Relationships between H2 photoproduction and different electron transport pathways in sulfur-deprived Chlamydomonas reinhardtii

international journal of hydrogen energy 34 (2009) 9087–9094

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Relationships between H2 photoproduction and different electron transport pathways in sulfur-deprived Chlamydomonas reinhardtii Taras K. Antal*, Alena A. Volgusheva, Galina P. Kukarskih, Tatyana E. Krendeleva, Andrej B. Rubin Faculty of Biology, Moscow State University, Vorobyevi Gory 119992, Moscow, Russia

article info

abstract

Article history:

In this study the relationships between photosystem (PS) II dependent and independent

Received 10 June 2009

pathways of H2 photoproduction, cyclic electron transport around PS I, chloro- and

Received in revised form

mitorespiration, and transmembrane DpH were examined by inhibitor analysis in S

25 August 2009

deprived Chlamydomonas reinhardtii. The rate of non-photochemical reduction of plasto-

Accepted 7 September 2009

quinones in photosynthetic membranes was significantly diminished under starvation

Available online 9 October 2009

which may explain the minor contribution of the PS II independent pathway of H2 photoproduction in starved cells. The suppressive effect of the herbicide 3-(3,4-dichlor-

Keywords:

ophenyl)-1,1-dimethylurea on the long-term H2 photoproduction was shown to be entirely

Sulfur deprivation

attributed to the inhibition of electron transport in PS II, whereas non-specific interactions

Hydrogen photoproduction

did not take place. Ferredoxin-quinone reductase – dependent cyclic electron transport

Chlamydomonas reinhardtii

around PS I slowed down H2 photoproduction more than two fold. This result was related to the competition between ferredoxin-quinone reductase and hydrogenase for the reduced ferredoxin and to the decrease in transmembrane DpH induced by the cyclic electron flow. The DpH gradient was shown to down regulate the PS II independent pathway of H2 photoproduction in starved cells. ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

In anaerobic conditions, green algae are able to produce H2 during first minutes of illumination due to the activity of hydrogenase (H2ase) [1]. Recently, the effects of several photosynthetic inhibitors on the transient H2 photoproduction by Chlamydomonas reinhardtii have been investigated in order to clarify pathways of electron transport to the H2ase [2]. As was shown in [2], such reagents as: 1) dibromothymoquinone (DBMIB), inhibitor of electron transport via cytochrome b6/f [3], 2) 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PS II inhibitor [4,5], and 3) N-ethylmaleimide which

inactivates NAD(P)H dehydrogenase (NDH) [6] suppressed strongly photoevolution of H2. In accordance with this, H2 photoproduction was suggested to result from two pathways of plastoquinone (PQ) reduction, viz. a PS II dependent pathway coupled to water splitting, and a PS II independent pathway mediated by NDH. Decoupling agents dissipating the electrochemical gradient across the thylakoid membrane were shown to enhance the PS II independent pathway of H2 evolution by an unknown mechanism [7]. It has been shown that sulfur (S) deprived C. reinhardtii incubated hetero- or autotrophically is capable of sustained H2 photoproduction under continuous illumination [8,9]. In

* Corresponding author. Fax: þ7 495 939 1115. E-mail address: [email protected] (T.K. Antal). 0360-3199/$ – see front matter ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.09.011

