Planta 9 Springer-Verlag 1984
Planta (1984) 160:235 241
Oxidation of cysteine to cystine by membrane fractions of Chlorella fusca Elisabeth Kr/imer and Ahlert Schmidt Botanisches Institut der Universitfit, Menzinger Strasse 67, D-8000 Mfinchen 19, Federal Republic of Germany
Abstract. Isolated m e m b r a n e fractions of Chlorella fusca 211-8b obtained by french-press treatment and sonication catalyzed the oxidation of L-cysteine to L-cystine. The p H - o p t i m u m of this reaction was determined to be a r o u n d 8-8.5 and a stoichiometry of 4 SH-groups oxidized for one O a consumed was obtained. This thiol-oxidation system w a s specific for D- and L-cysteine; oL-homocysteine and cysteamine were oxidized at a b o u t half the rate whereas all other thiols tested including glutathione, mercaptoethanol, mercaptopropionic acid and dithioerythritol were n o t oxidized by these m e m b r a n e fractions. The apparent K m for L-cysteine was determined as 3.3 m m o l 1 1. Rates of 200 gmol cysteine oxidized m g - 1 chlorophyll h - 1 were normally obtained. Extremely high rates of oxygen uptake were measured using L-cysteine methyl ester and L-cysteine ethyl ester. This thioloxidation system was not inhibited by mitochondrial electron-transport inhibitors such as rotenone or antimycin A, nor by the chloroplast electrontransport inhibitors 2,5-dibromothymochinone and 2,4-dinitrophenylether of iodonitrothymol. The cysteine oxidation catalyzed by C. fusca membranes was inhibited, however, by salicylhydroxamic acid, o-phenanthrolin, N,N'-disalicyliden1,3-diaminopropane-5,5'-disulfonic acid, ethylenediaminetetraacetic acid, high K C N levels and by the buffers, N-[2-hydroxyl-l,l-bis(hydroxymethyl) ethyl] glycine and phosphate. This cysteine-oxidation system seems to function as a counterpart of thioredoxin-mediated light activation of enzymes, allowing reduced thiol groups to be oxidized again by 0 2 (dark inactivation). Key words: ChloreIIa (membrane, thiol respiration) Cysteine oxidation - M e m b r a n e (thiol respiration) - Oxygenase - Thiol respiration. Abbreviation: DTNB=5,5'-dithio-bis(-2-nitrobenzoic acid), Ellmann reagent
Introduction Certain enzymes of the reductive pentose-phosphate cycle are activated in the light (Anderson 1979) by the ferredoxin-thioredoxin system (Buchanan 1980). During the light period, thioredoxin is reduced by the ferredoxin-thioredoxin reductase (De la Torre et al. 1979). The reduced thioredoxin activates oxidized enzymes by reduction of specific thiol groups (Buchanan et al. 1979). In the d a r k these enzymes are inactivated again, but this dark inactivation is not yet understood. Evidence has been obtained that molecular oxygen seems to be involved, since no inactivation occurs under anaerobic conditions (Leegood and Walker 1980, 1982). In this paper a thiol-oxidizing system, which is m e m b r a n e - b o u n d and utilizes 0 2 as an electron acceptor, is described for the green alga Chlorella fusca.
Material and methods Plant material. Chlorellafusca strain 211-8b was obtained from the algal collection of G6ttingen and grown as axenic culture as described earlier (Schmidt 1972). Preparation of membranefractions. Cells were harvested by centrifugation and the pellet was suspended in 0.1 M 2-amino-2(hydroxymethyl)-l,3-propanediol (Tris)-HC1 buffer (pH 8.0) containing 10 retool-1-1 MgC12 and 10 mmol.1-1 mercaptoethanol. Cells were broken in a french-press at 12,000 psi (1 psi ~ 7 kPa). The membrane fractions were collected by centrifugation and the pellet was washed once with 0.05 tool. 1-1 imidazol buffer (pH 8.0). Afterwards the pellet was solubilized by sonication (three times, 10 s, using a Branson S-125 sonicpower model). The membrane fractions obtained in this way were used for the experiments to be described. These membrane fractions contained 62% thylakoid membranes; 18% envelope membranes; 7% cytoplasmic membranes; 6% mitochondrial membranes; 6% nucleic membranes and about 1% endoplasmic reticulum and tonoplast membranes. These data were obtained from electron-microscopy pictures using Chlorella cells from the logarithmic growth phase before french-press treatment.
236
E. Kr/imer and A. Schmidt: Oxidation of cysteine to cystine in Chlorella / k E436
Chlorophyll determination. The method of Arnon (1949) was used.
Thiol-oxidation measurements. Thiol-group determinations were performed using the Ellmann reagent, 5,5'-dithio-bis(-2nitrobenzoic acid) (DTNB; Ellmann 1959). For these measurements a Zeiss (Oberkochen, FRG) filter photometer was used at 436 rim; the ~ for DTNB at this wavelength was calculated as 10.9.106 (Schmidt and Krfimer 1983). Thiol oxidation in the presence of membranes and a control without addition of membrane fractions were run in parallel to correct for thiol autoxidation.
o.9.
ne
~'"-----~
0.7
Oxygen measurements. Oxygen uptake was followed with a Hansatech Clark-type electrode (Bachofer, Reutlingen, FRG). Chemicals. Thiol compounds including DTNB were purchased from Sigma (Mfinchen, FRG); all other chemicals were obtained from Merck (Darmstadt, FRG).
