[FeFe]-hydrogenase from green algae (HydA1) is the most efficient hydrogen (H2) producing enzyme in nature and of prime interest for (bio)technology. Its active site is a unique six-iron center (H-cluster) composed of a cubane cluster, [4Fe4S]H, cysteine-linked to a diiron unit, [2Fe]H, which carries unusual carbon monoxide (CO) and cyanide ligands and a bridging azadithiolate group. We have probed the molecular and electronic configurations of the H-cluster in functional oxidized, reduced, and super-reduced or CO-inhibited HydA1 protein, in particular searching for intermediates with iron-hydride bonds. Site-selective X-ray absorption and emission spectroscopy were used to distinguish between low- and high-spin iron sites in the two subcomplexes of the H-cluster. The experimental methods and spectral simulations were calibrated using synthetic model complexes with ligand variations and bound hydride species. Distinct X-ray spectroscopic signatures of electronic excitation or decay transitions in [4Fe4S]H and [2Fe]H were obtained, which were quantitatively reproduced by density functional theory calculations, thereby leading to specific H-cluster model structures. We show that iron-hydride bonds are absent in the reduced state, whereas only in the super-reduced state, ligand rotation facilitates hydride binding presumably to the Fe-Fe bridging position at [2Fe]H. These results are in agreement with a catalytic cycle involving three main intermediates and at least two protonation and electron transfer steps prior to the H2 formation chemistry in [FeFe]-hydrogenases.

The amount of light energy received by the photosynthetic reaction centers photosystem II (PSII) and photosystem I (PSI) is balanced through state transitions. Reversible phosphorylation of a light-harvesting antenna trimer (L-LHCII) orchestrates the association between L-LHCII and the photosystems, thus adjusting the amount of excitation energy received by the reaction centers. In this study, we identified the enzyme NUCLEAR SHUTTLE INTERACTING (NSI; AT1G32070) as an active lysine acetyltransferase in the chloroplasts of Intriguingly, knockout mutant plants were defective in state transitions, even though they had a similar LHCII phosphorylation pattern as the wild type. Accordingly, plants were not able to accumulate the PSI-LHCII state transition complex, even though the LHCII docking site of PSI and the overall amounts of photosynthetic protein complexes remained unchanged. Instead, the mutants showed a decreased Lys acetylation status of specific photosynthetic proteins including PSI, PSII, and LHCII subunits. Our work demonstrates that the chloroplast acetyltransferase NSI is needed for the dynamic reorganization of thylakoid protein complexes during photosynthetic state transitions.

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-a-acetylation (NTA) and e-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5-related N-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities.

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.

In humans and plants, N-terminal acetylation plays a central role in protein homeostasis, affects 80% of proteins in the cytoplasm and is catalyzed by five ribosome-associated Nacetyltransferases (NatA-E). Humans also possess a Golgi-associated NatF (HsNAA60) that is essential for Golgi integrity. Remarkably, NAA60 is absent in fungi and has not been identified in plants. Here we identify and characterize the first plasma membrane-anchored post-translationally acting N-acetyltransferase AtNAA60 in the reference plant Arabidopsis thaliana by the combined application of reverse genetics, global proteomics, live-cell imaging, microscale thermophoresis, circular dichroism spectroscopy, nano-differential scanning fluorometry, intrinsic tryptophan fluorescence and X-ray crystallography. We demonstrate that AtNAA60, like HsNAA60, is membrane-localized in vivo by an a-helical membrane anchor at its C-terminus, but in contrast to HsNAA60, AtNAA60 localizes to the plasma membrane. The AtNAA60 crystal structure provides insights into substrate-binding, the broad substrate specificity and the catalytic mechanism probed by structure-based mutagenesis. Characterization of the NAA60 loss-of-function mutants (naa60-1 and naa60-2) uncovers a plasma membrane-localized substrate of AtNAA60 and the importance of NAA60 during high salt stress. Our findings provide evidence for the plant-specific evolution of a plasma membrane-anchored N-acetyltransferase that is vital for adaptation to stress.

conceived and designed the experiments; A.W. and G.W.v.E. performed the field experiments; A.W. conducted the phenotypic analyses; A.W. and G.W.v.E. conducted the RNA sequencing experiment and analyzed the data; G.G. contributed the characterization of the mbd mutant plant; A.W. performed allelism tests, gene resequencing, quantitative PCR experiments, and phylogenetic analysis; G.K.K. conducted in situ hybridization and protein localization experiments; A.B. and I.F. performed protein activity tests; and A.W. wrote the article with help from G.W.v.E. and M.v.K. [OPEN] Articles can be viewed without a subscription.

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

N a-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. In plants, the biological function of NTA remains enigmatic. The dominant N-acetyltransferase (Nat) in Arabidopsis (Arabidopsis thaliana) is NatA, which cotranslationally catalyzes acetylation of ;40% of the proteome. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. In human (Homo sapiens), fruit fly (Drosophila melanogaster), and yeast (Saccharomyces cerevisiae), this core NatA complex interacts with NAA50 to form the NatE complex. While in metazoa, NAA50 has N-acetyltransferase activity, yeast NAA50 is catalytically inactive and positions NatA at the ribosome tunnel exit. Here, we report the identification and characterization of Arabidopsis NAA50 (AT5G11340). Consistent with its putative function as a cotranslationally acting Nat, AtNAA50-EYFP localized to the cytosol and the endoplasmic reticulum but also to the nuclei. We demonstrate that purified AtNAA50 displays N a-terminal acetyltransferase and lysine-«-autoacetyltransferase activity in vitro. Global N-acetylome profiling of Escherichia coli cells expressing AtNAA50 revealed conservation of NatE substrate specificity between plants and humans. Unlike the embryo-lethal phenotype caused by the absence of AtNAA10 and AtNAA15, loss of NAA50 expression resulted in severe growth retardation and infertility in two Arabidopsis transfer DNA insertion lines (naa50-1 and naa50-2). The phenotype of naa50-2 was rescued by the expression of HsNAA50 or AtNAA50. In contrast, the inactive ScNAA50 failed to complement naa50-2. Remarkably, loss of NAA50 expression did not affect NTA of known NatA substrates and caused the accumulation of proteins involved in stress responses. Overall, our results emphasize a relevant role of AtNAA50 in plant defense and development, which is independent of the essential NatA activity.