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gas-proof bioreactors a starved culture passes through five subsequent physiological phases: O2 evolution, O2 consumption, anaerobic phase, H2 production phase, and a termination phase [10]. During the aerobic phase, stress induces specific physiological responses in the cell, such as accumulation of starch, reduction of PS II photochemistry and, consequently, the decline of photosynthetic O2 evolution. When O2 evolution falls below the level required for respiration, anoxia is reached, followed by the activation of an O2-sensitive FeH2ase and photoproduction of H2. However, mechanisms of long-term H2 photoproduction by S deprived green algae are still not clear. Analysis of the effects of different inhibitors helps to understand H2 photoproduction. Recently, the herbicide DCMU was reported to essentially slow down H2 evolution in S starved C. reinhardtii [11,12] suggesting an important role of water splitting during the anoxic H2 production phase. Fig. 1 shows a tentative scheme of electron transport pathways in S starved green algae during H2 production phase. Besides residual activity of PS II, fermentation of the stored starch serves as a source of the reducing equivalents for the H2ase reaction via the PS II independent pathway catalyzed by NDH and succinate dehydrogenase (not shown). Moreover, cyclic electron transport around PS I (CET), mediated by the ferredoxin-plastoquinone reductase (FQR), was proposed to influence H2 production [13]. Electrons derived into the PQ pool from either the residual water splitting reaction of PS II, NDH, or FQR are passed along the photosynthetic electron transport chain via cytochrome b6/f, plastocyanin (PC), PS I, and ferredoxin (Fd) before being used in the H2ase reaction or in the CET. CO2 assimilation and electron transport from Fd to NADP via ferredoxin-NADP reductase (FNR) were assumed to be negligible under S defficiency [8,14]. In mitochondria the electrons are transferred from Complex I (NADH dehydrogenase) or Complex II (succinate dehydrogenase, not indicated) to ubiquinone (not indicated), and then subsequently to Complex III (cytochrome bc1) and Complex IV (cytochrome c oxidase) or to the alternative oxidase (AOX). Oxidases including the chloroplastic oxidase (PQ oxidase) remove photosynthetically produced O2 and, hence, maintain anoxic conditions. Electron transfer into the PQ pool and the residual water splitting activity are coupled to the generation of transmembrane DpH which was shown to negatively regulate transient H2 photoproduction in non-starved cells [7]. Despite of recent advances in mechanisms of H2 photoproduction, still some points remain to be clarified. It is not clear whether the inhibitory effect of DCMU on H2 production can be entirely attributed to the specific inhibition of PS II or whether non-specific interactions, e.g. between DCMU and H2ase, also take place during prolonged incubation with an inhibitor. As mentioned above, sulfur deficiency induces significant inactivation and degradation of PS II [8]. In this respect the observation that PS II dependent H2 production contributes 50–60% in non-starved culture and about 70% in S starved cells [2,12] requires explanation. The relationships between H2 photoproduction and cyclic electron transport around PS I, chloro- and mitorespiration, and transmembrane DpH generation, have not been studied by inhibitor analysis in S starved C. reinhardtii. To reveal the nature of these interactions is important to find new ways for the optimization of H2

photoproduction. The present work is addressed to answer these questions.

2.

Materials and methods

2.1.

Culture conditions and sulfur depletion

C. reinhardtii strain Dang 137D WT was grown photoheterotrophically in tris-acetate-phosphate medium (TAP), pH 7.0, in Erlenmeyer flasks at 30  C under continuous illumination (photosynthetic photon flux density (PPFD) 150 mE m2 s1), constant shaking and aeration. Late-log cells (107 cells ml1) were centrifugated and resuspended in complete (standard TAP, pH 7.7) medium or S-depleted TAP medium (pH 7.7) containing MgCl2 instead of MgSO4. Cells in concentration of about 4  106 ml1 were aerobically incubated in 1.5 l bioreactor at 30  C under moderate continuous illumination (100 mE m2 s1) and constant mixing.

2.2.

Thylakoid preparation

C. reinhardtii cultures were incubated for 60 h either in complete or S-free medium as described in the previous section. The following procedures of thylakoid extraction were carried out in total darkness, and the samples were kept on ice throughout the process. Cultures were harvested by centrifugation at 3000 g for 5 min and washed once in buffer A (25 mM Tris, pH 7.2, 10 mM NaCl, 5 mM MgCl2). The resulting pellet was resuspended in the same buffer, and the cells were broken by repeated sonication for 5  20 s. Unbroken cells, cell debris and a bulk of starch were removed by centrifugation at 2000 g (4  C) for 15 min. Membranes were harvested by centrifugation at 20,000 g (4  C) for 25 min and resuspended in buffer B (25 mM Tris, pH 7.2, 400 mM sucrose, 10 mM NaCl, 5 mM MgCl2). This procedure was repeated two times. Finally thylakoids were resuspended in buffer B at 0.5 mg Chl ml1 and stored at 80  C.

2.3.

