o t o p h o s p h o r y l a t i o n by chromatophores of Rps.
r
N22.
Photophosphorylation was assayed as
described in the legend to Fig. i.
SF 6847 was
added to the Concentrations indicated ( O , 9 9 9
Where shown, the chromatophores were preincubated
with oligomycin at a final concentration of 0.i
Ug/ml ( 9
or 0.2 ~g/ml ( 9
occurrence of a significant chromatophore heterogeneity (as regards
energy-coupling properties), has been discussed at length elsewhere
( H i t c h e n s & K e l l , 1982).
We do not therefore reiterate these
arguments here.
T h a t oligornycin enhances the inhibitory e f f e c t on photophosphorylation of Antirnycin A (Fig. t ) , and vice versa (Fig. 2), shows very
clearly that the coupling between electron transport and phosphorylation in this system is quite strictly localized in nature, and cannot
thus be e f f e c t e d via a deloca}ized intermediate such as a bulk-to-bulk
phase e l e c t r o c h e m i c a l p r o t o n i c p o t e n t i a l difference.
The slight
difference between the data observed with oligornycin in Fig. } and
t h a t reported earlier in analogous experiments with DCCD (Hitchens &
Kell, 1982) is to be expected, and is due to the fact that oligornycin,
although a tight-binding inhibitor (see Linnett & Beechey, 1979)9 is
not quite as tightly bound in the steady state as is the covalent
modifier DCCD.
In c o n s i d e r a t i o n s based on the delocalized-chemiosmotic-coupling
t h e o r y of the r e l a t i o n s h i p b e t w e e n the a p p a r e n t or theoretical
bulk-phase protonmotive force and the rate of phosphorylation (see
714-8
HITCHENS & KELL
e.g. 3encks, 1980; 3ackson, "1982; Schlodder et al., 1982), it is taken
t h a t this r e l a t i o n s h i p , although highly non-linear, is monotonic in
nature. It is also to be assumed that partial restriction of the rate of
phosphorylation by an H+-ATP synthase inhibitor such as oligomycin
will not decrease the supposed protonmotive force in the absence of
added uncoupler.
On this basis, therefore, a delocalized coupling
model can predict only that the presence of a partially inhibitory
concentration of oligomycin will either have no e f f e c t on, or will
cause a decrease in, the efficiency of uncoupling that is displayed by
a p a r t i c u l a r uncoupler in the a b s e n c e of the H+-ATP synthase
inhibitor, whatever the presumed relationship between the protonmotive
f o r c e and the r a t e of ATP synthesis.
The observation that the
addition of a partially inhibitory concentration of oligomycin caused an
increase
in the uncoupling effectiveness of SF 6847 is explicable only
within the framework of a model in which uncoupler molecules shuttle
rapidly between the 'energized' and localized coupling sites present~
and in which the rate-limiting step in the uncoupler's action lies in
t h e uncoupling step i t s e l f , and not in the rate of diffusion of
uncoupler molecules between their sites of uncoupling action.
In conclusion, the present results, obtained with the dual inhibitor
t i t r a t i o n a p p r o a c h in b a c t e r i a l chromatophores, show clearly that
energy coupling in photophosph0rylation is strictly localized in nature,
and are in harmony with other data obtained using this approach in
chromatophores (Melandri et al., 1981; Venturoli & Melandri, 1982;
Hitchens & Kell, 1982), in submitochondrial particles (Baum et al.,
1971; Westerhoff et al., 1982a,b), and in phosphorylating membrane
vesicles from P a r a c o c c u s d e n i t r i f i c a n s
(Kell et al., 1979).
Acknowledgements
D.B.K. thanks Dr. Hans Westerh0ff for many Stimulating discussions
and for making available material prior to publication. This work was
supported by the Science and Engineering Research Council, U.K.
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CHROMATOPHORE
PHOTOPHOSPHORYLATION
749
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