Improved oxygen tolerance of the Synechocystis sp. PCC 6803 bidirectional hydrogenase by site-directed mutagenesis of putative residues of the gas diffusion channel
Introduction
Cyanobacteria are promising organisms to develop clean and sustainable hydrogen production, because several of these photosynthetic organisms, which essentially rely on solar energy as a source of energy and water as a source of electrons, possess H2-evolving enzymes, namely hydrogenases or nitrogenases [1], [2], [3], [4], [5], [6]. Hydrogenases catalyze the simplest chemical reaction, the interconversion of dihydrogen into two protons and two electrons [7]. Cyanobacteria may contain two types of hydrogenases, an uptake [NiFe] hydrogenase catalyzing the consumption of hydrogen (predominantly found in N2-fixing species where it recycles H2 produced by nitrogenase), and a bidirectional [NiFe] hydrogenase which has the capacity to both oxidize and produce hydrogen. Synechocystis sp. PCC 6803, a facultative non-N2-fixing photosynthetic cyanobacterium, holds only the bidirectional [NiFe] hydrogenase which has been studied for more than a decade [8], [9], [10]. The Synechocystis genome is fully sequenced [11] and numerous molecular tools are available [12], [13], [14], [15], [16], making it a good model for studies related to photosynthesis mechanisms and hydrogen production. However, the high sensitivity of the bidirectional hydrogenase towards dioxygen is a major hurdle impairing biotechnological applications in the field of biohydrogen photoproduction. Present research efforts aim at improving O2 tolerance of cyanobacterial hydrogenases [5], [17] or at expressing O2 tolerant hydrogenases in these organisms [18], [19].
Recent progress in O2 tolerance improvement of [NiFe] hydrogenases stemmed from comparative structural analysis and site-directed mutagenesis studies. Crystallographic analysis of the Desulfovibrio fructosovorans [NiFe] hydrogenase [20] showed the existence of a hydrophobic gas channel that could be a leading path from the external medium to the active site deeply buried within the hydrogenase. Structural studies resulted in the identification of critical amino acids involved in the conformation and the reactivity of the active site. Residues Val74, Val117 and Leu122 from the catalytic subunit of the D. fructosovorans dimeric hydrogenase were found to delineate the hydrophobic channel at close vicinity of the active site [20] and were identified as interesting targets for site-directed mutagenesis [21], [22], [23], [24]. The purple bacterium Rhodobacter capsulatus holds a protein (HupUV) that exhibits typical features of [NiFe] hydrogenases. It has very low enzymatic activity, but is remarkably resistant to inactivation by O2 [25], [26]. HupUV functions as an H2 sensor which regulates expression of the uptake hydrogenase HupSL. Sequence alignments between the large subunit (HupV) and the D. fructosovorans hydrogenase large subunit (HydB) revealed that Val74 and Leu122 of HydB are substituted by Ile65 and Phe113, respectively, in HupV. Substitution with these bulkier residues was proposed to narrow the gas channel [20], creating a “molecular sieve” effect responsible for part of the O2 tolerance of the sensor [27]. In line with this hypothesis, substitutions of Ile65 and Phe113 to Val and Leu in HupV generated a still functional, but O2-sensitive HupUV hydrogenase, which was attributed to an enlargement of the gas channel [28]. The same results were obtained with studies of the corresponding mutants of the O2-tolerant H2 sensor of Ralstonia eutropha [29]. Conversely, introduction of Ile and Phe at positions 74 and 122 in D. fructosovorans HydB restricted hydrogen diffusion [27] but did not relieve enzyme sensitivity towards O2 [23]. Different types of amino acid substitutions have been achieved in the D. fructosovorans enzyme at these positions, and their effects on gas diffusion and O2 sensitivity assessed [24]. Interestingly, introduction of a methionine residue at position 74 allowed a prolonged activity under aerobic conditions. It was hypothesized that two mechanisms could altogether contribute to this increased tolerance: (i) creation of a molecular sieve effect, reducing the access of O2 to the active site and (ii) chemical interaction of oxygen species with methionine sulfur, which might facilitate oxygen scavenging from the active site [23], [27]. Additionally, the presence of Met at position 122 produced a stimulation of the spontaneous enzyme reactivation after O2 exposure [23]. Furthermore, the modification of gas diffusion characteristics was found to alter the directionality of the enzyme, as the gas channel restriction induced a stronger decrease in H2 production than in H2 uptake rate [30]. Although the original declared purposes of this approach was indeed to target cyanobacterial hydrogenases, D. fructosovorans was the only model on which its feasibility was shown, and the actual demonstration that it could be transposed to the cyanobacterial model has never been made up to now.