DCIP reduction in isolated membranes

The reduction of DCIP from water (photoreduction) or from NAD(P)H was measured spectrophotometrically at 600 nm. Thylakoids were suspended in a reaction mixture in a final volume of 2 ml containing buffer B, 50 mM 2,6-dichlorophenolindophenol (DCIP), and thylakoids equivalent to 5 mg Chl ml1. When indicated, NADP and NADPH were added at the concentration 500 mM. Direct reduction of DCIP by NAD(P)H was taken into account when NAD(P)H – dependent dark reduction of DCIP was calculated. The photoreduction of DCIP was measured upon illumination with saturating (PPFD ¼ 1300 mE m2 s1) white light during 2 min. The rate of DCIP reduction was calculated using an extinction coefficient of 21 mM1 cm1.

2.4.

Measurement of H2 production and H2ase activity

Suspension of S deprived C. reinhardtii (8 ml) was poured into 14 ml gas tight glass vials. To remove air, the gas phase in each vial was flushed for 30 min with argon at the rate 5 ml s1

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international journal of hydrogen energy 34 (2009) 9087–9094

H2O

AOX

Mitochondria 6 I

O2 IV

III

H2O

Starch reserves

3,5

NAD(P)/ NAD(P)H

CO2 assimilation H2

pool

reactions of fermentation

FNR

ΔpH

7

PS II

2

PQ pool

FQR

1

Stroma PS I

Cyt b6/f

4

H+ H+ H+

PQ oxidase

4H+

+ O2

H+

Fd pool

3 NDH

H2ase

Lumen

PC

H+

2H2O

Chloroplast

O2 H2O

Fig. 1 – Electron transfer pathways in S starved green algae under anaerobic conditions. Sites of action of inhibitors are indicated by the corresponding numbers: 1 – DBMIB, 2 – DCMU, atrazine, 3, 5 – antimycin A, 4 – propyl gallate, 5 – myxothiazol, 6 – SHAM, 7 - nigericin, FCCP.

methyl viologen and 20 mM sodium dithionite in the anoxic atmosphere. The reaction mixture was constantly shaken at a temperature of 30  C, and H2 production was measured by gas chromatography after 20 min of cell incubation with reduced methyl viologen. H2se activity is expressed as mmoles of H2 evolved per hour per mg of Chl. The reagents were used in the following concentrations: 2  105 M DCMU, 2  105 M atrazine, 6  106 M DBMIB, 4  106 M antimycin A, 2  106 M myxothiazol, 9  104 M salicyl hydroxamic acid (SHAM), 103 M propyl gallate, 107 M nigericin, and 8  107 carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP).

100

H2 production, μmol mgChl-1

using the inlet and outlet needles. After this, in order to activate H2ase, cells were incubated for 3 h under constant shaking at low intensity of light (PPFD 25 mE m2 s1) which was shown to be optimal for H2 photoproduction by S deprived C. reinhardtii [10]. Then, H2 was removed by a short blowing with argon, and samples were exposed for 3 h to the effects of different reagents. Thereafter, H2 content in gas phase was estimated in percents of volume using a gas chromatograph ‘Gazokhrom-2000’ (Zchrom, Moscow, Russia) complete with the detector of thermoconductivity and using argon as a carrier gas. For this, 200 ml of gas sample was taken from each vial with a gas tight Hamilton syringe (Hamilton Company, USA) and analyzed in the gas chromatograph. H2 content was calculated using a calibration between signal amplitude and different volumes of H2 injected into the vial containing 8 ml of medium. During 3 h incubation, the effects of uncouplers and antimycin A, an inhibitor of FQR-mediated reduction of PQ [15], reached maximum. For example, Fig. 2 shows time courses of H2 production measured in S deprived C. reinhardtii during different periods of incubation with antimycin A. As seen in the figure, the stimulation of H2 production by this reagent was maximal during 3 h of incubation and decreased gradually at longer periods of incubation. The decrease of stimulation can be explained by the development of toxic action of the reagent. The total H2ase activity was measured as evolution of H2 in vivo in the presence of methyl viologen reduced by sodium dithionite [16]. Suspension of S deprived C. reinhardtii (4 ml) was poured into 14 ml gas tight vials, flushed during 30 min with argon, and incubated anaerobically in the dark under constant shaking in the presence or in the absence of reagents as indicated in text. After that, samples were gassed shortly with argon to remove H2 followed by the injection of 2 mM

no additives 4 μM antimycin A

80

60

40

20

0 0

5

10

15

20

25

Time, h Fig. 2 – Time courses of H2 photoproduction measured in S deprived C. reinhardtii during incubation in gas tight vials in the presence of antimycin A. Time of antimycin A injection is indicated by arrow.

