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BIOCHIMICA ET BIOPHYSICAACTA
249
BBA 45010
S T U D I E S ON L I G H T - I N D U C E D I N H I B I T I O N O F R E S P I R A T I O N
IN PURPLE BACTERIA: ACTION SPECTRA FOR
RHODOSPIRILL UM R UBR UM
AND RHODOPSEUDOMONAS
SPHEROIDES*
D. C. FORK** AND J. C. GOEDHEER
Biophysical Research Group, Physical Institute, State University, Utrecht (The Netherlands)
(Received September 2oth, 1963)
SUMMARY
I. A reversible light-induced inhibition of respiration has been studied in the
purple bacteria Rhodospirillum rubrum and Rhodopseudomonas spheroides by measuring 0 3 uptake with a Teflon-covered Pt electrode. The action spectra follow the
absorption of bacteriocb_lorophyll and show a partial participation of carotenoids
which was higher in Rhodopseudomonas than in Rhodospirillum.
2. Light saturation of the inhibition effect occurs at a much lower light intensity
than saturation of photosynthesis. Respiration stimulation after the end of illumination, similar to that occurring in red algae, could be observed. The measurements
suggest an intimate coupling between photosynthesis and respiration. The inhibition
effect seems to be brought about by a competition for electrons (or H +) by intermediates common to both processes.
3. NoMethylphenazonium methosulfate stimulates dark 0 2 uptake and inhibits
the light suppression of respiration.
INTRODUCTION
There are various indications of an interaction between photosynthesis and respiration
in photosynthesizing cells containing chlorophyll a 1-4. Photosynthesis in purple bacteria does not result in evolution of O 3 (ref. 5). Here the changes in O 3 concentration
are primarily due to changes in respiration. This makes these bacteria especially
suitable for measurements of the interaction of respiration and photosynthesis.
The observation of a light-induced suppression of respiration in a purple bacterium reported by NAKAMURAs has since been confirmed and extended by others 5,7-11.
KATOHTM has recently shown the inhibition effect to be located in the chromatophore.
HoRIo AND KAMENn suggested that a chain of electron-transport carriers operating
Abbreviations: DCMU, 3-(3,4-dichlorophenyl)-i,i-dimethylurea; PMS, N-methylphenazonium methosulfate.
"This work was performed at the Biophysical Research Group of the State University at
Utrecht during a visit by D.F. who is a member of the Carnegie Institution of Washington.
**
Present address : Department of Plant Biology, Carnegie Institution of Washington, Stanford,
Calif. (U.S.A.).
Biochim. Biophys. Acta, 79 (1964) 249-256
250
D . C . FORK, J. C. GOEDHEER
in cyclic fashion with the photoactive pigments and heme proteins provide competitive hydrogen accepters. If substrate hydrogen which normally reduces O 2 via the
respiratory chain is diverted, upon illumination, to the reduction of CO 2 via the photochemical apparatus then the action spectrum for this effect would be expected to
follow the absorption of the photosynthetically active pigments. This expectation was
borne out by the action spectra determined here.
With green plants two pigment systems are known to be needed to perform complete photosynthesis. With photosynthetic bacteria there is as yet no indication of
participation of more than one pigment system in photosynthesis. Action spectra
for light-induced inhibition of respiration might give an indication of a possible
participation of more than one pigment system in bacterial photosynthesis.
MATERIALS AND METHODS
Rhodospirillum rubrum cultures were grown in a I °/o peptone-o.5 % NaC1 medium
at pH 7, and Rhodopseudomonas spheroides in 0.5 % yeast extract, 0.5 % MgS04,
0.3 % L-malic acid and 0.02 M phosphate buffer at a pH of 6.8. Both media were
made with tap water. The anaerobic cultures were grown with continuous incandescent illumination around 25 ° . Cells were used for action spectra determinations
after I day's growth.