Therefore, the present study aims at improving the O2 tolerance of the Synechocystis [NiFe] hydrogenase by developing a strategy inspired by that previously developed for D. fructosovorans. Based on sequence alignments between the catalytic subunits HoxH from the Synechocystis hydrogenase, HydB from D. fructosovorans and HupV from R. capsulatus, a structural modeling of the Synechocystis hydrogenase was carried out, resulting in the identification of three conserved amino acid residues (Ile64, Leu107 and Leu112) putatively involved in shaping the terminus of the hydrophobic channel inside the Synechocystis enzyme. These residues were chosen as targets for amino acid substitutions in order to improve O2 tolerance of the Synechocystis hydrogenase. We show that mutations of these conserved residues affect internal gas diffusion and that a Met substitution at position I64 significantly improves O2 tolerance of the cyanobacterial hydrogenase. To our knowledge, this is the first successful report of a site-directed mutagenesis study to improve O2 tolerance of a cyanobacterial hydrogenase.
Section snippets
Cell growth
Escherichia coli strains were grown on LB medium at 37 °C. Wild-type and mutant strains of Synechocystis sp. PCC 6803 were grown autotrophically in modified liquid Allen's medium [31], [32] or ready-to-use BG11 medium (Cyanobacteria BG-11 Freshwater Solution, Sigma–Aldrich, St Louis, Missouri, USA) supplemented with 20 mM Hepes pH 8.2, in 250 mL flasks containing the appropriate antibiotic (chloramphenicol (Cm): 25 μg mL−1; spectinomycin (Sp): 25 μg mL−1; streptomycin (Sm): 5 μg mL−1). Flasks
Structural modeling of Synechocystis hydrogenase
HoxH and HoxY subunits of the Synechocystis sp. PCC 6803 hydrogenase are part of a pentameric HoxEFUHY complex in which the HoxF subunit has diaphorase activity. The HoxEFUHY subunits display significant sequence homology with subunits 2, 1, 3, 4 and 6 of the NADH:ubiquinone oxidoreductase (complex 1), respectively. Because the structure of the latter is known at medium resolution, it may be used as a basis to model the pentameric Synechocystis complex [37]. In addition, high-resolution
Discussion
Mutagenesis of targeted amino acids at the vicinity of the active site has been shown to widely modify internal gas diffusion and reactivity towards O2 in D. fructosovorans, R. eutropha and R. capsulatus [NiFe] hydrogenases [21], [22], [23], [24], [28], [29], [43]. In the present work, based on homology modeling of the Synechocystis [NiFe] hydrogenase, the structure of which is not available so far, we identified and further targeted for site-directed mutagenesis residues of the Synechocystis
Acknowledgments
We thank Marc Rousset and Sébastien Dementin (BIP-CNRS, Marseille) for helpful discussions, Alexandra Dubini (NREL, Colorado, USA) for critical reading of the manuscript and Jean-Marc Adriano for technical support. Support was provided by the HélioBiotec platform, funded by the European Union (European Regional Development Fund, N° 1944-32670), the Région Provence Alpes Côte d’Azur (DEB 09-621), the French Ministry of Research, and the CEA (Commissariat à l’Energie Atomique et aux Energies
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Present address: IRD, UMR Eco&Sols, Bat 12, 2 Place Viala, 34060 Montpellier Cedex 2, France.