0.5
Results
0.3
Demonstration of a cysteine oxidase M e m b r a n e fractions o f Chlorella catalyzed the disa p p e a r a n c e o f thiol groups as s h o w n in Fig. 1. These d a t a were obtained using the Ellman reagent ( D T N B ) for the d e t e r m i n a t i o n o f thiol groups. A c o n t r o l w i t h o u t addition o f m e m b r a n e fractions was r u n to analyze for a u t o x i d a t i o n o f thiols. It is evident that a rapid disappearance o f thiol groups is catalyzed by this Chlorella m e m b r a n e fraction. Since n o electron acceptor had been added and the vessels were m a i n t a i n e d in n o r m a l air, molecular oxygen was considered as a possible electron acceptor. This was tested using a Clarktype electrode for m e a s u r e m e n t s o f oxygen uptake. These d a t a are shown in Fig. 2. A d d i t i o n o f the pellet caused a rapid u p t a k e o f oxygen; this rate was not altered by addition o f 100 units o f catalase, indicating that no H 2 0 2 accunmlated. This could be the consequence o f a reduction leading directly to H 2 0 or to m e m b r a n e - b o u n d catalase activity. A d d i t i o n o f H 2 0 2 t o m e m b r a n e fractions produced O 2 at a rate sufficient to destroy all H 2 0 2 possibly formed. Therefore, a pellet fraction was heated for 3 rain to 100 ~ C to destroy the catalase activity. Afterwards this catalase-free pellet fraction had lost 80% o f its thiol-oxidation capacity; however, the rate was again not influenced by catalase, indicating that H z O z is not an intermediate in this o x y g e n - u p t a k e reaction. T h e stoichiometry o f thiols oxidized and O2 c o n s u m e d is given in Table 1. It was calculated that 44 nmol o f oxygen were c o n s u m e d to oxidize 179 nmol o f thiol groups leading to a ratio o f 1 O2 to 4.07 thiol groups using L-cysteine. T h e ratio for D-cysteine was calculated to be 1 0 2 to 3.97 thiol
9 >
i0
is
i0
is
30
rnJn
Fig. 1. Membrane-catalyzed cysteine oxidation. The reaction vessel contained in a total volume of 3 ml: imidazol buffer pH 8.0 300 gmol; L-cysteine 3 ~mol; and Chlorella membranes containing 13.2 p.g of chlorophyll. A control was run without addition of membrane fractions to measure the autoxidation of thiols. At the time intervals indicated 100-~tl aliquots were withdrawn and pipetted into a cuvettc containing 900 gl of water and 0.1 gmol of DTNB. The optical density was read at 436 nm and plotted against time
groups oxidized; thus for one 0 2 c o n s u m e d four thiols are oxidized leading to the following sequence: O2+4(~)
cysteine
membrane
2H20+2
(~)cystin e
This d e m o n s t r a t e s that m e m b r a n e fractions o f Chlorella contain an oxidase which catalyzes the oxidation o f cysteine to cystine using molecular oxygen and with the p r o d u c t i o n o f water. By addition o f excess dithioerythritol, reduced cysteine could be recovered, indicating that oxidized cystine a n d n o t cysteine sulfinic acid was the product. This was c o n f i r m e d using a T r i s a c r y l - G F 0 5 - c o l u m n technique, which separates cysteine sulfinic-sulfonic acids f r o m cysteine-cystine (Krauss and Schmidt 1983).
Properties of the membrane-bound cysteine oxidase 1. p H optimum. The p H o p t i m u m for this system was determined using either the Clark-type oxygen electrode or the D T N B method. These data are
E. Kr/imer and A. Schmidt: Oxidation of cysteine to cystine in Chlorella
237
25 84
x ~ - - - x 20
ase
,2 15
,.o
o~ E
--r
10 I
I
1 rain
Fig. 2. L-Cysteine-dependent oxygen uptake by membrane fractions of Chlorella. The experimental conditions were identical to those described in Table 1. The arrow indicates addition of 100 units of catalase
J 71s
Table 1. Comparison of oxygen consumption and thiol groups oxidized using L- or D-cysteine. A Clark-type electrode was immersed in a total volume of 2 ml : 200 gmol imidazolbuffer (pH 8.0); 3 gmol D- or L-cysteine or 4.4 gmol H 2 0 a and Chlorella membrane fractions containing J9.3 gg chlorophyll. Incubation was at 37 ~ C. The rates of the first 3 5 min were listed. Thiol oxidation rates were determined according to the legend to Fig. I
C o m p o u n d added
Oxygen consumed or produced (nmol min 1)
Thiols oxidized (nmol m i n - z )
L-Cysteine D-Cysteine HzOz
-- 44 --38 + 80
179 151 -
shown in Fig. 3. For both methods, the pH optimum is found in the range between 8 and 8.5, and the ratio of SH-groups oxidized to oxygen consumed was in the range of 3.3 to 3.9; however these data were not obtained using pellets incubated side by side, they were collected within ] d using the same pellet fraction.
2. K,,-determination for r-cysteine. The K m for Lcysteine was determined using the oxygen electrode. In a first set of experiments the autoxidation of L-cysteine was determined at increasing L-cysteine concentrations. Afterwards the pellet-catalyzed oxygen-uptake rate was measured at the same L-cysteine concentration. The rates corrected for autoxidation are given in Fig. 4. L-Cysteine oxidation by these Chlorella membranes follows Michaelis-Menten kinetics and from these data the
d.o
e.s
9.0
pH
Fig. 3. Determination of pH optimum for L-cysteine oxidation and oxygen uptake by membrane fractions of Chlorella. The experimental conditions were as described in Table l ; however, 30.7 gg of chlorophyll was added and the temperature was set to 27 ~ C. The rates are expressed as gmol L-cysteine oxidized or 0 2 consumed m g - 1 chlorophyll (10 m i n ) - 1 x-x, Disappearance o f - S H groups; zx-,,, oxygen consumed
apparent K m for L-cysteine was calculated as 3.3 mmol l- 1 (insert of Fig. 4). For routine measurements, however, the thiol concentration was kept to J or 1.5 mmol 1-1, since this is the range of the cysteine concentration within the plant Lemna minor (Brunold and Schmidt 1978).
3. Temperature dependence. The Q-10 value of cysteine autoxidation and the membrane-cytalyzed Lcysteine oxidation was determined between 2 7 ~ and 37 ~ C. The following data were obtained as a Q-10 value" a) L-cysteine autoxidation: 2.2 __0.43 (n= 5); b) membrane-catalyzed L-Cysteine oxidation: 1.57_+0.16 (n=5). This demonstrates that measurements at the lower temperature should be preferred since the rate of membrane-catalyzed oxidation, compared with autoxidation, is favoured at the lower temperature. However, this system was discovered using incubation temperatures of 37 ~ C; therefore, this temperature was used for comparison with the data obtained earlier. 4. Thiol specificity. The specificity of this system towards different artificial and natural thiols was tested using the D T N B method for the measurements of membrane-catalyzed thiol oxidation.
238
E. Kr/imer and A. Schmidt: Oxidation of cysteine to cystine in Chlorella
50
C
E
4o E O U
c~ O
E
X
30.
.
