Inhibitor sensitivity of light-dependent oxygen reduction in chromatophores from wild-type and an oxidase-deficient mutant of Rhodopseudomonas capsulata

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 209, No. 1, June, pp. 2’76-233, 1981

Inhibitor Sensitivity of Light-Dependent Oxygen Reduction in Chromatophores from Wild-Type and an Oxidase-Deficient Mutant of Rhodopseudomonas Capsulata RAYA BITTAN,

AYALA

HOCHMAN, EZRA YAGIL, AND CHANOCH CARMELI

Department of Biochemistry, Tel-Aviv University, Ram&Aviv,

Tel-Aviv, Israel

Received September 12, 1936 Chromatophores from Rhodopseudomonae capeulatu cells grown semiaerobically in the dark oxidize NADH, succinate, and dichlorophenolindophenol. In the presence of NT these activities are inhibited, but light induces oxidation of dichlorophenolindophenol with 0, as a terminal electron acceptor. Cyanide also inhibits electron transport but much higher concentrations are required to inhibit the photooxidation than the dark oxidation. The photooxidation was studied in a mutant strain of Rhodqpseudomonas capeulata (YIV) which cannot grow anaerobically in the light, but similarly to the wild type, grows in the presence of oxygen. Chromatophores from YIV mutant catalyze photophosphorylation and dark oxidation activities with the same properties as those of the wild type. However, the rate of photooxidation in the mutant is only one-third that of the wild type. Based on the differential inhibitor sensitivity and on the mutation it is suggested that the photooxidase is different from the two respiratory oxidases and that this photooxidation activity might be essential for growth of the cells under anaerobic conditions in the light.

Rhodopseudomonus capsulata is a facultative photosynthetic bacterial species of the Rhodospirillaceae group which can grow both aerobically in the dark or anaerobically in the light. The energy transducing processes in this species of bacteria contain respiratory chains which catalyze oxidative phosphorylation and a cyclic electron transport chain which catalyzes photophosphorylation. In analogy to the photosynthetic process in plants it is reasonable to assume that the photosynthetic electron transport chain in bacteria would reduce pyridine nucleotides which are required in addition to ATP for CO2fixation. However, direct light-driven reduction of pyridine nucleotides is yet to be demonstrated. Similarly to what was found in other Rhodospirillaceae we have demonstrated that chromatophores from the photosynthetic bacteria Rhodopseudomonas capsulata catalyze both cyclic and noncyclic light-induced electron transport (1). The 1 This work was supported by grant 314 from the Israel-USA Binational Foundation. 0003-9861/81/0’70276-03$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

noncyclic electron transport is mediated by electron carriers, some of which are also part of the cyclic pathway. Electrons are donated to this pathway by various externally added reducing agents such as ascorbic acid, reduced dichlorophenolindophenol and reduced cytochrome c. Molecular oxygen is the electron acceptor and it is reduced to water. This noncyclic electron transport is of interest because it might represent a segment of the postulated path of pyridine nucleotide reduction. Chromatophores from Rhodospirillum rubrum are also capable of reducing oxygen in a light dependent electron transport from external electron donors. S. del-Valle-Tascon et al. (3) isolated a nonphototrophic mutant of Rhodos@-illum rubrum which catalyzed at a decreased rate the photoreduction of oxygen and tetrazolium blue by exogenous electron donors. They suggested that the defect in the mutant is in a photooxidase, and that this electron carrier is essential for growth of the cells anaerobically in the light because it helps to prevent overreduction of the electron carriers. 276

Rhodopseudomonas

capsulata

FIG. 1. Effect of N; and CN- on the light-induced electron transport from reduced dichlorophenolindophenol to 0, in chromatophores from cells grown under semiaerobic conditions in the dark. Reaction conditions were as described under Materials and Methods. The reaction mixture contained: tricineNaOH (pH 8), 33 mM; dichlorophenolindophenol, 10 pM; ascorbate, 1.5 mM; and chromatophores containing 80 pg bacteriochlorophyll in a final volume of 4.5 ml. The reaction was carried out in the presence of NT (0) or CN- (A).

