Limiting steps of hydrogen production in Chlamydomonas reinhardtii and Synechocystis PCC 6803 as analysed by light-induced gas exchange transients

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Abstract

In the cyanobacterium Synechocystis PCC 6803 and in the microalga Chlamydomonas reinhardtii, transient hydrogen photo-production is observed when cells are exposed to light in anoxia. We measured changes in H2, O2, and CO2 concentrations using time-resolved mass spectrometry in wild-type and mutant strains of Chlamydomonas and Synechocystis. In both organisms, non-photochemical reduction of the plastoquinone pool is shown to contribute to the initial H2 photo-production. This pathway, which does not produce O2, exhibits a low rate in normal conditions. From the effect of the uncoupler FCCP, we conclude that PS II-independent H2 production in Chlamydomonas is limited by the trans-thylakoidal proton gradient. In Synechocystis, from the study of a mutant deficient in the NDH-1 complex (M55), we conclude that PS II-independent H2 production is limited by recycling of NAD(P)H through the NDH-1 complex. Based on these conclusions, we propose strategies for optimising H2 photo-production in these organisms.

Introduction

Among oxygenic photosynthetic organisms, many species of microalgae and cyanobacteria are able to produce H2 thanks to the activity of an hydrogenase. In Chlamydomonas reinhardtii, the hydrogenase is of the Fe-only type [1], [2], [3]; it is nuclear-encoded, located in the chloroplast and interacts in a reversible manner with ferredoxin (Fd) as a redox partner [3]. In Chlamydomonas, transcription of the hydrogenase gene is induced in anaerobiosis, [3], and the enzyme needs anaerobiosis to become active. H2 driven CO2 photo-reduction [4] or H2 photo-production have long been described in microalgae [5]. H2 photo-production is a transitory process which is inhibited by O2 produced by photosystem II (PS II). The pioneering work of Gaffron and Rubin [5] has proposed that H2 photo-production might derive either from the photolysis of water or from stored organic material. The existence of a water photolysis-independent pathway was further confirmed by the effect of PS II inhibitors such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) [6] or uncouplers [7]. By studying the effects of DCMU, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and respiratory substrates on 33 algal strains, Healey showed that the relative contribution of PS II-dependent and -independent pathways of H2 photo-production could be very different from one species to the other [8]. The activity of the PS II-independent pathway has been related to the mobilisation of carbohydrates [9] and in some cases of protein reserves [10]. This pathway does not produce O2 and therefore allows sustained H2 production [10]. In the absence of PS II, H2 production occurs, thanks to the existence of alternative ways of plastoquinone (PQ) reduction by stromal components [10], [11], [12]. It has been recently reported that Chlamydomonas cells deprived from sulfur could sustain hydrogen production in the light due to these alternative pathways [10]. Different electron carriers such as NAD(P)H [11], succinate [13] or reduced ferredoxin [14] have been proposed to mediate the non-photochemical reduction of the plastoquinone pool, and therefore to be potential electron sources for PS II-independent H2 production in chloroplasts. In higher plants, the discovery of chloroplast genes (ndh genes) showing homologies with genes encoding subunits of the mitochondrial NADH-ubiquinone oxidoreductase (complex I or NDH-1), was taken as an evidence for the existence of a NADH dehydrogenase complex in chloroplasts [12, and references therein]. Interestingly, although non-photochemical reduction of PQ occurring during chlororespiration has been extensively studied in the green alga Chlamydomonas, ndh genes are absent from its chloroplast genome [12]. It has been proposed that alternative NADH dehydrogenase (NDH-2) activities such as the one evidenced in chloroplasts of higher plants [15] could account for PS II-independent PQ reduction in Chlamydomonas [12], [16].

