Sigrun Rumpel

Hybrid [FeFe]-Hydrogenases with Modified Active Sites Show Remarkable Residual Enzymatic Activity

[FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme.

Enhancing hydrogen production of microalgae by redirecting electrons from photosystem I to hydrogenase

Photohydrogen generation in microalgae is catalysed by hydrogenases, which receive electrons from photosystem I via the ferredoxin PETF. The dominant acceptor of photosynthetic electrons is, however, ferredoxin-NADP+-oxidoreductase (FNR). By utilizing targeted ferredoxin and FNR variants in a light-dependent competition assay, electrons can be redirected to the hydrogenase yielding a five-fold enhanced hydrogen evolution activity.

Structure and DNA-binding properties of the cytolysin regulator CylR2 from Enterococcus faecalis

Enterococcus faecalis is one of the major causes for hospital‐acquired antibiotic‐resistant infections. It produces an exotoxin, called cytolysin, which is lethal for a wide range of Gram‐positive bacteria and is toxic to higher organisms. Recently, the regulation of the cytolysin operon was connected to autoinduction by a quorum‐sensing mechanism involving the CylR1/CylR2 two‐component regulatory system. We report here the crystal structure of CylR2 and its properties in solution as determined by heteronuclear NMR spectroscopy. The structure reveals a rigid dimer containing a helix–turn–helix DNA‐binding motif as part of a five‐helix bundle that is extended by an antiparallel β‐sheet. We show that CylR2 is a DNA‐binding protein that binds specifically to a 22 bp fragment of the cytolysin promoter region. NMR chemical shift perturbation experiments identify surfaces involved in DNA binding and are in agreement with a model for the CylR2/DNA complex that attributes binding specificity to a complex network of CylR2/DNA interactions. Our results propose a mechanism where repression is achieved by CylR2 obstruction of the promoter preventing biosynthesis of the cytolysin operon transcript.

Structural Insight into the Complex of Ferredoxin and [FeFe] Hydrogenase from Chlamydomonas reinhardtii.

The transfer of photosynthetic electrons by the ferredoxin PetF to the [FeFe] hydrogenase HydA1 in the microalga Chlamydomonas reinhardtii is a key step in hydrogen production. Electron delivery requires a specific interaction between PetF and HydA1. However, because of the transient nature of the electron‐transfer complex, a crystal structure remains elusive. Therefore, we performed protein–protein docking based on new experimental data from a solution NMR spectroscopy investigation of native and gallium‐substituted PetF. This provides valuable information about residues crucial for complex formation and electron transfer. The derived complex model might help to pinpoint residue substitution targets for improved hydrogen production.

Assignment-free solution NMR method reveals CesT as an unswapped homodimer

The X‐ray structure of the homodimeric chaperone CesT is the only structure among the type three secretion system (TTSS) chaperones that shows a domain swap. This swap has potential importance for the mechanism of effector translocation through a TTSS. Here we present two nuclear magnetic resonance strategies exploiting pre‐existing structural models and residual dipolar couplings (RDCs), which reveal the unswapped 35.4‐kDa dimer to be present in solution. Particularly efficient is the discrimination of a swapped and unswapped structural state performed simultaneously to automatic backbone assignment using only HN‐RDCs and carbonyl backbone chemical shifts. This direct approach may prove to be generally useful to rapidly differentiate two structural models.

Spectroscopic investigations of a semi-synthetic [FeFe] hydrogenase with propane di-selenol as bridging ligand in the binuclear subsite: comparison to the wild type and propane di-thiol variants