Respiratory changes were followed by means of a Teflon-covered Pt electrode
used in conjunction with the liquid-circulating and gas-exchange system described
previously 12. The liquid-circulating system was not used for action spectra determinations. Instead, the bacteria after centrifugation were resuspended as a thin suspension in a fresh sample of medium. A drop of this suspension was placed on the
electrode and held in place with another piece of 6/~ thick Teflon-covered membrane.
Air was passed at a constant rate over the bacteria on the electrode.
For action spectra measurements the bacteria were illuminated with a 5oo-mm
focal length Bausch and Lomb monochromator having a IOO > ioo-mm grating ruled
with 6oo grooves/mm. Each action spectrum was done in three parts with the slits
set to pass a beam having a half width of 3.3 m/,. From 94o to 74 ° m/~, a 6oo-m/~
cut-off filter to remove second-order wavelengths plus a 48 °"o transmission neutral
density filter were inserted in tile monochromator beam; from 65o to 55o m/~ only
the 48 % transmission filter was used; and from 55o to 45o m/~ no filters were used
in the monochromator beam. The precision of measurement was lower in this region
than for the other portions of the action spectrum because the light intensities were
low and the resulting responses small. The wavelength dial was turned manually at
I m/~ per IO sec while inhibition of respiration was being recorded. The resulting
record was then corrected for equal incident quanta and for loss of activity with time
(if any) and replotted at 5-m/~ intervals.
RESULTS
A time course for inhibition of respiration of Rhodospirillum exposed to 88o-m~ light
is given in Fig. I. Decreased respiration is indicated by deflection of the pen above
the dark baseline because more 0 2, diffusing from the circulating medium, can be
reduced at the electrode when the respiratory uptake is lower. The electrode measures
only changes in respiratory 02 uptake since these bacteria do not evolve 0 2 (ref. 5)Biochim. Biophvs. ,4cla, 79 (1964) 249-256
251
LIGHT INHIBITION OF RESPIRATION IN BACTERIA
That these bacteria are not evolving 0 8 is also seen b y the disappearance of the
inhibition of respiration effect when the cells are made anaerobic (unlike green-plant
0 8 evolution which m a y continue under anaerobic conditions). Also, adding the
inhibitor of green-plant 0 8 evolution, DCMU, to a final concentration of 6.5" lO -5 M
did not have an appreciable effect on light-induced inhibition.
20
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Fig. I. Time course of inhibition of respiration in Rhodospirillum rubrum u p o n exposure to 88o-m/z
light h a v i n g an intensity of 497 ergs. cm-~.sec -1 and a half b a n d w i d t h of io m/~. The cells,
harvested after 2 days' growth, were resuspended in fresh m e d i u m and gassed w i t h 5 % CO2 in air.
JOHNSTON AND BROWN5 found m a x i m u m light-induced respiratory inhibitions
to range from 60 to 85 %. A similar magnitude of inhibition was noted in the present
study. The time course of inhibition given in Fig. i shows that the inhibition becomes
constant after about 2 rain in 88o-m/~ light. Darkening the cells causes the recorder
tracing to dip below the dark baseline established previously. It regains its former
level in about 12 min which suggests that an exposure to 88o-m/~ light causes a
temporary respiratory stimulation (compare respiratory stimulation observed in the
red alga Porphyridium cruentum b y FRENCH AND FORKS). A similar stimulation of
20
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Fig. 2. I n h i b i t i o n of respiration as a function of light intensity. F o r R h o d o p s e u d o m o n a s the
85o-m/* light used h a d a half b a n d w i d t h of 3.3 m/z. Cells from a 1-day-old culture were resuspended
in fresh m e d i u m a n d gassed w i t h air. This sample was used for d e t e r m i n a t i o n of the action spect r u m . F o r Rhodospirillum the 88o-m~u light used had a half b a n d w i d t h of IO m/z. 2-day-old
culture gassed w i t h 5 % CO2 in air.