X
200.10'
x
10-
/
;
i*
1/s
),
i
i
~
Z mM
g
6
§
i
g
Fig. 4. K m determination for L-cysteine oxidation. The experimental conditions were as described in Table 1 ; however, 12.5 gg of chlorophyll was added and the temperature was 27 ~ C. The L-cysteine concentration was varied as indicated; for other details see text
1'o
L- Cysteine
These data are summarized in Table 2. It can be seen that D- and L-cysteine are good substrates for this thiol-oxidation system, whereas glutathione is not oxidized at all. Other artificial thiols including mercaptoethanol and dithioerythritol are not oxidized with the exception of cysteine derivatives with a blocked carboxylic group such as O-methylL-cysteine. Some activity was detected using either DL-homocysteine or cysteamine. 5. Inhibitor studies. The inhibitor studies for the membrane-bound cysteine-oxidation system were performed using a Clark-type oxygen electrode. The reaction was always started by addition of the pellet and the inhibitor was added after a linear rate of oxygen uptake had been achieved. The inhibition rate is given as the percentage of the oxygen uptake rate without addition of inhibitor, cor-
rected for L-cysteine autoxidation. From these data, it is evident that certain buffers (phosphate and N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine (Tricine)) inhibited L-cysteine oxidation; thus these buffers had to be avoided. Inhibitors of the respiratory chain of mitochondria had no effect; therefore, it is concluded that the electron-transport chain of mitochondria is not involved in this L-cysteine oxidation. Inhibitors of the chloroplast electron-transport chain after the plastoquinone site (2,5-dibromothymochinone and 2,4-dinitrophenylether of iodonitrothymol) did not inhibit this thiol oxidation, raising the question of the involvement of electron carriers. Complexing agents such as ethylenediaminetetraacetic acid (EDTA), o-phenanthroline, salicylhydroxamic acid, and N,N'-disalicyliden-l,3-diaminopropane5,5'-disulfonic acid (Sulfo-DSPD) inhibited this
E. KrS.mer and A. Schmidt: Oxidation of cysteine to cystine in ChIoretIa Table 2. Specificity of the membrane-catalyzed thiol oxidation. The experimental conditions were as described in the legend to Fig. 1; however, cysteine was replaced by the thiol compound indicated, with the exception of dithioerythritol, which was used at a concentration of 0.5 mM. Membrane fractions containing 13.3 pg chlorophyll were used; 100% represents 225.6 gmol -SH oxidized mg-1 chlorophyll h-1. All thiol compounds were analyzed with a control without addition of membranes, and the corrected rates are given
Table 3. Effect of different inhibitors on L-cysteine-dependent oxygen uptake using Chlorella membranes. The data were obtained using membrane fractions containing 20 70 ~tg chlorophyll. Oxygen uptake was measured at 27~ using imidazol buffer (pH 8.0) and 1.5 mM L-cysteine. The reaction was started with the membrane fraction; after a steady state was achieved, the compound to be tested was added and the % inhibition was calculated
Concentration (pmol.l-1)
% inhibition of oxygen uptake
5,000
100
5,000
100
Ethylenediaminetetraacetic acid (EDTA) o-Phenanthroline
1,000
79
1,000
Salicylhydroxamic acid
1,000
79 94
290
75
10 2,000 10,000
t2 95 0
10 250
0 0
10
0
10
0
Inhibitor tested Thiol compound added
SH-groups oxidized (% of L-cysteine)
L-Cysteine D-Cysteine Glutathione Cysteamine DL-Homocysteine Dithioerythritol Mercaptoethanol Mercaptopropionic acid Thioglycerol N-Acetyl-L-cysteine O-Methyl-L-cysteine
100
117 0 57 31 0 0 0 0 0 349
system strongly indicating that perhaps a metal is involved in this thiol oxidation. Washing the membrane fractions with E D T A did not change this oxidation capacity, therefore, the postulated metal must be bound firmly to the membrane fraction.
Discussion
Plants can regulate key enzymes of the reductive pentose-phosphate cycle by the "light switch", thioredoxin (Buchanan 1980). It is coupled to photosystems II and I by ferredoxin-thioredoxin reductase (De la Torre et al. 1979). Reduced thioredoxins are activating thiol-dependent enzymes such as the C-1 phosphatases (Buchanan et al. 1979), whereas glucose-6-phosphate dehydrogenase is inhibited by that system (Ashton et al. 1980). Thiol-dependent regulation of enzyme activity has been detected in animals as well (Ziegler and Poulsen 1977; Steven et al. 1981 ; Gilbert 1982) indicating that a thiol-disulfide regulation is common to living systems. In the dark, light-activated enzymes have to be inactivated again in a way which is not yet not understood. Evidence obtained by Leegood and Walker (1980, 1982) indicates that 02 is involved in dark inactivation, since no loss of light-activated enzyme activity was observed in the dark under anaerobic conditions. Thus, the pathway from reduced thiols to 0 2 has to be postulated as being either direct or indirect.
239
a) Buffers Phosphate buffer pH 8.0 N-[2-Hydroxy-l,lbis(hydroxymethyl) ethyl glycine (Tricine) pH 8.0 b) Complexing agents
N,N'-Disalicyliden-l,3diaminopropane-5,5'disulfonic acid c) Inhibitors of respiration KCN Azide Antimycin A Rotenone
d) Inhibitors of photosynthesis 2,5-Dibromothymochinone 2,4-Dinitrophenylether of iodonitrothymol
We demonstrate in this paper that the green alga Chlorella fusca contains a membrane-bound system capable of oxidizing thiols at the expense of 0 2. Such a system was previously detected in cyanobacteria by us (Schmidt and Kr/imer 1983). For one 0 2 consumed four thiol groups disappeared and the evidence presented indicates that water is formed and not H 2 0 2. Thus the membranes catalyze an oxidation of cysteine to cystine forming water with 0 2 as the electron acceptor. This membrane-bound thiol oxidase was specific for D- and L-cysteine, whereas glutathione and other (artificial) thiols were not oxidized. The structure for good activity needs a three-carbon skeleton containing an SH-group in the fl-position and an amino group in the a-position to a carboxylic group. If the amino group is acylated (N-acetyl-L-cysteine) or not present (3-mercaptopro-
240
E. Kr/imer and A. Schmidt: Oxidation of cysteine to cystine in Chlorella