Abstract[FeFe] Hydrogenases catalyze the reversible conversion of H2 into electrons and protons. Their catalytic site, the H-cluster, contains a generic [4Fe–4S]H cluster coupled to a [2Fe]H subsite [Fe2(ADT)(CO)3(CN)2]2−, ADT = µ(SCH2)2NH. Heterologously expressed [FeFe] hydrogenases (apo-hydrogenase) lack the [2Fe]H unit, but this can be incorporated through artificial maturation with a synthetic precursor [Fe2(ADT)(CO)4(CN)2]2−. Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH2 (PDT = propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with simplified redox behavior. Here, we study the effect of sulfur-to-selenium (S-to-Se) substitution in the bridging PDT ligand incorporated in the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii using magnetic resonance (EPR, NMR), FTIR and spectroelectrochemistry. The resulting HydA1-PDSe enzyme shows the same redox behavior as the parent HydA1-PDT. In addition, a state is observed in which extraneous CO is bound to the open coordination site of the [2Fe]H unit. This state was previously observed only in the native enzyme HydA1-ADT and not in HydA1-PDT. The spectroscopic features and redox behavior of HydA1-PDSe, resulting from maturation with [Fe2(PDSe)(CO)4(CN)2]2−, are discussed in terms of spin and charge density shifts and provide interesting insight into the electronic structure of the H-cluster. We also studied the effect of S-to-Se substitution in the [4Fe–4S] subcluster. The reduced form of HydA1 containing only the [4Fe–4Se]H cluster shows a characteristic S = 7/2 spin state which converts back into the S = 1/2 spin state upon maturation with a [2Fe]–PDT/ADT complex.

1H NMR Spectroscopy of [FeFe] Hydrogenase: Insight into the Electronic Structure of the Active Site

The [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii has been studied using 1H NMR spectroscopy identifying the paramagnetically shifted 1H resonances associated with both the [4Fe-4S]H and the [2Fe]H subclusters of the active site “H-cluster”. The signal pattern of the unmaturated HydA1 containing only [4Fe-4S]H is reminiscent of bacterial-type ferredoxins. The spectra of maturated HydA1, with a complete H-cluster in the active Hox and the CO-inhibited Hox–CO state, reveal additional upfield and downfield shifted 1H resonances originating from the four methylene protons of the azadithiolate ligand in the [2Fe]H subsite. The two axial protons are affected by positive spin density, while the two equatorial protons experience negative spin density. These protons can be used as important probes sensing the effects of ligand-binding to the catalytic site of the H-cluster.

Direct Detection of the Terminal Hydride Intermediate in [FeFe] Hydrogenase by NMR Spectroscopy

Hydride state intermediates are known to occur in various hydrogen conversion enzymes, including the highly efficient [FeFe] hydrogenases. The intermediate state involving a terminal iron-bound hydride has been recognized as crucial for the catalytic mechanism, but its occurrence has up to now eluded unequivocal proof under (near) physiological conditions. Here we show that the terminal hydride in the [FeFe] hydrogenase from Chlamydomonas reinhardtii can be directly detected using solution 1H NMR spectroscopy at room temperature, opening new avenues for detailed in situ investigations under catalytic conditions.

Rational redesign of the ferredoxin-NADP+-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production

Utilization of electrons from the photosynthetic water splitting reaction for the generation of biofuels, commodities as well as application in biotransformations requires a partial rerouting of the photosynthetic electron transport chain. Due to its rather negative redox potential and its bifurcational function, ferredoxin at the acceptor side of Photosystem 1 is one of the focal points for such an engineering. With hydrogen production as model system, we show here the impact and potential of redox partner design involving ferredoxin (Fd), ferredoxin-oxido-reductase (FNR) and [FeFe]‑hydrogenase HydA1 on electron transport in a future cyanobacterial design cell of Synechocystis PCC 6803. X-ray-structure-based rational design and the allocation of specific interaction residues by NMR-analysis led to the construction of Fd- and FNR-mutants, which in appropriate combination enabled an about 18-fold enhanced electron flow from Fd to HydA1 (in competition with equimolar amounts of FNR) in in vitro assays. The negative impact of these mutations on the Fd-FNR electron transport which indirectly facilitates H2 production (with a contribution of ≤42% by FNR variants and ≤23% by Fd-variants) and the direct positive impact on the Fd-HydA1 electron transport (≤23% by Fd-mutants) provide an excellent basis for the construction of a hydrogen-producing design cell and the study of photosynthetic efficiency-optimization with cyanobacteria.