Biochim. Biophys. Acta, 79 (1964) 249-256
252
D . C . FORK, J . C . GOEDHEER
respiration following illumination of Rhodopseudomonas with 85o-mt~ light has also
been observed. In some instances no stimulation of respiration follows illumination.
Light-saturation curves of respiration inhibition, measured for Rhodopseudomonas with incident light of 85o mff and for Rhodospirillum with light of 88o mF,
are given in Fig. 2. Since these curves start to bend even at low light intensities
the action spectra were determined by keeping the intensities as low as possible. The
Rhodopseudomonas sample used for the saturation curve given in Fig. 2 was also
used to determine the action spectrum given in Fig. 3- At the 85o-mff peak in the
action spectrum the intensity used was 64. 3 ergs.cm-2.sec -1. At this intensity the
effect per unit of intensity is 16 % less titan at very low intensity. Since the calculations were made by assuming that a linear relationship existed between inhibition
of respiration and light intensity the action spectrum would be flattened somewhat
in this region. The action spectrum for relative inhibition of respiration in Rhodopseudomonas has peaks at 85o, 8oo, 59 o, 51o, and 48o mff and a shoulder around
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Fig. 3. L o w e r half : A c t i o n s p e c t r u m for relative i n h i b i t i o n of r e s p i r a t i o n b y l i g h t in Rhodopseudomonas spheroides. 1-day-old c u l t u r e in g r o w t h m e d i u m , gas p h a s e air. U p p e r half: A b s o r p t i o n
s p e c t r u m of c h r o m a t o p h o r e s in p h o s p h a t e buffer w h i c h were p r e p a r e d f r o m a different s a m p l e
t h a n u s e d for a c t i o n s p e c t r u m .
Biochim. Biophys. Acta, 79 (1964) 249-256
LIGttT INHIBITION OF RESPIRATION IN BACTERIA
253
880 m/,. The action spectrum for Rhodospirillum (Fig. 4) has peaks at 88o, 81o, 595,
approx. 520, and approx. 485 m~.
KATOH1° has studied the effect of a number of inhibitors on photoinhibition of
respiration but found none which specifically affected the photoinhibition in question.
He noted, however, that the effect was sensitive to high temperature and could be
abolished b y a 5-min treatment at 4 o°. HORIO AND KAMEN11 discovered that 3 M
methanol, I M ethanol, and 0.05 M isobutanol inhibited almost all of the lightsensitive respiration. This was attributed to a disruption in the coupling between
the photoactive pigments.
A disruption of light inhibition of respiration was noted with the redox dye, PMS.
Fig. 5 shows that in Rhodospirillum, PMS stimulates respiration in the dark and
inhibits the effect of light on respiration. When cells in phosphate buffer and sodium
butyrate are exposed to 88o-m/, light a 75 % inhibition of respiration results. After
the 88o-m F exposure a transient respiratory stimulation of 25 % results. Addition of
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Fig. 4. Lower half: Action spectrum for relative inhibition of respiration by light in Rhodospirillura
rubrum. I-day-old culture in growth medium, gas phase air. A different sample used for the action
spectrum from t h a t used for the saturation curve of Fig. 2. Upper half: Absorption spectrum of
chromatophores in phosphate buffer which were prepared from a different sample than used for
action spectrum.
Biochim. Biophy$. Acta, 79 (I964) 249-256
254
D . C . FORK, J. c. GOEDHEER
~
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Fig. 5. Effect of PMS on inhibition of respiration by light in Rhodospirillum. Cells from a 7-dayold culture in o.oi M sodium butyrate and o.o2 M Na2HPO4-KH2PO 4 buffer (pH 7.5). Gas phase,
air. The 88o-m/~ light (half band width io m#) had an intensity of 497 ergs.cm -2.sec -1. The
same intensity 88o-m# light used after addition of PMS (to a final concentration of 3-3" io 4 M).