pionic acid), no activity was detected. If the chain length is varied, e.g. by the addition of - C H 2group in homocysteine or shortened by a decarboxylation to cysteamine, the activity is drastically reduced, whereas methylation of the carboxylic group enhanced the activity, indicating that the acidity of the SH-group is of importance. The possible involvement of components of mitochondrial or chloroplast electron-transport chains was analyzed using specific inhibitors. The system was not inhibited by rotenone, antimycin A or low concentrations of KCN, therefore, electron transport through the normal mitochondrial chain does not seem to be involved (Slater 1967). The same statement applies to the chloroplast system since neither 2,5-dibromothymochinone (Trebst et al. 1970) nor 2,4-dinitrophenylether of iodonitrothymol (Trebst et al. 1978) inhibited oxygen uptake. This leads to the question of which electron carrier could possibly be involved in the electron transfer from the thiol group of cysteine to 0 2 forming water. On the basis of indirect measurements, quinones have recently been suggested to be coupled to oxygen consumption in spinach chloroplasts (Bennoun 1982) or to a lipid-peroxydation cycle in mitochondria (Rustin et al. 1983). An NAD dehydrogenase linked to a plastoquinone pool has been demonstrated for Chlamydomonas (Godde and Trebst 1980); a similar enzyme system specific for cysteine could possibly function in a membrane-bound system coupling cysteine oxidation to quinone reduction. The evidence obtained does not rule out such a system since i) inhibition by high KCN could act at the quinone site; ii) the redox properties of cysteine ( - 3 4 0 mV) are such that quinones could be reduced; and iii) the inhibitors used do not exclude a branching of the pathway at the quinone site to an alternative pathway (John 1981). The term' thiol respiration' is suggested for this oxygen-dependent cysteine oxidation by O 2 . This thiol respiration could function as the electron shuttle for the postulated chlororespiration necessary for starch metabolism in the dark (Wah et al. 1982). Oxygen reduction observed using chloroplasts in the light could be explained by the same system (Egneus et al. 1975; Marsho and Behrens 1979; Foyer and Hall 1980). We see the main function of this cysteine respiration as a regulatory system concerning the thioldisulfide status of the cell. The ferredoxin-thioredoxin system activates enzymes by reduction of thiol groups in the light, and these thiol groups are oxidized again (dark inactivation) by the thiol
respiration system, either by coupling the reduction of cysteine directly to the activated proteins or by coupling to thioredoxin as a redox carrier. Enzyme activation would, therefore, be a balance between the reducing capacity of thiol groups by the electron-transport chain and the cysteine oxidation capacity; the cysteine concentration would be of critical importance for such a balance. It is an open question whether a thiol transhydrogenase is involved or if cysteine is reduced directly by thioredoxins, although a direct reduction of cystine to cysteine by thioredoxins is possible (Holmgren 1979). The thiol concentration used in these studies was in the range of 1 to 1.5mmol1-1 which is actually the cysteine concentration found in Lemna minor (Brunold and Schmidt 1978); thus the measurements were made close to natural systems, although the apparent K m determination for L-cysteine gave a value of 3.3 mmol 1- ~. Enhanced cysteine concentrations are, therefore, critical for a cell since they increase the thiol-oxidation capacity, thereby distorting the thiol-disulfide status. Evidence for such a role of cysteine in vivo comes from our observation that cysteine concentrations above 0.5 mmol 1-1 are toxic for Chlorella and Synechococcus growth (Schmidt et al. 1982) and high cysteine concentrations also inhibited plant tissue cultures, in contrast to glutathione which is less toxic (Rennenberg 198l). It has been suggested that thioredoxin and glutathione regulate photosynthesis (Wolosiuk and Buchanan 1977); we would like to change this statement and suggest that thioredoxin and cysteine regulate photosynthesis. This work was supported by a grant from the Deutsche Forschungsgemeinschaft. We are indebted to Professor Elstner and Dr. Youngman (Miinchen, FRG) for providing facilities for inhibitor studies correlating to HzO 2 and to Dr. Lockau (Regensburg, FRG) for providing inhibitors and facilities for the study of electron transport inhibitors.
References Anderson, L.E. (1979) Interaction between photochemistry and activity of enzymes. In: Encyclopedia of plant physiology, N.S., vol. 6, Photosynthesis II, pp. 27/-28], Gibbs, M., Latzko, E., eds. Springer, Berlin Heidelberg New York Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 Ashton, A.R., Brennan, T., Anderson, L.E. (1980) Thioredoxin like activity of thylakoid membranes. Thioredoxin catalyzing the reductive inactivation of glucose-6-phosphate dehydrogenase occurs in both soluble and membrane-bound form. Plant Physiol. 66, 605-608 Bennoun, P. (1982) Evidence for a respiratory chain in the chloroplast. Proc. Natl. Acad. Sci. USA 79, 4352 4356 Brunold, C., Schmidt, A. (1978) Regulation of sulfate assimilation in plants. 7. Cysteine inactivation of adenosine-5'-
E. Kr/imer and A. Schmidt: Oxidation of cysteine to eystine in Chlorella phosphosulfate sulfotransferase in Lemna minor. Plant Physiol. 61, 342-347 Buchanan, B.B. (1980) Role of light in the regulation of chloroplast enzymes. Annu. Rev. Plant Physiol. 31,341-374 Buchanan, B.B.., Wolosiuk, R.A., Schfirmann, P. (1979) Thioredoxin and enzyme regulation. Trends Biochem. Sci. 4, 93-96 De la Torre, A., Lara, C., Wolosiuk, R.A., Buchanan, B.B. (1979) Ferredoxin-thioredoxin reductase: a chromophorefree protein of chloroplasts. FEBS Lett. 107, 141-145 Egneus, H., Heber, U., Mathiesen, U., Kirk, M. (1975) Reduction of oxygen by the electron transport chain of chloroplasts during assimilation of carbon dioxide. Biochim. Biophys. Acta 408, 252-268 Ellmann, G.H. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-77 Foyer, C.H., Hall, D.O. (1980) Oxygen metabolism in the active chloroplast. Trends Biochem. Sci. 5, 188-190 Gilbert, H.F. (1982) Biological disulfides: the third messenger? Modulation of phosphofructokinase activity by thiol/disulfide exchange. J. Biol. Chem. 257, 12086-12091 Godde, D., Trebst, A. (1980) NADH as electron donor for the photosynthetic membrane of Chlamydomonas reinhardii. Arch. Microbiol. 127, 245 252 Holmgren, A. (1979) Reduction of disulfides by thioredoxin. Exceptional reactivity of insulin and suggested functions of thioredoxin in mechanism of hormone action. J. Biol. Chem. 254, 9113-9119 John, P. (1981) Schematic representation of branched respiratory chains. Trends Biochem. Sci. 6, 8-10 Krauss, F., Schmidt, A. (1983) Separation of sulphur-containing substances, amino acids and nucleotides during gel filtration on Trisacryl GF05 by specific gel interaction. J. Chromatogr. 264, 111-118 Leegood, R.C., Walker, D.A. (1980) Regulation of fructose-l,6bisphosphatase activity in intact chloroplasts. Studies of the mechanism of inactivation. Biochim. Biophys. Acta 593, 362-370 Leegood, R.C., Walker, D.A. (1982) Regulation of fructose-l,6bisphosphatase activity in leaves. Planta 156, 449-456 Marsho, T.U., Behrens, P.W. (1979) Photosynthetic oxygen reduction in isolated intact chloroplasts and cells from spinach. Plant Physiol. 64, 656-659
