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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 Dark 8Bo m~ I > v L- I ' ~ ' ~ ' ~ Time (rain) ' I ~o ~6 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 ~ ~ , i ' -: Rhodopseudomonas r / Rhodospirillum .-c ~,_~ ,o ~ .[.5 5 -- , 0 ~ I00 200 I t 300 I 400 i 500 Lighf infensity(ergs cm-2sec-I) 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 r Absor phon spec!rum 6O i I I, li I i 2ol i --L 550 ' 40 >~ T A 650 750 Wavelength (m/s) • T • q 850 ' ~- Achon specfrum 950 " [ \ l I ' ~2oF i '°i 0450 , //'~ ' 550 . . . . 650 750 Wavelength ( m~ ) ~] t i 850 950 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 I Absorp÷ion I ~ " - spec+rum 6C 8 u 2O 450 i I i 550 4O 1 ~ 750 I 850 950 (m]J) Achsopnecurtm~ I L I 650 Wavelength I J I a. ~2c .o = 450 I 550 a ] .... ~0 Wavelength ~" j " I 750 (m,/J) r I 850 950 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 ~ 1 16 I I I P I I [ I ] I I ; Dark currenf for zero respira+ion ~- I'~ 880m~ '1 Dark I'-- 88o.,~, '1 Dark / 14 I- / / - - ~ - - ~ " ,21- i ,° 8 [-J Dark PMS added l '( ,/ 6k off fT---q , f' / On 0[ [ 0 I [ 4 I I I 12 16 I i 20 i 24 Time(rain) 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