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2.5.

international journal of hydrogen energy 34 (2009) 9087–9094

Other methods

The variable (FV) to maximal (FM) Chl fluorescence yield ratio FV/FM (¼(FM–FO)/FM) was measured with a Pulse Amplitude Modulated fluorometer (PAM 2000) (Walz, Effeltrich, Germany). Duplicate samples of Chl (a þ b) were determined spectrophotometrically following the extraction of pigments with 95% ethanol [17]. Data are mean values of experiments done in triplicate. Error bars represent standard deviation.

3.

centers able to reduce QA but incapable of transferring electrons further to the PQ pool, in agreement with previous observations [18]. The NAD(P)H dependent dark reduction of DCIP was also significantly reduced in these preparations reaching only 44% of the control. Membranes from cells incubated without sulfur for 60 h showed a further decrease in the rate of DCIP photoreduction and NAD(P)H dependent dark reduction which attained only 22 and 27% of the control, respectively. For this reason, S depletion slows down the rates of both photochemical and non-photochemical reduction of the PQ pool, which may explain the major contribution of the PS II dependent pathway of H2 photoproduction under normal conditions and S starvation stress.

Results and discussion

3.1. PS II dependent and NAD(P)H dependent reduction of DCIP in thylakoid membranes from S starved C. reinhardtii

3.2. Evaluation of H2 photoproduction and H2ase activity in S deprived C. reinhardtii in the presence of inhibitors of photosynthetic electron flow

As was shown earlier [8,18], S deficiency induces significant inactivation of PS II. Therefore the contribution of the PS II dependent pathway of H2 evolution can be expected to be lower in starved cultures than in normal ones. However, PS II dependent H2 production in C. reinhardtii remains a major pathway irrespective of the starvation status of the alga [7,12]. In order to explain these observations, we compared the rates of photoreduction and NAD(P)H dependent dark reduction of DCIP in thylakoid membranes extracted from non-starved and S starved C. reinhardtii (Table 1). Thylakoids prepared from non-starved cells showed rapid reduction of DCIP at the initial rate of 59.4 mmol mg Chl1 h1 when exposed to saturating light. In C. reinhardtii the NADPH and NADH were shown to be the main substrates for the nonphotochemical electron transport to PQ mediated by NDH [2]. The addition of 500 mM of each of these substrates led to the DCIP reduction in the dark at the rate of 23.0 mmol mg Chl1 h1. The proportion of DCIP photoreduction in the sum of photoreduction and NAD(P)H dependent dark reduction reached 72%, which is in line with the observed major contribution of PS II dependent pathway in H2 production in non-starved cells. In membranes from cells incubated for 48 h in S-depleted medium, the initial rate of DCIP photoreduction was only 31% of that value in non-starved cells (Table 1). In this condition, the maximal quantum yield of PS II primary photochemistry, FV/FM ((FM–FO)/FM) [19], was diminished to a much lesser extent reaching 76% of control. This result indicates the appearance of a great number of PS II