PHOTOOXIDASE

277

tures were grown in &liter bottles which contained 15 liters of medium and were aerated by bubbling air. The rest of the growth conditions and harvesting of the cells were as previously described (1). The growth of the bacteria was monitored by measuring the turbidity of the cultures with a Klett-Somerson photometer equipped with a red (No. 66) filter. Preparation of the chromatophores was as described earlier (1). Bacteriochlorophyll was determined according to the method of Clayton (6). Photosynthetic mutants were isolated after treatment of the wild-type cells with N-methyl-N-nitro-Nnitrosoguanidine (10 mgiml for 7 min) and enrichment with penicillin (500 units/ml for 1 h). Cells surviving penicillin treatment were plated on aerobic plates and the colonies obtained were streaked both on aerobic plates incubated in the dark and on anaerobic plates incubated in the light. Colonies which failed to grow after a week in the illuminated anaerobic plates were selected as possible photosynthetic mutants. Photophosphorylation and noncyclic electron transport to Ozwere measured essentially as described earlier (1). Illumination for the measurement of light-induced electron transport was provided by a 300-W slide projector equipped with a CS 2-64 Corning glass filter (light intensity 4.2 x 105 ergs x cm-’ X SK’). Dehydrogenase activities were measured spectrophotometrically with a Gilford spectrophotometer. NADH dehydrogenase was measured by following the decrease in absorbance at 340 nm when O2was the electron acceptor, or at 420 nm with &Fe (CN), as the terminal electron acceptor. Succinate dehydrogenase was measured with dichlorophenolindophenol and phenazinemethosulfate as the terminal electron acceptor and by following the decrease in absorbance at 600 nm. Bacteriochlorophyll spectra were measured with a Cary Model 14 spectrophotometer and the different spectra of cytochromes b and c were measured with a Gary Model 118. Antimycin A, NADH, and ADP were purchased from Sigma. All other chemicals were of Analar grade.

The possible involvement of one of the two respiratory chain oxidases of Rhodopseudomonas capsulata, in the process of photooxidation, was recently studied with the aid of the mutant strains M6 and M7, each deficient in one of these oxidases (4). It was suggested that the photooxidation is mediated through the cyanide-insensitive oxidase which is the only one present in the M7 strain since this mutant catalyzed higher rates than M6 of the lightinduced oxidation of external donors. In contrast, however, in the present work we show that the photooxidation activity in Rhodopseudomonas capsulata can be differentiated from the respiratory oxidases by its specific sensitivity to inhibitors RESULTS and by the isolation of a photosynthetic mutant deficient in this activity. It is sugWe have previously shown (1, 2) that regested that the process is catalyzed by a duced dichlorophenolindophenol is oxidized photooxidase which differs from the two by chromatophores from Rhodopseudorespiratory oxidases. monas capsulata. This activity was completely inhibited by NT. In the presence of MATERIALS AND METHODS the inhibitor light induced an electron transport from the external donor to 0,. Rhodopseudomonas capsulata cells were grown anaerobically in the light and semiaerobically in the dark Although this photooxidation was inhibited on a medium described by Ormerod et al. (5) supple- by high concentrations of CN- (Fig. 1) it mented with 0.002% thiamine-HCl. For anaerobic was resistant to similar concentrations of NT. Baccarini-Melandri et al. (7) and La growth the culture was incubated in 11-liter bottles completely filled with medium. The semiaerobic cul- Monica and Marrs (8) have suggested that

278

BITTAN ET AL.