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Rennenberg, H. (1981) Differences in the use of cysteine and glutathione as sulfur source in photoheterotrophic tobacco suspension cultures. Z. Pflanzenphysiol. 105, 31-40 Rustin, P., Dupont, J., Lance, C. (1983) A role for fatty acid peroxy radicals in the cyanide-insensitive pathway of plant mitochondria? Trends Biochem. Sci. 8, 155-157 Schmidt, A. (1972) l~ber Teilreaktionen der photosynthetischen Sulfatreduktion in zellfreien Systemen aus Spinatchloroplasten und Chlorella. Z. Naturforseh. Teil B 27, 183-192 Schmidt, A., Erdle, I., K6st, H.-P. (1982) Changes of C-phycocyanin in Synechococeus 6301 in relation to growth on various sulfur compounds. Z. Naturforsch. Teil C 37, 870-876 Schmidt, A., Krfimer, E. (1983) A membrane-bound cysteine oxydase from the cyanobacterium Synechococcus 6301. Z. Naturforsch. Teil C 38, 446-450 Slater, E.C. (1967) Application of inhibitors and uncouplers for a study of oxidative phosphorylation. Methods Enzymol. 10, 48 57 Steven, F.S., Griffin, M.M., Smith, R.H. (1981) Disulfide exchange reactions in the control of enzymatic activity. Evidence for the participation of dimethyldisulfide in exchanges. Eur. J. Biochem. 119, 75 78 Trebst, A., Harth, E., Draber, W. (1970) On a new inhibitor of photosynthetic electron transport in isolated chloroplasts. Z. Naturforsch. Teil B 25, 1157-1159 Trebst, A., Wietoska, H., Draba, W., Knops, H.J. (1978) The inhibition of photosynthetic electron flow in chloroplasts by dinitrophenylether of bromo- or iodo-nitrothymol. Z. Naturforsch. Teil C 33, 919-927 Wah, Y., Erbes, D.L., Gibbs, M. (1982) Chloroplast respiration. A means for supplying oxidized pyridine nucleotides for dark chloroplastic metabolism. Plant Physiol. 69, 442-447 Wolosiuk, R.A., Buchanan, B.B. (1977) Thioredoxin and glutathione regulate photosynthesis in chloroplasts. Nature (London) 266, 565-567 Ziegler, D.M., Poulsen, L.L. (1977) Protein disulfide bond synthesis : a possible intracellular mechanism. Trends Biochem. Sci. 2, 79 91
Received 5 July; accepted 25 September 1983
Planta (1984) 160:235 241
Oxidation of cysteine to cystine by membrane fractions of Chlorella fusca Elisabeth Kr/imer and Ahlert Schmidt Botanisches Institut der Universitfit, Menzinger Strasse 67, D-8000 Mfinchen 19, Federal Republic of Germany
Abstract. Isolated m e m b r a n e fractions of Chlorella fusca 211-8b obtained by french-press treatment and sonication catalyzed the oxidation of L-cysteine to L-cystine. The p H - o p t i m u m of this reaction was determined to be a r o u n d 8-8.5 and a stoichiometry of 4 SH-groups oxidized for one O a consumed was obtained. This thiol-oxidation system w a s specific for D- and L-cysteine; oL-homocysteine and cysteamine were oxidized at a b o u t half the rate whereas all other thiols tested including glutathione, mercaptoethanol, mercaptopropionic acid and dithioerythritol were n o t oxidized by these m e m b r a n e fractions. The apparent K m for L-cysteine was determined as 3.3 m m o l 1 1. Rates of 200 gmol cysteine oxidized m g - 1 chlorophyll h - 1 were normally obtained. Extremely high rates of oxygen uptake were measured using L-cysteine methyl ester and L-cysteine ethyl ester. This thioloxidation system was not inhibited by mitochondrial electron-transport inhibitors such as rotenone or antimycin A, nor by the chloroplast electrontransport inhibitors 2,5-dibromothymochinone and 2,4-dinitrophenylether of iodonitrothymol. The cysteine oxidation catalyzed by C. fusca membranes was inhibited, however, by salicylhydroxamic acid, o-phenanthrolin, N,N'-disalicyliden1,3-diaminopropane-5,5'-disulfonic acid, ethylenediaminetetraacetic acid, high K C N levels and by the buffers, N-[2-hydroxyl-l,l-bis(hydroxymethyl) ethyl] glycine and phosphate. This cysteine-oxidation system seems to function as a counterpart of thioredoxin-mediated light activation of enzymes, allowing reduced thiol groups to be oxidized again by 0 2 (dark inactivation). Key words: ChloreIIa (membrane, thiol respiration) Cysteine oxidation - M e m b r a n e (thiol respiration) - Oxygenase - Thiol respiration. Abbreviation: DTNB=5,5'-dithio-bis(-2-nitrobenzoic acid), Ellmann reagent
Introduction Certain enzymes of the reductive pentose-phosphate cycle are activated in the light (Anderson 1979) by the ferredoxin-thioredoxin system (Buchanan 1980). During the light period, thioredoxin is reduced by the ferredoxin-thioredoxin reductase (De la Torre et al. 1979). The reduced thioredoxin activates oxidized enzymes by reduction of specific thiol groups (Buchanan et al. 1979). In the d a r k these enzymes are inactivated again, but this dark inactivation is not yet understood. Evidence has been obtained that molecular oxygen seems to be involved, since no inactivation occurs under anaerobic conditions (Leegood and Walker 1980, 1982). In this paper a thiol-oxidizing system, which is m e m b r a n e - b o u n d and utilizes 0 2 as an electron acceptor, is described for the green alga Chlorella fusca.
Material and methods Plant material. Chlorellafusca strain 211-8b was obtained from the algal collection of G6ttingen and grown as axenic culture as described earlier (Schmidt 1972). Preparation of membranefractions. Cells were harvested by centrifugation and the pellet was suspended in 0.1 M 2-amino-2(hydroxymethyl)-l,3-propanediol (Tris)-HC1 buffer (pH 8.0) containing 10 retool-1-1 MgC12 and 10 mmol.1-1 mercaptoethanol. Cells were broken in a french-press at 12,000 psi (1 psi ~ 7 kPa). The membrane fractions were collected by centrifugation and the pellet was washed once with 0.05 tool. 1-1 imidazol buffer (pH 8.0). Afterwards the pellet was solubilized by sonication (three times, 10 s, using a Branson S-125 sonicpower model). The membrane fractions obtained in this way were used for the experiments to be described. These membrane fractions contained 62% thylakoid membranes; 18% envelope membranes; 7% cytoplasmic membranes; 6% mitochondrial membranes; 6% nucleic membranes and about 1% endoplasmic reticulum and tonoplast membranes. These data were obtained from electron-microscopy pictures using Chlorella cells from the logarithmic growth phase before french-press treatment.
236
E. Kr/imer and A. Schmidt: Oxidation of cysteine to cystine in Chlorella / k E436
Chlorophyll determination. The method of Arnon (1949) was used.
Thiol-oxidation measurements. Thiol-group determinations were performed using the Ellmann reagent, 5,5'-dithio-bis(-2nitrobenzoic acid) (DTNB; Ellmann 1959). For these measurements a Zeiss (Oberkochen, FRG) filter photometer was used at 436 rim; the ~ for DTNB at this wavelength was calculated as 10.9.106 (Schmidt and Krfimer 1983). Thiol oxidation in the presence of membranes and a control without addition of membrane fractions were run in parallel to correct for thiol autoxidation.
o.9.
ne
~'"-----~
0.7
Oxygen measurements. Oxygen uptake was followed with a Hansatech Clark-type electrode (Bachofer, Reutlingen, FRG). Chemicals. Thiol compounds including DTNB were purchased from Sigma (Mfinchen, FRG); all other chemicals were obtained from Merck (Darmstadt, FRG).