Starved cells incubated without additives generated H2 at the mean rate of 7.7 mmol H2 (mg Chl h)1 (Fig. 3A). As known, the artificial quinone DBMIB inhibits photosynthetic electron transport between PQ pool and PS I by replacing plastoquinol (PQH2) from the place of binding in the Qo site of cytochrome b6/f [3] (the action sites of the reagents are shown in Fig. 1). Incubation of samples for 3 h with 5 mM DBMIB led to almost complete suppression of H2 production by 97% (Fig. 3A) suggesting that electron transport via cytochrome b6/f is necessary for the generation of H2. In the presence of 20 mM DCMU, an urea class inhibitor of PS II [5], H2 yield declined by about 70% as compared to control cells (Fig. 3A), that was explained by the inhibition of PS II [11,12]. However, prolonged incubation with DCMU may also lead to non-specific interactions of the inhibitor with other electron transfer steps in cell. To test this possibility we examined the effect of a triazine-type family of PS II inhibitors: 6-chloro-N-ethyl-N0 -(1-methylethyl)1,3,5-triazine-2,4-diamine (atrazine) [5], on H2 evolution. Both DCMU and atrazine function by competing with plastoquinone for binding to the QB niche in D1 protein of PS II [5,20]. However, there is no cross-resistance to DCMU in atrazine resistant algae and vice versa, suggesting that spatial domains for their binding to PS II are not the same (see e.g. [21]). We found that in the presence of 20 mM atrazine, H2 evolution by starved cells decreased to the same extent as in the presence of DCMU (Fig. 3A), indicating that both herbicides influence H2 photoproduction by inhibiting electron transport in PS II. We tested the possibility that prolonged incubation of cells with DCMU may itself decrease H2ase activity. The latter was estimated in samples after 6 h of anaerobic incubation without additives or with 20 mM DCMU. In the control activity of H2ase was 102 mmol H2 (mg Chl h)1 (Fig. 3B) while samples incubated with DCMU showed a noticeable increase in activity by almost 20%. The elevated H2ase activity can be explained by the DCMU-induced inhibition of PS II and photosynthetic evolution of oxygen, the latter being a strong suppressor of H2ase activity [22]. Indeed, S starved C. reinhardtii cells retain residual activity of PS II during the H2 production phase [12], and O2 production by this residual PS II activity may still cause partial inactivation of the H2ase. Thus, according to the obtained data, prolonged incubation of cells with DCMU does

Table 1 – PS II - dependent and NAD(P)H – dependent reduction of DCIP in thylakoid membranes extracted from C. reinhardtii after different periods of incubation in S-free medium. Numbers in parentheses show percentage of the control value. Time, h 0 48 60

FV/FM 0.63 (100%) 0.48 (76%) 0.39 (62%)

H2O / DCIP, NAD(P)H / DCIP, mmol mg Chl1 h1 mmol mg Chl1 h1 59.4  3.1 (100%) 18.7  2.2 (31%) 13.2  2.6 (22%)

23.0  1.8 (100%) 10.1  1.4 (44%) 6.2  0.8 (27%)

international journal of hydrogen energy 34 (2009) 9087–9094

A

140

B

120 8 100 6

80 60

4

40 2

DCMU

Ctrl

Atrazine

DCMU

DBMIB

0

Ctrl

20

H2ase activity, μmol (mgChl h)-1

H2 production, μmol (mgChl h)-1

10

9091

0

Fig. 3 – Effect of DCMU, atrazine, and DBMIB on the rate of H2 photoproduction and on the H2ase activity in S deprived C. reinhardtii.

not down regulate H2ase activity and its action on H2 yield is entirely caused by inhibition of electron transport in PS II.

3.3. Effect of inhibitors of CET, mito- and chlororespiration, and of the uncouplers on PS II dependent and PS II independent H2 photoproduction by S starved C. reinhardtii In green microalgae S deprivation significantly impairs CO2 assimilation and promotes the accumulation of starch in cell, which results in more reduced state of photosynthetic electron carriers, including the PQ pool [8,12,23,24]. Moreover, under anoxic conditions established upon incubation of S deprived C. reinhardtii in a bioreactor, additional increase in the intracellular reducing power occurs. The reduced state of PQ in starved C. reinhardtii induces a transition of photosynthetic membranes from state 1 to state 2 [18] which favors the excitation of PS I [25]. In its turn, the state transition triggers the switching of the electron transport from linear to cyclic [26]; the latter is mediated by a membrane bound enzyme FQR [15] (see Fig. 1). Additionally, electron transport from Fd to PQ can be driven subsequently by FNR and by type 2 NDH which functions as NAD(P)H-PQ oxidoreductase in C. reinhardtii [2,27]. However, NDH mediated CET plays a rather minor role in starved cells, since the electron flow from NAD(P)H to PQ is saturated due to the over-accumulation of the reductant. Therefore, during the H2 production phase the FQR-mediated CET and H2ase reaction may compete with each other for the reduced Fd, as suggested by Kruse et al. [13]. In fact, as shown in [13], C. reinhardtii strain stm6 blocked in state 1 and thus unable to maintain efficient CET produced much higher amount of H2 than the wild type. On the other hand, the mutant also showed modified respiratory metabolism which resulted in a) accumulation of large amounts of starch, an important prerequisite for H2 production, and b) elevated rate of respiration that might efficiently remove O2 and stimulate H2ase activity. It is unclear, whether all of these factors or only some of them are responsible for the high H2 output by stm6.