PMS (arrow) in the d a r k increases 0 2 u p t a k e b y 8o %. The time course of inhibition
of respiration b y light in the presence of PMS is m a r k e d l y slowed down. A f t e r 4 min
in the light, inhibition is o n l y I8 % as c o m p a r e d to 75 % w i t h o u t PMS. The t i m e
course of r e c o v e r y of respiration in darkness is complex a n d shows a fast c o m p o n e n t
followed b y a slower one. No s t i m u l a t i o n of respiration follows i l l u m i n a t i o n in tile
presence of PMS. R e p e a t e d exposures to 88o-m/~ light resulted in a g r a d u a l decrease
in the a m o u n t of l i g h t - i n d u c e d inhibition of respiration as well as a g r a d u a l r e t a r d a t i o n
in the t i m e course. (The zero-respiration line is the electrode d a r k current after the
cells were killed b y a d d i n g f o r m a l d e h y d e solution to a final c o n c e n t r a t i o n of a b o u t
4 % in the circulating system.)
DISCUSSION
A scheme p r o p o s e d b y ~'~ISHIMURAla suggests t h a t electron t r a n s p o r t for b o t h p h o t o synthesis a n d respiration passes t h r o u g h a c o m m o n c y t o c h r o m e . A similar idea has
been p r o p o s e d b y HoRIo AND KAMEN n who e x p l a i n e d l i g h t - i n d u c e d inhibition of
respiration on the basis of a c o m p e t i t i o n between t h e p h o t o a c t i v e p i g m e n t s a n d an
i n t e r m e d i a t e in t h e r e s p i r a t o r y e l e c t r o n - t r a n s p o r t chain.
A c t i o n s p e c t r a for l i g h t - i n d u c e d inhibition of respiration in Rhodospirillum
rubrum a n d Rhodopseudomonas spheroides which we h a v e d e t e r m i n e d in the range
from 45 ° to 95o m/~ indicate a close correspondence between this inhibition effect
Bioehim. Biophys. Acta, 79 (I964) 249-256
LIGHT INHIBITION OF RESPIRATION IN BACTERIA
255
a n d the spectral absorption of the photosynthetic pigments, and suggest an intimate
coupling between photosynthesis and respiration. This substantiates the assumption
of NAKAMURAs that under certain conditions it is reasonable to study some aspects
of bacterial photosynthesis by measuring the light-induced changes of respiration of
the organisms'. The inhibition effect, however, saturates at a light intensity of only
a few per cent of that of photosynthesis. HORIO et al. 15 are reporting an action spectrum
for light inhibition of respiration in Rhodospirillum r u b r u m in the 4io-6io-m/z region
which appears to be similar to that reported here.
A comparison of the relative activity of the carotenoids in sensitizing inhibition
in Rhodopseudomonas and Rhodospirillum shows carotenoid activity to be higher
in Rhodopseudomonas. It is interesting to note, in this regard, that GOEDHEERle has
found a higher efficiency in the transfer of energy from carotenoids to bacteriochlorophyll in Rhodopseudomonas than in Rhodospirillum. These action spectra are
similar to the action spectra for phototaxis of a young culture of Rhodospirillum
r u b r u m reported b y DUYSENS14.
The similarity of the spectra of inhibition effect and absorption in the near
infrared, both with Rhodopseudomonas and Rhodospirillum, and the absence of a
measurable "long-wavelength decline" indicate that all bacteriochlorophyll types
"B 800, B 850 etc." participate in this reaction, either directly or via energy transfer.
This appears to be another indication that bacterial photosynthesis acts via a single
pigment system.
A disruption of photometabolism of Rhodospirillum b y 5" lO-4 M PMS was noted
by GEST et al. 17 who found it to inhibit completely the endogenous and substratedependent H z evolution. It also caused the cells to ferment their endogenous reserves
with the formation of f a t t y acids even though they were in continuous light. This
fermentation was attributed to an inhibition of photophosphorylation by PMS.