I lo-’

I 5rlr7

I 10-6 Antimycin

I 5X10-6

I 10-S

I

I

I

5.!0-5

IO-’

5.W’

, 10-J

A(M)

FIG. 2. Effect of antimycin A on the dark oxidation of NADH and light-induced electron transport from dichlorophenolindophenol to OSin chromatophores from cells grown under semiaerobic conditions in the dark. Reaction conditions were as described under Materials and Methods. The reaction mixture contained: tricine-NaOH (pH 8), 33 mM and chromatophores containing 79 wg bacteriochlorophyll in a final volume of 4.5 ml. The reaction was carried out in the dark with 0.3 mM NADH (0) or with 10 PM dichlorophenolindophenol and 1.5 mM ascorbate (0) in the light.

the respiratory electron transport chain of Rhodopseudomonas capsulata is branched and includes two terminal oxidases. The resistance of the photooxidation to N, indicates that it is different from the two dark oxidations since a high concentration of aside (10 mM) inhibited oxidation of NADH, succinate, and reduced dichlorophenolindophenol in the dark. This pattern is also true for other inhibitors. Figure 2 compares the effects of the electron transport inhibitor antimycin A on the electron transport from NADH to 0, in the dark and on the photooxidation of ascorbate reduced dichlorophenolindophenol. It can be seen that at an inhibitor concentration of low4 M there was 82.4% inhibition of NADH oxidation while the photooxidation activity is 228% relative to the control. The same effect is seen with CN- which is classical inhibitor of oxidases (Fig. 3). In chromatophores from cells grown semiaerobically in the dark, lop4 M CN- completely inhibits the dark oxidation of reduced dichlorophenolindophenol and 80% of the oxidation of NADH. But at this concentration of the inhibitor the light-induced oxidation of dichlorophenolindophenol was hardly affected. Antimycin A (0.5 PM) was added to

the last assay to block the light-dependent cyclic electron transport. At this concentration it hardly had any effect on the dark oxidation (Fig. 2). The pattern of inhibition in chromatophores from photosynthetically grown cells is similar to the one found in membranes from semiaerobically grown cells (Fig. 3). The degree of inhibition by CN- of the photooxidation of reduced dichlorophenolindophenol or of the oxidation of succinate and NADH was hardly changed when the chromatophores were illuminated either with white light or with the routinely used red light. Neither of the light sources had any significant effect on the rate of the autooxidation of 10 j..&M dichlorophenolindophenol in the presence of 1.5 InM ascorbate which was used in these experiments (data not shown). The fact that high concentrations of KCN completely inhibited the light-induced oxidation (Figs. 3,4) also indicates that there was little contribution of 0, uptake from light-stimulated autooxidation of dichlorophenolindophenol since KCN does not inhibit the chemical reaction. The rate of the photooxidation activity in chromatophores from photosynthetically grown bacteria was higher than the rate of

279

Rhodopseudomonas capsulata PHOTOOXIDASE

1,,

0

10-8

I IL

10-7

10"

10-s

Ah

10"

10-S

10-2

I

FIG. 3. Effect of CN- on oxidation of NADH and dichlorophenolindophenol in chromatophores from cells grown under semiaerobic conditions in the dark. Reaction conditions were as described under Materials and Methods. The reaction mixture contained: tritine-NaOH (pH 31, 33 mM and chromatophores containing 40 pM bacteriochlorophyll in a final volume of 4.5 ml. The reaction was carried out either in the presence of 0.45 mM NADH in the dark (0) or in the presence of 40 pg dichlorophenolindophenol and 1.5 mM ascorbate in the dark (A) or with 40 pM dichlorophenolindophenol, 1.5 mM ascorbate, and 5 X lo-’ M antimycin A in the light (A). These. rates were calculated by subtracting the rate of O2 uptake in the dark from the apparent rates in the light under the same conditions.

NADH oxidase, indicating that the noninhibited fraction of the NADH oxidase could not support the rate required for photooxidation of reduced dichlorophenolindophenol. It is interesting to note that the rate of oxidation of both dichlorophenolindophenol and of NADH in the dark was three to six times as fast while the rate of the photooxidation was only slightly lower in chromatophores from semiaerobically grown cells when compared to the rates obtained in membranes from photosynthetically grown bacteria (Figs. 3,4). The shape of the curves for both photosynthetically and semiaerobically grown

cells is in agreement with the findings of Baccarini-Melandri et al. (7) whose data show that the inhibition curve of NADH oxidation by CN- is biphasic in aerobically grown cells. The light-dependent and the dark electron transports were interrelated with each other. It can be seen from Table I that illumination of chromatophores in the presence of reduced dichlorophenolindophenol did not result in an increase in the 0, uptake. The results were similar also in the presence of lo+ M KCN which inhibits 73.2% of the dark activity. Increasing of the KCN concentration to lo+ M which caused 94.5% inhibition of the dark reaction, enabled light to induce an electron transport. Addition of antimycin A in the presence of 10e5M KCN completely inhibited the activity in the dark, and resulted in a much higher activity of photooxidation. 110 t