In order to elucidate the relationships between H2ase and FQR driven CET, we examined the effect of antimycin A, an inhibitor of FQR [15], on the rate of H2 evolution by S deprived C. reinhardtii. The mitochondrial respiration at the level of Complex III (CoQH2-cyt c oxidoreductase) is also sensitive to the antimycin A (see sites of inhibitors action in Fig. 1). Therefore, we tested effect of the myxothiazol, specific inhibitor of Complex III. As demonstrated in Fig. 4, incubation of cells with 4 mM antimycin A led to increase in H2 evolution by 128%, i.e. more than by two fold as compared to the control cells incubated under the same conditions without additions. Incubation with 2 mM myxothiazol enhanced H2 production to a lesser degree (by about 30%). This result suggests that antimycin A facilitates H2 production mainly due to the inhibition of the FQR activity while inhibition of mitorespiration at the level of the Complex III contributes to the lesser extent. To understand how the inhibition of FQR influences PS II dependent and PS II independent H2 production, the effect of antimycin A was examined in the presence of DCMU. Added together with DCMU, antimycin A enhanced H2 output by 124% as compared to the sample treated with DCMU alone (Fig. 4) indicating that PS II independent H2 production is stimulated approximately to the same extent as total (PS II dependent plus PS II independent) H2 production. Thus, the action site of antimycin A on H2 production is located along the photosynthetic electron transport chain between PQ pool and Fd, in agreement with the suggestion about competition between H2ase and FQR for the reduced Fd. CET may also maintain an elevated trans-thylakoid proton gradient; the latter was shown to slow down H2 photoevolution in normal (non-starved) C. reinhardtii [7]. To test this hypothesis, we studied the effect of the protonophore nigericin and the proton-ionophore FCCP on H2 yield in S starved cells. As shown in Fig. 4, both uncouplers caused an increase in H2 production by about 30%, which is much less than the effect of antimycin A. Importantly, that incubation of algae with both animycin A and nigericin affected H2 production to the same extent as incubation with antimycin A alone (data not shown), suggesting that the stimulatory effect of

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international journal of hydrogen energy 34 (2009) 9087–9094

H2 production, μmol (mgChl h)-1

20 228

18 16 14 12 10

138

127

137

134 115

100

8 224

6 4

214

195

204

177

100

2 FCCP

D CM U +F C C P

Nigericin

DCMU+Nigericin

Propyl Gallate

Myxothiazol + SHAM

DCMU+Myxothiazol+SHAM

Myxothiazol

DCMU+Myxothiazol

Antimycin A

DCMU+Antimycin A

DCMU

Ctrl

0

Fig. 4 – Effects of photosynthetic inhibitors and uncouplers on the rate of H2 photoproduction by S deprived C. reinhardtii. H2 production measured in the presence of DCMU is indicated by grey color. Changes of H2 evolution (in % of control or of control with DCMU) are shown over the bars.