0.5
Results
0.3
Demonstration of a cysteine oxidase M e m b r a n e fractions o f Chlorella catalyzed the disa p p e a r a n c e o f thiol groups as s h o w n in Fig. 1. These d a t a were obtained using the Ellman reagent ( D T N B ) for the d e t e r m i n a t i o n o f thiol groups. A c o n t r o l w i t h o u t addition o f m e m b r a n e fractions was r u n to analyze for a u t o x i d a t i o n o f thiols. It is evident that a rapid disappearance o f thiol groups is catalyzed by this Chlorella m e m b r a n e fraction. Since n o electron acceptor had been added and the vessels were m a i n t a i n e d in n o r m a l air, molecular oxygen was considered as a possible electron acceptor. This was tested using a Clarktype electrode for m e a s u r e m e n t s o f oxygen uptake. These d a t a are shown in Fig. 2. A d d i t i o n o f the pellet caused a rapid u p t a k e o f oxygen; this rate was not altered by addition o f 100 units o f catalase, indicating that no H 2 0 2 accunmlated. This could be the consequence o f a reduction leading directly to H 2 0 or to m e m b r a n e - b o u n d catalase activity. A d d i t i o n o f H 2 0 2 t o m e m b r a n e fractions produced O 2 at a rate sufficient to destroy all H 2 0 2 possibly formed. Therefore, a pellet fraction was heated for 3 rain to 100 ~ C to destroy the catalase activity. Afterwards this catalase-free pellet fraction had lost 80% o f its thiol-oxidation capacity; however, the rate was again not influenced by catalase, indicating that H z O z is not an intermediate in this o x y g e n - u p t a k e reaction. T h e stoichiometry o f thiols oxidized and O2 c o n s u m e d is given in Table 1. It was calculated that 44 nmol o f oxygen were c o n s u m e d to oxidize 179 nmol o f thiol groups leading to a ratio o f 1 O2 to 4.07 thiol groups using L-cysteine. T h e ratio for D-cysteine was calculated to be 1 0 2 to 3.97 thiol
9 >
i0
is
i0
is
30
rnJn
Fig. 1. Membrane-catalyzed cysteine oxidation. The reaction vessel contained in a total volume of 3 ml: imidazol buffer pH 8.0 300 gmol; L-cysteine 3 ~mol; and Chlorella membranes containing 13.2 p.g of chlorophyll. A control was run without addition of membrane fractions to measure the autoxidation of thiols. At the time intervals indicated 100-~tl aliquots were withdrawn and pipetted into a cuvettc containing 900 gl of water and 0.1 gmol of DTNB. The optical density was read at 436 nm and plotted against time
groups oxidized; thus for one 0 2 c o n s u m e d four thiols are oxidized leading to the following sequence: O2+4(~)
cysteine
membrane
2H20+2
(~)cystin e
This d e m o n s t r a t e s that m e m b r a n e fractions o f Chlorella contain an oxidase which catalyzes the oxidation o f cysteine to cystine using molecular oxygen and with the p r o d u c t i o n o f water. By addition o f excess dithioerythritol, reduced cysteine could be recovered, indicating that oxidized cystine a n d n o t cysteine sulfinic acid was the product. This was c o n f i r m e d using a T r i s a c r y l - G F 0 5 - c o l u m n technique, which separates cysteine sulfinic-sulfonic acids f r o m cysteine-cystine (Krauss and Schmidt 1983).
Properties of the membrane-bound cysteine oxidase 1. p H optimum. The p H o p t i m u m for this system was determined using either the Clark-type oxygen electrode or the D T N B method. These data are
E. Kr/imer and A. Schmidt: Oxidation of cysteine to cystine in Chlorella
237
25 84
x ~ - - - x 20
ase
,2 15
,.o
o~ E
--r
10 I
I
1 rain
Fig. 2. L-Cysteine-dependent oxygen uptake by membrane fractions of Chlorella. The experimental conditions were identical to those described in Table 1. The arrow indicates addition of 100 units of catalase
J 71s
Table 1. Comparison of oxygen consumption and thiol groups oxidized using L- or D-cysteine. A Clark-type electrode was immersed in a total volume of 2 ml : 200 gmol imidazolbuffer (pH 8.0); 3 gmol D- or L-cysteine or 4.4 gmol H 2 0 a and Chlorella membrane fractions containing J9.3 gg chlorophyll. Incubation was at 37 ~ C. The rates of the first 3 5 min were listed. Thiol oxidation rates were determined according to the legend to Fig. I
C o m p o u n d added
Oxygen consumed or produced (nmol min 1)
Thiols oxidized (nmol m i n - z )
L-Cysteine D-Cysteine HzOz
-- 44 --38 + 80
179 151 -
shown in Fig. 3. For both methods, the pH optimum is found in the range between 8 and 8.5, and the ratio of SH-groups oxidized to oxygen consumed was in the range of 3.3 to 3.9; however these data were not obtained using pellets incubated side by side, they were collected within ] d using the same pellet fraction.
2. K,,-determination for r-cysteine. The K m for Lcysteine was determined using the oxygen electrode. In a first set of experiments the autoxidation of L-cysteine was determined at increasing L-cysteine concentrations. Afterwards the pellet-catalyzed oxygen-uptake rate was measured at the same L-cysteine concentration. The rates corrected for autoxidation are given in Fig. 4. L-Cysteine oxidation by these Chlorella membranes follows Michaelis-Menten kinetics and from these data the
d.o
e.s
9.0
pH
Fig. 3. Determination of pH optimum for L-cysteine oxidation and oxygen uptake by membrane fractions of Chlorella. The experimental conditions were as described in Table l ; however, 30.7 gg of chlorophyll was added and the temperature was set to 27 ~ C. The rates are expressed as gmol L-cysteine oxidized or 0 2 consumed m g - 1 chlorophyll (10 m i n ) - 1 x-x, Disappearance o f - S H groups; zx-,,, oxygen consumed
apparent K m for L-cysteine was calculated as 3.3 mmol l- 1 (insert of Fig. 4). For routine measurements, however, the thiol concentration was kept to J or 1.5 mmol 1-1, since this is the range of the cysteine concentration within the plant Lemna minor (Brunold and Schmidt 1978).