KCN

(Ml

FIG. 4. Effect of CN- on oxidation of NADH and dichlorophenolindophenol in chromatophores from cells grown anaerobically in the light. Reaction conditions were as described in Fig. 3. The reaction was carried out in the presence of 0.4 mu NADH in the dark (0) or in the presence of 40 pM dichlorophenolindophenol and 1.5 mM ascorbate in the dark (A) or with 40 PM dichlorophenolindophenol, 1.5 mM ascorbate, and 5 x lo-’ M antimycin A in the light (minus dark) (AL).

280

BITTAN ET AL. TABLE I

INTERRELATIONSHIP BETWEEN DARK AND LIGHT OXIDATION OF DICHL~ROPHENOLINWPHENOL IN CHROMA~PHORES FROM CELLS GROWN IN THE DARK UNDER SEMIAEROBIC CONDITIONS

Conditions bmol Op consumed x mg BChl-’ x h-l) Addition (M) None KCN (1O-s) KCN (lo+) KCN (lo-+) + antimycin A (5 x lo-‘)

Dark

Light

Light minus dark

181.1” 48.5 4.6

181.1 48.5 29.1

0 0 24.5

0

60.7

60.7

a The reaction was carried out as described under Materials and Methods. The reaction mixture contained: tricine-NaOH (pH 8), 33 mM; dichlorophenolindophenol, 10 pM; ascorbate, 1.5 mM; and chromatophores containing 3’7pg bacteriochlorophyll in a total volume of 4.5 ml. Tricine, N-tris(hydroxymethyl)methyl-glycine; BChl, bacteriochlorophyll.

We have selected a mutant strain of Rhodopseudomonas capsulata (YIV), which in contrast to the wild type, could not grow under anaerobic conditions in the light (Fig. 5). The mutant grew under aerobic or semiaerobic conditions in the dark, with the same generation time as the wild type. This mutation is probably a point mutation since it was found that the reversion frequency is at the order of lo+. The absorption spectrum of the pigments in chromatophores from mutant YIV is the same as that of the wild type grown under semiaerobic conditions in the dark (not shown). Chromatophores from cells of the mutant contain a higher ratio of cytochromes of b and c type to bacteriochlorophyll in comparison to the wild type (Table II). Chromatophores from the mutant YIV showed photophosphorylation activity (Table III). Photophosphorylation in both the wild type and the mutant respond in a similar manner to different experimental conditions which affect the rate of cyclic phosphorylation. Chromatophores from YIV catalyzed

FIG. 5. Growth curve of a wild-type and a mutant strain YIV of Rhdopseudommas capsulata. Cells of the wild type strain (A) or YIV (A) were grown anaerobically in the light in test tubes filled completely with growth medium. Cells of wild type (0) or YIV (0) were also grown semiaerobically in the dark with constant shaking in Erlenmayer flasks equipped with a side arm and filled up to 40% of their volume with growth medium. The growth of the bacteria was monitored by measuring the turbidity of the culture with a Klett-Somerson photometer equipped with a red (No. 66) filter.

photooxidation of reduced dichlorophenolindophenol at a rate which was 35.7% of the wild type (Table IV). However, the residual activity was enhanced by antimycin A in accord with the effect of this inhibitor on the photooxidation in the wild type. The enhanced rate in the mutant was also lower than the rate of photooxidase activity in the TABLE II CYTOCHROME CONTENT IN CHROMATOPHORES FROM WILD-TYPE AND MUTANT YIV

Strain

b-Type cytochrome: BChl

c-Type cytochrome : BChl

Wild type YIV

1:19” 1:9

1:13 1:6

a The ratio was calculated from the difference spectra of dithionite reduced versus & (Fe(CN),) oxidized chromatophores. b-Type cytochromes were measured at 560 nm and c-type cytochromes were measured at 550 nm. The extinction coefficient was taken at 15 cm-’ mM-’ for both types of cytochromes according to Ref. (10).