Synechocystis sp. strain PCC 6803 contains a reversible/bidirectional hydrogenase able to both take up and produce H2. This multimeric cytoplasmic reversible NAD(P)-reducing [NiFe]-hydrogenase is composed of two moieties [17]. The diaphorase moiety, encoded by the hoxE, hoxU and hoxF genes, is homologous to complex I subunits of mitochondrial and bacterial respiratory chains and contains NAD(P), FMN and FeS binding sites. The [NiFe]-hydrogenase moiety is encoded by the hoxY and hoxH genes [17], [18], [19]. Since this strain has no nitrogenase [19], no hydrogenase of the HupSL type [20] and since no hydrogenase activity could be detected in a hoxH deletion mutant [21], the bidirectional HoxEFUYH hydrogenase is probably the only enzyme involved in H2 uptake or H2 production in this organism. In cyanobacteria, photosynthetic electron transport takes place in thylakoid membranes, while respiratory electron transport occurs in both thylakoid and cytoplasmic membranes [22], [23]. A transitory H2 photo-production linked to the activity of hydrogenase has been observed in Oscillatoria chalybea [24] and Synechocystis PCC 6803 [25]. This production was of small amplitude and occurred on a very short time scale in the absence of chemicals addition, but was stimulated by the uncoupler CCCP [25].

In this study, we have used an on-line mass spectrometer to measure H2, CO2, and O2 exchange during dark-light transients in whole cells of Synechocystis sp. PCC 6803 and Chlamydomonas reinhardtii with the aim to determine the limiting steps of hydrogen production in these organisms. From the effects of DCMU, uncouplers and mutations, we conclude that in Chlamydomonas PS II-independent H2 production is limited by the proton electrochemical gradient and likely involves a NDH-2 dehydrogenase, while in Synechocystis this activity is limited by recycling of NAD(P)H through the NDH-1 complex.

Section snippets

Strains and growth conditions

Cyanobacterial cells (Synechocystis PCC 6803 and the mutant strain M55) were grown autotrophically in liquid aerated modified Allen's medium [26] at 34°C, under continuous illumination using two fluorescent tubes providing an average light intensity of 70μEm−2s−1. The high CO2 requiring mutant M55 (ndhB::Kmr cartridge), kindly provided by Dr. T. Ogawa, was grown in a medium supplemented with 50μgml−1 kanamycin and 10mMNaHCO3 [27]. Chlamydomonas reinhardtii cells were grown on a

Anaerobic H2 production in the dark

When Synechocystis cells were placed in the dark in a closed vessel, anaerobiosis was rapidly reached due to the activity of respiration. A significant H2 production was immediately observed upon reaching anaerobiosis and a steady state rate was established after 15min of anaerobiosis (Fig. 1A). This is in line with the finding that Synechocystis hydrogenase is expressed under aerobic conditions [21]. When the same experiment was performed with Chlamydomonas cells, H2 production did not start

Discussion

Two types of oxygenic photosynthetic microorganisms, the green alga Chlamydomonas reinhardtii and the cyanobacterium Synechocystis PCC 6803, have been compared for their capacity to evolve H2 in vivo using a mass spectrometer and a membrane inlet system to measure H2, CO2, and O2 exchange. Both organisms were found to evolve H2 upon illumination of anaerobic cell suspensions, but the production was low and rapidly stopped in the light. We have used mutant strains and electron transport

References (38)

  • H. Gaffron et al.

    Fermentative and photochemical production of hydrogen in algae

    J Gen Physiol

    (1942)
  • F.B. Abeles

    Cell-free hydrogenase from Chlamydomonas

    Plant Physiol

    (1964)
  • H. Kaltwasser et al.

    Light-dependent hydrogen evolution by Scenedesmus

    Planta

    (1969)
  • F.P. Healey

    Hydrogen evolution by several algae

    Planta

    (1970)
  • U. Klein et al.

    Fermentative metabolism of hydrogen-evolving Chlamydomonas moewusii

    Plant Physiol

    (1978)
  • A. Melis et al.

    Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii

    Plant Physiol

    (2000)
  • D. Godde et al.

    NADH as electron donor for the photosynthetic membrane of Chlamydomonas reinhardtii

    Arch Microbiol

    (1980)
  • G. Peltier et al.

    Chlororespiration

    Annu Rev Plant Physiol Plant Mol Biol

    (2002)
  • K.O. Willeford et al.

    Evidence for chloroplastic succinate dehydrogenase participating in the chloroplastic respiratory and photosynthetic electron transport chains of Chlamydomonas reinhardtii

    Plant Physiol

    (1989)
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