Some microalgae, such as , harbor a highly flexible photosynthetic apparatus capable of using different electron acceptors, including carbon dioxide (CO), protons, or oxygen (O), allowing survival in diverse habitats. During anaerobic induction of photosynthesis, molecular O is produced at photosystem II, while at the photosystem I acceptor side, the reduction of protons into hydrogen (H) by the plastidial [FeFe]-hydrogenases primes CO fixation. Although the interaction between H production and CO fixation has been studied extensively, their interplay with O produced by photosynthesis has not been considered. By simultaneously measuring gas exchange and chlorophyll fluorescence, we identified an O photoreduction mechanism that functions during anaerobic dark-to-light transitions and demonstrate that flavodiiron proteins (Flvs) are the major players involved in light-dependent O uptake. We further show that Flv-mediated O uptake is critical for the rapid induction of CO fixation but is not involved in the creation of the micro-oxic niches proposed previously to protect the [FeFe]-hydrogenase from O By studying a mutant lacking both hydrogenases (HYDA1 and HYDA2) and both Flvs (FLVA and FLVB), we show that the induction of photosynthesis is strongly delayed in the absence of both sets of proteins. Based on these data, we propose that Flvs are involved in an important intracellular O recycling process, which acts as a relay between H production and CO fixation.

SummaryThe global population is predicted to increase from ~7.3 billion to over 9 billion people by 2050. Together with rising economic growth, this is forecast to result in a 50% increase in fuel demand, which will have to be met while reducing carbon dioxide (CO 2) emissions by 50–80% to maintain social, political, energy and climate security. This tension between rising fuel demand and the requirement for rapid global decarbonization highlights the need to fast‐track the coordinated development and deployment of efficient cost‐effective renewable technologies for the production of CO 2 neutral energy. Currently, only 20% of global energy is provided as electricity, while 80% is provided as fuel. Hydrogen (H2) is the most advanced CO 2‐free fuel and provides a ‘common’ energy currency as it can be produced via a range of renewable technologies, including photovoltaic (PV), wind, wave and biological systems such as microalgae, to power the next generation of H2 fuel cells. Microalgae production systems for carbon‐based fuel (oil and ethanol) are now at the demonstration scale. This review focuses on evaluating the potential of microalgal technologies for the commercial production of solar‐driven H2 from water. It summarizes key global technology drivers, the potential and theoretical limits of microalgal H2 production systems, emerging strategies to engineer next‐generation systems and how these fit into an evolving H2 economy.

Ferredoxins are soluble iron sulphur proteins which function as electron donors in a number of metabolic pathways in a broad range of organisms. In photosynthetic organisms, PETF, or ferredoxin 1 (FDX1), is the most studied ferredoxin due to its essential role in photosynthesis, where it transfers electrons from photosystem I to ferredoxin-NADP oxidoreductase. However, PETF can also transfer electrons to a large number of other proteins. One important PETF electron acceptor found in green microalgae is the biologically and biotechnologically important [FeFe]-hydrogenase HYDA, which catalyses the production of molecular hydrogen (H) from protons and electrons. The interaction between PETF and HYDA is of considerable interest, as PETF is the primary electron donor to HYDA and electron supply is one of the main limiting factors for H production on a commercial scale. Although there is no three dimensional structure of the PETF-HYDA complex available, protein variants, nuclear magnetic resonance titration studies, molecular dynamics and modelling have provided considerable insight into the residues essential for forming and maintaining the interaction. In this review, we discuss the most recent findings with regard to ferredoxin-HYDA interactions and the evolution of the various Chlamydomonas reinhardtii ferredoxin isoforms. Finally, we provide an outlook on new PETF-based biotechnological approaches for improved H production efficiencies.

The [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii is particularly amenable to biochemical and biophysical characterization because the H-cluster in the active site is the only inorganic cofactor present. Herein, we present the complete chemical incorporation of the H-cluster into the HYDA1-apoprotein scaffold and, furthermore, the successful replacement of sulfur in the native [4FeH ] cluster with selenium. The crystal structure of the reconstituted pre-mature HYDA1[4Fe4Se]H protein was determined, and a catalytically intact artificial H-cluster variant was generated upon in vitro maturation. Full hydrogen evolution activity as well as native-like composition and behavior of the redesigned enzyme were verified through kinetic assays, FTIR spectroscopy, and X-ray structure analysis. These findings reveal that even a bioinorganic active site with exceptional complexity can exhibit a surprising level of compositional plasticity.

The [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii is particularly amenable to biochemical and biophysical characterization because the H-cluster in the active site is the only inorganic cofactor present. Herein, we present the complete chemical incorporation of the H-cluster into the HYDA1-apoprotein scaffold and, furthermore,t he successful replacement of sulfur in the native [4Fe H ]cluster with selenium. The crystal structure of the reconstituted pre-mature HYDA1-[4Fe4Se] H protein was determined, and ac atalytically intact artificial H-cluster variant was generated upon in vitro maturation. Full hydrogen evolution activity as well as native-like composition and behavior of the redesigned enzyme were verified through kinetic assays,FTIR spectroscopy, and X-ray structure analysis.T hese findings reveal that even ab ioinorganic active site with exceptional complexity can exhibit asurprising level of compositional plasticity.With turnover frequencies of up to 10 4 molecules of dihydrogen (H 2 )p er second, [FeFe]-hydrogenases are the fastest known biocatalysts for the reduction of protons to H 2 . [1] There are several types of [FeFe]-hydrogenases,w hich differ not only in protein structure and composition, but also in the number of accessory [FeS]-clusters,w hich act as an electron (e À )relay between the e À -mediator docking site and the active center (H-cluster) of the enzyme. [2] TheH-cluster is ac omplex inorganic cofactor, consisting of ac ysteine-coordinated [4Fe4S]-cluster ([4Fe H ]) linked to au nique [2Fe2S]subcluster ([2Fe H ]) with three CO and two CN À ligands.A n azadithiolate (adt =-S-CH 2 -NH-CH 2 -S-) ligand, bridging both Fe sites in [2Fe H ]( proximal (Fe p )a nd distal (Fe d )r elative to the [4Fe H ]-moiety) is essential for fast proton shuttling to and from the vacant ligand site at Fe d , [3] where proton reduction and H 2 oxidation occur ( Figure 1A). [1c, 4] Owing to its structural simplicity,t he monomeric hydrogenase,H YDA1, from the unicellular green alga Chlamydomonas reinhardtii,is amodel enzyme for studying the catalytic features of [FeFe]hydrogenases.A st he H-cluster is the only bioinorganic