Rhodopseudomonas capsulata

PHOTOOXIDASE

281

concentration range of IO-7-1O-2 M. It was PH~T~PHO~PHORYLATIONACTIVITY IN CHROMATO- also seen that NADH dehydrogenase activity was higher in the mutant than in the YIV PHORESFROMWILD-TYPEANDMUTANT wild type. GROWNSEMIAEROBICALLYINTHE DARK TABLE III

Chromatophores from hmol Pi esterified x mg BChlF x h-‘)

DISCUSSION

Two lines of evidence which were pursued in this work support our suggestion (2) that photooxidation of external electron donors is catalyzed by a third oxidase which is different from the two aerobic oxiConcentration Wild Gas dases found in Rhodopseudomonas capsuAddition phase YIV Cm@ type Zata chromatophores (7, 8). One line of evidence is based on the different sensitivity 99.8 None 108.2” N2 of the three oxidation pathways to inhibi9 533.2 Ascorbate 427.5 N* tors of electron transport and to specific inNone air 146.1 231.4 hibitors of oxidases. The other involves a o The reaction was assayed as described earlier (1) selection of a photosynthetic mutant defiin a reaction mixture containing tricine-NaOH (pH 81, cient in photooxidation activity. 33nu.q &H”PO, (pH 8), 3.3 mM (containing 10scpm); The existence of a branched chain of oxiMgCI, ,8 mu; ADP, 1.6 ~IU; and chromatophores con- dative electron transport in chromatotaining 86 pg bacteriochlorophyll. phores from both aerobically and photosynthetically grown cells was well presence of antimycin A in chromatophores established by showing differential inhibifrom the wild type. The decrease in the oxi- tors sensitivity and by selection of respiradation of an external donor was specific for tory mutants (7-9). With the aid of two oxthe photooxidation. The rates of dark oxiTABLE IV dation of NADH, succinate, and dichlorophenolindophenol were even slightly DARKOXIDASEANDPH~T~~XIDATION ACTIVITIES higher in the mutant relative to the wild IN CHROMATOPHORESFROY SEMIAEROBICALLY type (Table IV). Studies of the inhibitory GROWNBACTERIAOFWILD-TYPE AND effect of CN- on NADH oxidation in the MUTANT YIV dark and on dichlorophenolindophenol phoChromatophores tooxidation by chromatophores from Y IV wnol O2 reveal a behavior similar to that of the wild consumed x mg BChl-’ x h-‘) type (Fig. 6). At lo-* M CN-, NADH oxidase activity is 70% of the control while Electron donor Addition and Condi- Wild dichlorophenolindophenol photooxidation and concentration concentration tions YIV type was hardly affected. This curve also shows NADH, 0.45 m!d Dark 252.9 316.1 the biphasic behavior of NADH oxidation Succinate, 20 “IP Dark 163.0 355.5 in response to increasing concentrations of Dichlomphenolindophenol” 40 p&l 1.5 nm N; Dark 199.5 363.i CN-. 1.5 rn~ N; + Light’ 21.6 60.4 @PM The suggestion that the photooxidase is 40 PM 0.5 /.m different from the two oxidases of the res159.0 X2.3 antimycin A Lighta piratory chain is based, among other Note. Oxidase activity was measured with an oxygen electrode as things, on studies of the inhibitory effect of described earlier (2). The reaction mixture contained tricine-NaOH (pH 81, 33 mid, and chromatophores containing 60 fig bacterioehloroCN-. Therefore, it was important to verify phyll. Sue&ate dehydrogenase was activated by preincubation of the whether CN- inhibits the oxidase rather preparation with 50 IIIY succinate in 30°C for 30 min. Both the wild than the dehydrogenation activity. It was type and the mutant were grown semiaerobically in the dark. a Diehlorophenolindophenol was kept in the reduced state with 1.5 mu found (Table V) that both in chromatophores from wild type and YIV, NADH de- ascorbate. a Calculated rates in the light were obtained by subtracting the rate hydrogenase is not affected by CN- in a in the dark from the apparent rates in the light.

