Discovery of Two Novel Radical S-Adenosylmethionine Proteins Required for the Assembly of an Active [Fe] Hydrogenase

Abstract

To identify genes necessary for the photoproduction of H 2 in Chlamydomonas reinhardtii, random insertional mutants were screened for clones unable to produce H 2 . One of the identified mutants, denoted hydEF-1, is incapable of assembling an active [Fe] hydrogenase. Although the hydEF-1 mutant transcribes both hydrogenase genes and accumulates full-length hydrogenase protein, H 2 production activity is not observed. The HydEF protein contains two unique domains that are homologous to two distinct prokaryotic proteins, HydE and HydF, which are found exclusively in organisms containing [Fe] hydrogenase. In the C. reinhardtii genome, the HydEF gene is adjacent to another hydrogenase-related gene, HydG. Hydrogen has enormous potential to serve as a non-polluting fuel, alleviating the environmental and political concerns associated with fossil energy utilization. Among the most efficient H 2 -generating catalysts known are the [Fe] hydrogenase enzymes, which are found in numerous microorganisms, including the photosynthetic green alga Chlamydomonas reinhardtii (1, 2). Our research, aimed at identifying accessory proteins required for H 2 production in C. reinhardtii, has resulted in the discovery of two [Fe] hydrogenase assembly genes. These genes are essential for the formation of an active [Fe] hydrogenase and are conserved among sequenced organisms containing [Fe] hydrogenase enzymes.Algal H 2 production was first reported by Hans Gaffron and co-workers in seminal experiments performed over 60 years ago (3). Photosynthetic green algae are unique in that H 2 production by [Fe] hydrogenases is coupled directly to water oxidation through photosystem II and the photosynthetic electron transport chain, providing the means to generate H 2 using sunlight. In the past 5 years, great strides have been made in sustaining H 2 photoproduction activity in C. reinhardtii (4, 5), and intensive research continues to probe novel ways to use this and other photosynthetic organisms to generate renewable H 2 .Hydrogenases containing metallo-catalytic clusters occur as [NiFe] or [Fe]-only enzymes (2, 6, 7). These two forms are phylogenetically distinct (8), which suggests that hydrogenase function is the result of convergent evolution (2). Although [NiFe] and [Fe] hydrogenases are genetically unrelated, similarities between the proteins do exist. First, the active sites of both enzymes contain CO and CN ligands, and, second, each active site contains a binuclear metal center. In general, [NiFe] hydrogenases catalyze H 2 uptake, whereas [Fe] hydrogenases tend to catalyze H 2 evolution (9). Furthermore, the turnover number of [Fe] hydrogenases is 10 -100 times higher than that of [NiFe] hydrogenases (9), making the former one of the most efficient H 2 production catalysts known. Hydrogenases containing NiFeSe (10) and a unique class of hydrogenases containing a single Fe atom bound to a co-factor have also been reported (11).The [NiFe] hydrogenases are found in a variety of anaerobic and facultative archaea, eubacteria, a...

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii

“…Gas exchange measurements showed that H 2 evolution occurs prior to CO 2 fixation upon illumination (Cournac et al, 2002). At light onset after a prolonged period in dark anoxic conditions, the photosynthetic electron flow is mainly a LEF toward hydrogenase , and lack of hydrogenase activity in hydrogenase maturation factor EF (hydEF) mutant strain deficient in hydrogenases maturation (Posewitz et al, 2004) induces a lag in induction of PSII activity . In cyanobacteria, the bidirectional Ni-Fe hydrogenase might also work as an electron valve for disposal of electrons generated at the onset of illumination of cells (Cournac et al, 2004) or when excess electrons are generated during photosynthesis, preventing the slowing of the electron transport chain under stress conditions (Appel et al, 2000;Carrieri et al, 2011).…”

Metallocofactor assembly for [FeFe]-hydrogenases

“…HydE is a member of the RS family and is essential for [FeFe]-hydrogenase maturation [6,29]. Several years ago, structures of TmHydE were reported from a crystal form that diffracted to high resolution (1.25 Å) [30].…”

“…In contrast, the [2Fe] H subcluster is held by a single protein ligand, a cysteine residue, but contains three carbon monoxides, two cyanides and an azadithiolate ligand [5]. The contrast between the sub-clusters extends to their assembly (Figure 1a), as the [4Fe-4S] H subcluster is synthesized and inserted by general Fe-S cluster biosynthetic machinery, however biosynthesis and insertion of the [2Fe] H subcluster requires the activity of highly specific maturase enzymes, HydE, HydG and HydF [6].…”

In Vivo EPR Characterization of Semi‐Synthetic [FeFe] Hydrogenases

“…In the case of [FeFe] hydrogenase (HydA), the reaction is catalyzed by the H‐cluster, which features a [4Fe4S] cluster coupled to an organometallic [2Fe] subsite (Figure 1 a) 2, 3, 4, 5. It is now well established that the biosynthesis of the H‐cluster is a two‐step process, in which the assembly of the [2Fe] subsite requires three HydA specific maturation proteins 6, 7, 8. The catalytic mechanism of this unique cofactor has been intensively studied and several catalytically active and non‐active states of the H‐cluster have been characterized 1, 4, 9, 10, 11, 12.…”