However, KATOI-I1° ruled out the possibility that the photoinhibition effect could be
explained on the basis of a competition between photophosphorylation and oxidative
phosphorylation for a common phosphate acceptor since added ADP had little effect.
He also noted that o-phenanthroline or 2,6-dichlorophenolindophenol in concentrations effective in blocking photophosphorylation did not affect photoinhibition.
PMS m a y mediate a more rapid passage of electrons to 0 2 by acting as a "bypass"
of that intermediate which is common to both photosynthesis and respiration, resulting in an increased dark respiration and a loss of the inhibitory effect of light
on respiration.
GOEDHEER16 has suggested an interaction of respiration in a two-pigment system
for photosynthesis in green plants in order to explain chromatic transients, induction
effects, and certain other aspects of luminescence. His scheme also proposes a cytochrome common to both photosynthesis and respiration and suggests that excitation
of the long-wavelength chlorophyll reaction would result in an inhibition of 0 2 uptake.
It is interesting in this regard that HOCH et al. 3 noted an inhibition of respiration
in the bhie-green alga Anacystis when chlorophyll was excited and very little, if any,
when phycocyanin was excited.
Tile light-stimulated respiration of these bacteria which persists for some minutes
in the dark after the exposure is also analogous to respiratory stimulation observed
* A suggestion t h a t action spectra of p h o t o s y n t h e s i s could be measured in this w a y has been
m a d e by L. N. M. DUYSENS14 in his Stelling VI.
Biochim. Biophys. Acta, 79 (1964) 249-256
256
D . C . FORK, J. c. GOEDFIEER
after excitation of long-wavelength chlorophyll in Porphyridium 2. This m a y result
from the accumulation of a reduced intermediate such as pyridine nucleotide in the
light which is respired in the dark.
ACKNOWLEDGEMENTS
One ot the authors (D.F.) wishes to express his thanks to Professor J. B. THOMAS
who made all the facilities of the Biophysical Research Group of the State University
of Utrecht available to him, and to the Carnegie Institution of Washington which
made this visit possible.
REFERENCES
1 D. WEIS AND A. H. BROWN, Plant Physiol., 34 (1959) 235.
C. S. FRENCH AND D. C. FORK, Carnegie Inst. Wash. Yearbook, 60 (1961) 351.
a G. HocH, O. v. H. OWENS AND B. KOK, Arch. Biochem. Biophys., IOi (1963) 171.
4 j . C. GOEDIaEER, Bioehim. Biophys. Acta, 66 (1963) 61.
5 j . A. JOHNSTON AND A. H. BROWN, Plant Physiol., 29 (1954) 177.
tl H. NAKAMURA, Acta Phytoehim., 9 (1937) 189.
7 C. B. VAN NIEL, Advan. Enzymol., I (1941) 263.
C. B. VAN NIEL, Baeteriol. Rev., 8 (1944) I.
9 S. MORITA, J. Biochem., 42 (1955) 533.
10 S. KATOH, J. Bioehem., 49 (1961) 126.
11 T. HORIO AND M. D. KAMEN, Biochemistry, i (1962) 1141.
1~ D. C. FORK, Plant Physiol., 38 (1963) 323.
la M. NISHIMURA, Bioehim. Biophys. Aeta, 57 (1962) 88.
14 L. N. M. DUYSENS, Thesis, U t r e c h t , 1952.
15 T. HoRIO AND C. P. S. TAYLOR,J. Biol. Chem., in t h e press.
is j . c. GOEDHEER, Biochim. Biophys. Acta, 35 (1959) I.
17 H. GEST, J. G. ORMEROD AND K. S. ORMEROD, Arch. Biochem. Biophys., 97 (1962) 21.
Biochim. Biophys. Acta, 79 (1964) 2 4 9 - 2 5 6