282

BITTAN ET AL.

idase inhibitors CN- and NT which were used in our experiments it was possible to distinguish between these two oxidases and the photooxidase. The photooxidase was resistant even to high concentrations of azide (20 mM> which inhibited the two oxidases. On the other hand 10 InM KCN inhibited all three oxidation activities indicating that although the photooxidase has low affinity for the inhibitor, the light-induced oxygen uptake is not an artificial system. At lower concentrations (0.1 mM> a differential inhibition by KCN was observed. Thus, at 0.1 mM KCN 88% of NADH oxidation was inhibited while no inhibition of the photooxidation was observed in chromatophores from photosynthetically grown cells. Although a similar pattern of inhibi-

240

TABLE V EFFECT OF CN- ON NADH DEHYDR~GENASE ACTIVITY IN CHROMATOPHORES FROM WILD TYPE AND YIV GROWNSEMIAEROBICALLY IN THE DARK

Chromatophores from @mol NADH oxidized x lo-* x mg BChl-’ x h-‘) KCN concentration (Ml

Wild type

YIV

lo-’ 10-S 10-4 10-S 5 x 10-s 10-Z

8.1’ 8.9 9.0 9.0 8.6 10.0

17.2 17.6 16.8 18.8 17.6 18.3

a The reaction mixture contained: tricine-NaOH (pH 8), 33 mM; NADH, 0.6 mM; &,(Fe(CN),), 1.66 mM; rotenone, 10 pg/ml; and chromatophores containing 14.3 pg/ml bacteriochlorophyll in a ilnal volume of 3 ml. The activity was measured spectrophotometrically following decrease in absorbance in 340 nm for NADH oxidation, and as a control, the decrease in 420 nm for reduction of K,(Fe(CN&). The values in the table are an average of both measurements.

1

KCN(M1

FIG. 6. Effect of CN- on oxidation of NADH and dichlorophenolindophenol in chromatophores from mutant YIV. Reaction conditions were as described for Fig. 3. The reaction was carried out in the presence of 0.45 mu NADH in the dark (0) or in the presence of 40 PM dichlorophenolindophenol and 1.5 mM ascorbate in the dark (A) or with 40 PM dichlorophenohndophenol, 1.5 mM ascorbate, and 5 x lo-’ antimyein A in the light (light minus dark) (A).

tion was also observed in chromatophores from semiaerobically grown cells the rate of the residual activity of NADH oxidation was only slightly slower than the photooxidation activity in the presence of 0.1 lllM KCN. It would be incorrect however, to suggest that the residual oxidase could account for the photoxidation activity since, under similar degree of inhibition the rate of the photooxidation activity was nine times faster than that of the NADH oxidase in chromatophores from photosynthetically grown bacteria. One of the main reasons for the conclusion drawn by Zannoni et al. (4) that lightinduced oxidation is catalyzed through the cyanide-insensitive oxidase rather than a photooxidase was based on the observation that a mutant strain, M6, of Rhodopseudomonas capsulata which is deficient in this oxidase, catalyzed 10% of the rates of photooxidation of external electron donor relative to the mutant M7 (4). It should be noted that in their experiments 5 mM KCN