3. Temperature dependence. The Q-10 value of cysteine autoxidation and the membrane-cytalyzed Lcysteine oxidation was determined between 2 7 ~ and 37 ~ C. The following data were obtained as a Q-10 value" a) L-cysteine autoxidation: 2.2 __0.43 (n= 5); b) membrane-catalyzed L-Cysteine oxidation: 1.57_+0.16 (n=5). This demonstrates that measurements at the lower temperature should be preferred since the rate of membrane-catalyzed oxidation, compared with autoxidation, is favoured at the lower temperature. However, this system was discovered using incubation temperatures of 37 ~ C; therefore, this temperature was used for comparison with the data obtained earlier. 4. Thiol specificity. The specificity of this system towards different artificial and natural thiols was tested using the D T N B method for the measurements of membrane-catalyzed thiol oxidation.
238
E. Kr/imer and A. Schmidt: Oxidation of cysteine to cystine in Chlorella
50
C
E
4o E O U
c~ O
E
X
30.
.
X
200.10'
x
10-
/
;
i*
1/s
),
i
i
~
Z mM
g
6
§
i
g
Fig. 4. K m determination for L-cysteine oxidation. The experimental conditions were as described in Table 1 ; however, 12.5 gg of chlorophyll was added and the temperature was 27 ~ C. The L-cysteine concentration was varied as indicated; for other details see text
1'o
L- Cysteine
These data are summarized in Table 2. It can be seen that D- and L-cysteine are good substrates for this thiol-oxidation system, whereas glutathione is not oxidized at all. Other artificial thiols including mercaptoethanol and dithioerythritol are not oxidized with the exception of cysteine derivatives with a blocked carboxylic group such as O-methylL-cysteine. Some activity was detected using either DL-homocysteine or cysteamine. 5. Inhibitor studies. The inhibitor studies for the membrane-bound cysteine-oxidation system were performed using a Clark-type oxygen electrode. The reaction was always started by addition of the pellet and the inhibitor was added after a linear rate of oxygen uptake had been achieved. The inhibition rate is given as the percentage of the oxygen uptake rate without addition of inhibitor, cor-
rected for L-cysteine autoxidation. From these data, it is evident that certain buffers (phosphate and N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine (Tricine)) inhibited L-cysteine oxidation; thus these buffers had to be avoided. Inhibitors of the respiratory chain of mitochondria had no effect; therefore, it is concluded that the electron-transport chain of mitochondria is not involved in this L-cysteine oxidation. Inhibitors of the chloroplast electron-transport chain after the plastoquinone site (2,5-dibromothymochinone and 2,4-dinitrophenylether of iodonitrothymol) did not inhibit this thiol oxidation, raising the question of the involvement of electron carriers. Complexing agents such as ethylenediaminetetraacetic acid (EDTA), o-phenanthroline, salicylhydroxamic acid, and N,N'-disalicyliden-l,3-diaminopropane5,5'-disulfonic acid (Sulfo-DSPD) inhibited this
E. KrS.mer and A. Schmidt: Oxidation of cysteine to cystine in ChIoretIa Table 2. Specificity of the membrane-catalyzed thiol oxidation. The experimental conditions were as described in the legend to Fig. 1; however, cysteine was replaced by the thiol compound indicated, with the exception of dithioerythritol, which was used at a concentration of 0.5 mM. Membrane fractions containing 13.3 pg chlorophyll were used; 100% represents 225.6 gmol -SH oxidized mg-1 chlorophyll h-1. All thiol compounds were analyzed with a control without addition of membranes, and the corrected rates are given
Table 3. Effect of different inhibitors on L-cysteine-dependent oxygen uptake using Chlorella membranes. The data were obtained using membrane fractions containing 20 70 ~tg chlorophyll. Oxygen uptake was measured at 27~ using imidazol buffer (pH 8.0) and 1.5 mM L-cysteine. The reaction was started with the membrane fraction; after a steady state was achieved, the compound to be tested was added and the % inhibition was calculated
Concentration (pmol.l-1)
% inhibition of oxygen uptake
5,000
100
5,000
100
Ethylenediaminetetraacetic acid (EDTA) o-Phenanthroline
1,000
79
1,000
Salicylhydroxamic acid
1,000
79 94
290
75
10 2,000 10,000
t2 95 0
10 250
0 0
10
0
10
0
Inhibitor tested Thiol compound added
SH-groups oxidized (% of L-cysteine)
L-Cysteine D-Cysteine Glutathione Cysteamine DL-Homocysteine Dithioerythritol Mercaptoethanol Mercaptopropionic acid Thioglycerol N-Acetyl-L-cysteine O-Methyl-L-cysteine
100
117 0 57 31 0 0 0 0 0 349
system strongly indicating that perhaps a metal is involved in this thiol oxidation. Washing the membrane fractions with E D T A did not change this oxidation capacity, therefore, the postulated metal must be bound firmly to the membrane fraction.
Discussion
Plants can regulate key enzymes of the reductive pentose-phosphate cycle by the "light switch", thioredoxin (Buchanan 1980). It is coupled to photosystems II and I by ferredoxin-thioredoxin reductase (De la Torre et al. 1979). Reduced thioredoxins are activating thiol-dependent enzymes such as the C-1 phosphatases (Buchanan et al. 1979), whereas glucose-6-phosphate dehydrogenase is inhibited by that system (Ashton et al. 1980). Thiol-dependent regulation of enzyme activity has been detected in animals as well (Ziegler and Poulsen 1977; Steven et al. 1981 ; Gilbert 1982) indicating that a thiol-disulfide regulation is common to living systems. In the dark, light-activated enzymes have to be inactivated again in a way which is not yet not understood. Evidence obtained by Leegood and Walker (1980, 1982) indicates that 02 is involved in dark inactivation, since no loss of light-activated enzyme activity was observed in the dark under anaerobic conditions. Thus, the pathway from reduced thiols to 0 2 has to be postulated as being either direct or indirect.