antimycin A on H2 yield is partially related to the dissipation of CET-induced transmembrane DpH. The results lead to a conclusion that the observed antimycin A induced increase in H2 yield can be partly explained by inhibition of mitorespiration at the level of Complex III and by slowing down of the CET-related proton pumping into lumen. However, more than 50% of the effect was due to the redirection of the electron transport from FQR to H2ase. The negative effect of proton gradient on H2 production can be attributed to the suppression of the electron transport at the level of NDH or cytochrome b6/f as well as to the decrease in proton concentration in stroma, which would limit the H2ase reaction. As shown in Fig. 4, the uncouplers nigericin and FCCP enhanced total H2 output by about 30% while the PS II independent component of H2 production measured in the presence of DCMU was two fold stimulated by these reagents. This result indicates that DpH gradient mainly suppresses the PS II independent pathway, i.e. DpH dependent regulation occurs at the level of the NDH enzyme. Since type 2 NDH does not pump protons into lumen, the mechanisms of such regulation by DpH is not clear. The mechanism of regulation may involve, e.g. the decrease in the rate of NADPH oxidation at alkaline pH due to the electrostatic repulsion between the negative charges of the phosphate group of NADPH and the phospholipids of the membrane [28]. As was mentioned above, inhibition of mitorespiration at the level of Complex III by myxothiazol enhanced H2 output by about 30% (Fig. 4). Only the DCMU-insensitive component of H2 production was affected suggesting that the rate of nonphotochemical reduction of the PQ pool increases in the presence of this reagent. Inhibition of mitorespiration by the mixture of myxothiazol and SHAM, an inhibitor of the

mitochondrial alternative oxidase, stimulated the total H2 yield by 38% while PS II independent component was increased by 114%. It can be concluded that in S starved cells mitorespiration functions as an electron sink and its inhibition promotes redirection of electron flow from respiratory to photosynthetic chain and further to H2ase. In addition to mitorespiration, reductants in green algae can be also utilized via a chloroplast respiratory electron transport process [29]. The chlororespiratory chain involves transfer of high potential electrons from stroma to the PQ pool via the NAD(P)H-PQ oxidoreductase as well as oxidation of PQ by O2 through the action of a PQ oxidase [30,31]. Since the chloroplast PQ oxidase acts as an electron sink, its activity may reduce H2 output. As known, the chloroplast PQ oxidase is slightly sensitive to inhibitors of mitorespiration, but it can be inhibited specifically by propyl gallate [31]. As demonstrated in Fig. 4, anaerobic incubation of S starved cells with 1 mM propyl gallate resulted in the insignificant increase in H2 yield by 15% suggesting that chlororespiration competes with H2 production to a lesser extent than mitorespiration. These data are in line with the observation that chlororespiratory PQ oxidase contributes relatively little to O2 consumption during S deprivation [12].

4.

Conclusion

In this work the mechanisms underlying H2 photoproduction by S starved C. reinhardtii were studied. Despite the fact that deficiency of sulfur essentially impairs PS II centers, PS II dependent H2 photoproduction remains a major pathway in starved cells. We showed that this observation can be

international journal of hydrogen energy 34 (2009) 9087–9094

explained by the noticeable decrease in the activity of type 2 NDH in S deprived cell which mediates a PS II independent pathway of H2 production. It is important to note that PS II activity does not contribute per se to the net reducing power in a cell under anaerobic conditions, because generation of high potential electrons by PS II should be compensated by equimolar consumption of the reducing equivalents in the respiration required to maintain anoxia. The mitorespiration was shown to be the main pathway of O2 removal in starved culture. Generally, H2 production by S starved green algae appears to be the way for the indirect utilization of the reductant accumulated in reactions of starch fermentation mainly via residual PS II activity. Thus, H2 production is coupled to the starch catabolism via interaction between residual activity of PS II producing O2 and at the same time donating electrons to the H2ase, and respiration, which removes photosynthetic O2 using reductants released in fermentation (see Fig. 1). The FQR dependent CET slows down H2 photoproduction by S straved cells, which can be due to the competition between FQR and H2ase for the reduced Fd and to the negative regulation of NDH by the CET – induced transmembrane DpH. Construction of mutants lacking FQR seems to be important in order to improve H2 production by S starved C. reinhardtii.

Acknowledgement This work was supported by the Russian Foundation of Basic Research (07-04-00222) and by the MK - 702.2009.4. The authors would like to thank Dr. E. Tyystja¨rvi for help in preparation of the manuscript.

references

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