Rho&pseudomonas

capsulata

was used to inhibit the oxidation in M6 and M7 mutant. As seen in Fig. 4, at this concentration of inhibitor almost 90% of the photooxidation was inhibited. It seems that careful study of the effect of various concentrations of cyanide and azide on the oxidative activities in the M6 and M7 mutants has to be conducted before conclusions can be drawn. Such experiments might distinguish between photooxidase and oxidase activity in the mutants. The fact that CO which inhibits the cyanide-insensitive oxidase (4) also inhibited photooxidation activity in the M7 mutant which lacks cytochrome c oxidase does not necessarily mean that photooxidation is catalyzed through the cyanide-insensitive oxidase. It might merely be an indication that the photooxidase is also inhibited by CO. Indeed, it was demonstrated that there are several cytochromes which bind CO in these bacteria (9). Both the identity and the physiological role of the photooxidase are accentuated by a selection of a photosynthetic mutant (YIV) of Rhodopseudomonas capsulata which is deficient in the photooxidation activity. The pigment system, cytochrome band c-type content, and photophosphorylation activity were similar to the wild type while the dark oxidase activities were higher in the YIV mutant. If the photooxidation was catalyzed by the dark oxidases an increase in the rate of the latter would have been expected to result in a similar increase in the activity of the former. However, the YIV mutant catalyzed only one-third of the rate of photooxidation found in the wild type. Unlike M6 which is a respiratory mutant, YIV is a photosynthetic mutant. Another photosynthetic mutant selected from Rhodospirillum rubrum (10) is also deficient in the photooxidation activity and in rhodoquinone, but contains ubiquinone (11). Added rhodoquinone restored the photooxidation activity to this mutant. It was suggested that rhodoquinone is involved in the photooxidative pathway. However, it could not be part of the catalysis of this reaction in Rhodopseudomonas capsulata since these bacteria contain only ubiquinone (12). The inability

PHOTOOXIDASE

283

of these mutants to grow photosynthetically is by itself an indication of the physiological importance of this activity. Gimenez-Gallego et al. (10) suggested that the physiological function of the photooxidase is in regulating the redox level in the cells by the transfer of electrons to oxygen or some other endogenous acceptor. However, it can be speculated that the photooxidase represents also a shunt in the light-induced electron transport system which leads to photoreduction of NAD+ (13) under anaerobic atmosphere. Such an activity was found in Rhodospirillum ribum (14, 15) though its pathway is unknown. REFERENCES 1. HOCHMAN, A., AND CARMELI, C. (1977) Arch. Biochm. Biophys. 179, 349-359. 2. HOCHMAN, A., BEN-HAWIM, G., AND CARMELI, C. (1977) Arch. Biochem. Biophys. 184, 416422. 3. DEL-VALLE-TASCON, S., GIMENEZ-GALLEGO, G., AND RAMIREZ, J. M. (1975) Biochem. Biophys. Res. Commun. 66, 514-519. 4. ZANNONI, D., JASPER, P., AND MARRS, B. (1978) Arch. Biochem. Biophys. 191, 625-631. 5. ORMEROD, J. G., ORMEROD, K. S., AND GEST, H. (1961) Arch. Biochem. Biophys. 94, 449-463. 6. CLAYTON, R. K. (1963) Biochim. Biophys. Acta 75, 312-323. 7. BACCARINI-MELANDRI, A., ZANNONI, D., AND MELANDRI, B. A. (1973) Biochim. Biophys. Acta 314, 298-311. 8. LA MONICA, R. F., AND MARRS, B. L. (1976) Biochim. Biophys. Acta 423, 431-439. 9. ZANNONI, D., MELANDRI, B. A., AND BACCARINI-MELANDRI, A. (1976) Biochim. Biophys. Acta 449, 386-400. 10. GIMENEZ-GALLEGO, G., DEL-VALLE TASCON, S., AND RAMIREZ, J. M. (1976) Arch. Microbial. 109, 119-125. 11. RAMIREZ-PONCE, M. P., GIMENEZ-GALLEGO, G., AND RAMIREZ, J. (1980) FEBS Lett. 114, 319322. 12. BACCARINI-MELANDRI, A., AND MELANDRI, B.A. (1977) FEBS Lett. 80, 459-464. 13. FRENKEL, A. W. (1958)J. Amer. Chem Sot. 80, 3479-3480. 14. NOZAKI, M., TAGAWA, K., AND ARNON, D. I. (1961) Proc. Nat. Acad. Sci. lJ.S.A 47, 13341340. 15. BOSE, S. K., AND GEST, H. (1963). Proc. Nut. Acad. Sci. USA 49, 337-345.