239
a) Buffers Phosphate buffer pH 8.0 N-[2-Hydroxy-l,lbis(hydroxymethyl) ethyl glycine (Tricine) pH 8.0 b) Complexing agents
N,N'-Disalicyliden-l,3diaminopropane-5,5'disulfonic acid c) Inhibitors of respiration KCN Azide Antimycin A Rotenone
d) Inhibitors of photosynthesis 2,5-Dibromothymochinone 2,4-Dinitrophenylether of iodonitrothymol
We demonstrate in this paper that the green alga Chlorella fusca contains a membrane-bound system capable of oxidizing thiols at the expense of 0 2. Such a system was previously detected in cyanobacteria by us (Schmidt and Kr/imer 1983). For one 0 2 consumed four thiol groups disappeared and the evidence presented indicates that water is formed and not H 2 0 2. Thus the membranes catalyze an oxidation of cysteine to cystine forming water with 0 2 as the electron acceptor. This membrane-bound thiol oxidase was specific for D- and L-cysteine, whereas glutathione and other (artificial) thiols were not oxidized. The structure for good activity needs a three-carbon skeleton containing an SH-group in the fl-position and an amino group in the a-position to a carboxylic group. If the amino group is acylated (N-acetyl-L-cysteine) or not present (3-mercaptopro-
240
E. Kr/imer and A. Schmidt: Oxidation of cysteine to cystine in Chlorella
pionic acid), no activity was detected. If the chain length is varied, e.g. by the addition of - C H 2group in homocysteine or shortened by a decarboxylation to cysteamine, the activity is drastically reduced, whereas methylation of the carboxylic group enhanced the activity, indicating that the acidity of the SH-group is of importance. The possible involvement of components of mitochondrial or chloroplast electron-transport chains was analyzed using specific inhibitors. The system was not inhibited by rotenone, antimycin A or low concentrations of KCN, therefore, electron transport through the normal mitochondrial chain does not seem to be involved (Slater 1967). The same statement applies to the chloroplast system since neither 2,5-dibromothymochinone (Trebst et al. 1970) nor 2,4-dinitrophenylether of iodonitrothymol (Trebst et al. 1978) inhibited oxygen uptake. This leads to the question of which electron carrier could possibly be involved in the electron transfer from the thiol group of cysteine to 0 2 forming water. On the basis of indirect measurements, quinones have recently been suggested to be coupled to oxygen consumption in spinach chloroplasts (Bennoun 1982) or to a lipid-peroxydation cycle in mitochondria (Rustin et al. 1983). An NAD dehydrogenase linked to a plastoquinone pool has been demonstrated for Chlamydomonas (Godde and Trebst 1980); a similar enzyme system specific for cysteine could possibly function in a membrane-bound system coupling cysteine oxidation to quinone reduction. The evidence obtained does not rule out such a system since i) inhibition by high KCN could act at the quinone site; ii) the redox properties of cysteine ( - 3 4 0 mV) are such that quinones could be reduced; and iii) the inhibitors used do not exclude a branching of the pathway at the quinone site to an alternative pathway (John 1981). The term' thiol respiration' is suggested for this oxygen-dependent cysteine oxidation by O 2 . This thiol respiration could function as the electron shuttle for the postulated chlororespiration necessary for starch metabolism in the dark (Wah et al. 1982). Oxygen reduction observed using chloroplasts in the light could be explained by the same system (Egneus et al. 1975; Marsho and Behrens 1979; Foyer and Hall 1980). We see the main function of this cysteine respiration as a regulatory system concerning the thioldisulfide status of the cell. The ferredoxin-thioredoxin system activates enzymes by reduction of thiol groups in the light, and these thiol groups are oxidized again (dark inactivation) by the thiol
respiration system, either by coupling the reduction of cysteine directly to the activated proteins or by coupling to thioredoxin as a redox carrier. Enzyme activation would, therefore, be a balance between the reducing capacity of thiol groups by the electron-transport chain and the cysteine oxidation capacity; the cysteine concentration would be of critical importance for such a balance. It is an open question whether a thiol transhydrogenase is involved or if cysteine is reduced directly by thioredoxins, although a direct reduction of cystine to cysteine by thioredoxins is possible (Holmgren 1979). The thiol concentration used in these studies was in the range of 1 to 1.5mmol1-1 which is actually the cysteine concentration found in Lemna minor (Brunold and Schmidt 1978); thus the measurements were made close to natural systems, although the apparent K m determination for L-cysteine gave a value of 3.3 mmol 1- ~. Enhanced cysteine concentrations are, therefore, critical for a cell since they increase the thiol-oxidation capacity, thereby distorting the thiol-disulfide status. Evidence for such a role of cysteine in vivo comes from our observation that cysteine concentrations above 0.5 mmol 1-1 are toxic for Chlorella and Synechococcus growth (Schmidt et al. 1982) and high cysteine concentrations also inhibited plant tissue cultures, in contrast to glutathione which is less toxic (Rennenberg 198l). It has been suggested that thioredoxin and glutathione regulate photosynthesis (Wolosiuk and Buchanan 1977); we would like to change this statement and suggest that thioredoxin and cysteine regulate photosynthesis. This work was supported by a grant from the Deutsche Forschungsgemeinschaft. We are indebted to Professor Elstner and Dr. Youngman (Miinchen, FRG) for providing facilities for inhibitor studies correlating to HzO 2 and to Dr. Lockau (Regensburg, FRG) for providing inhibitors and facilities for the study of electron transport inhibitors.
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Rennenberg, H. (1981) Differences in the use of cysteine and glutathione as sulfur source in photoheterotrophic tobacco suspension cultures. Z. Pflanzenphysiol. 105, 31-40 Rustin, P., Dupont, J., Lance, C. (1983) A role for fatty acid peroxy radicals in the cyanide-insensitive pathway of plant mitochondria? Trends Biochem. Sci. 8, 155-157 Schmidt, A. (1972) l~ber Teilreaktionen der photosynthetischen Sulfatreduktion in zellfreien Systemen aus Spinatchloroplasten und Chlorella. Z. Naturforseh. Teil B 27, 183-192 Schmidt, A., Erdle, I., K6st, H.-P. (1982) Changes of C-phycocyanin in Synechococeus 6301 in relation to growth on various sulfur compounds. Z. Naturforsch. Teil C 37, 870-876 Schmidt, A., Krfimer, E. (1983) A membrane-bound cysteine oxydase from the cyanobacterium Synechococcus 6301. Z. Naturforsch. Teil C 38, 446-450 Slater, E.C. (1967) Application of inhibitors and uncouplers for a study of oxidative phosphorylation. Methods Enzymol. 10, 48 57 Steven, F.S., Griffin, M.M., Smith, R.H. (1981) Disulfide exchange reactions in the control of enzymatic activity. Evidence for the participation of dimethyldisulfide in exchanges. Eur. J. Biochem. 119, 75 78 Trebst, A., Harth, E., Draber, W. (1970) On a new inhibitor of photosynthetic electron transport in isolated chloroplasts. Z. Naturforsch. Teil B 25, 1157-1159 Trebst, A., Wietoska, H., Draba, W., Knops, H.J. (1978) The inhibition of photosynthetic electron flow in chloroplasts by dinitrophenylether of bromo- or iodo-nitrothymol. Z. Naturforsch. Teil C 33, 919-927 Wah, Y., Erbes, D.L., Gibbs, M. (1982) Chloroplast respiration. A means for supplying oxidized pyridine nucleotides for dark chloroplastic metabolism. Plant Physiol. 69, 442-447 Wolosiuk, R.A., Buchanan, B.B. (1977) Thioredoxin and glutathione regulate photosynthesis in chloroplasts. Nature (London) 266, 565-567 Ziegler, D.M., Poulsen, L.L. (1977) Protein disulfide bond synthesis : a possible intracellular mechanism. Trends Biochem. Sci. 2, 79 91
Received 5 July; accepted 25 September 1983
