Wasserstoffkatalyse in Mikroalgen

Hydrogenasen in Grünalgen katalysieren die Abgabe von Wasserstoff. Wie läuft das auf molekularer Ebene ab? Isotopenmarkierung und Infrarotspektroskopie helfen, diese Frage zu beantworten

The molecular proceedings of biological hydrogen turnover

Over the past two decades, the bioinorganic chemistry of hydrogenases has attracted much interest from basic and applied research. Hydrogenases are highly efficient metalloenzymes that catalyze the reversible reduction of protons to molecular hydrogen (H2) in all domains of life. Their iron- and nickel-based cofactors represent promising blueprints for the design of biomimetic, synthetic catalysts. In this Account, we address the molecular proceedings of hydrogen turnover with [FeFe]-hydrogenases. The active site cofactor of [FeFe]-hydrogenases (“H-cluster”) comprises a unique diiron complex linked to a [4Fe-4S] cluster via a single cysteine. Since it was discovered that a synthetic analogue of the diiron site can be incorporated into apoprotein in vitro to yield active enzyme, significant progress has been made toward a comprehensive understanding of hydrogenase catalysis. The diiron site carries three to four carbon monoxide (CO) and two cyanide (CN–) ligands that give rise to intense infrared (IR) absorption bands. These bands are sensitive reporters of the electron density across the H-cluster, which can be addressed by infrared spectroscopy to follow redox and protonation changes at the cofactor. Synthetic variation of the metal-bridging dithiolate ligand at the diiron site, as well as site-directed mutagenesis of amino acids, provides access to the proton pathways toward the cofactor. Quantum chemical calculations are employed to specifically assign IR bands to vibrational modes of the diatomic ligands and yield refined H-cluster geometries. Here, we provide an overview of recent research on [FeFe]-hydrogenases with emphasis on experimental and computational IR studies. We describe advances in attenuated total reflection Fourier transform infrared spectroscopy (ATR FTIR) and protein film electrochemistry, as well as density functional theory (DFT) calculations. Key cofactor species are discussed in terms of molecular geometry, redox state, and protonation. Isotope editing is introduced as a tool to evaluate the cofactor geometry beyond the limits of protein crystallography. In particular, the role of proton-coupled electron transfer (PCET) in the generation of catalytically relevant redox species is addressed. We propose that site-selective protonation of the H-cluster biases surplus electrons either to the [4Fe-4S] cluster or to the diiron site. Protonation of the [4Fe-4S] cluster prevents premature reduction at the diiron site and stabilizes a reactive, terminal hydride. The observed H-cluster species are assigned to rapid H2 conversion or to reactions possibly involved in activity regulation and cellular H2 sensing. In the catalytic cycle of [FeFe]-hydrogenases, an H-cluster geometry is preserved that features a bridging CO ligand. PCET levels the redox potential for two steps of sequential cofactor reduction. The concept of consecutive PCET at a geometrically inert cofactor with tight control of electron and proton localization may inspire the design of a novel generation of biomimetic catalysts for the production of H2 as a fuel

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis

Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H2). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN)2CO metallo-center and a small subunit with three iron–sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN)2CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor- containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase

biodiversity and spectroscopic investigations

Hydrogenases are redox enzymes that catalyze the conversion of protons and molecular hydrogen (H2). Based on the composition of the active site cofactor, the monometallic [Fe]-hydrogenase is distinguished from the bimetallic [NiFe]- or [FeFe]-hydrogenase. The latter has been reported with particularly high turnover activities for both H2 release and H2 oxidation, notably at neutral pH, ambient temperatures, and negligible electric overpotential. Due to these properties, [FeFe]-hydrogenase represents the “gold standard” in enzymatic hydrogen turnover. Understanding hydrogenase chemistry is crucial for the design of transition metal complexes that serve as potentially sustainable proton reduction or H2 oxidation catalysts, e.g., in electrolytic devices or fuel cells. However, even 20 years after the crystal structures of [FeFe]-hydrogenase have been published, several aspects of biological hydrogen turnover are heatedly discussed. In this perspective, we give an overview on how the diversity of naturally occurring and artificially prepared, semisynthetic [FeFe]-hydrogenases deepens our understanding of hydrogenase chemistry. In parallel, we cover recent results from biophysical techniques that go beyond the scope of conventional X-ray diffraction, EPR, and FTIR spectroscopy. Taking into account both proton transfer and electron transfer as well as the notorious sensitivity of [FeFe]-hydrogenase toward carbon monoxide, the discussion further touches upon the molecular proceedings of biological hydrogen turnover

Infrared Characterization of the Bidirectional Oxygen-Sensitive [NiFe]-Hydrogenase from E. coli

[NiFe]-hydrogenases are gas-processing metalloenzymes that catalyze the conversion of dihydrogen (H2) to protons and electrons in a broad range of microorganisms. Within the framework of green chemistry, the molecular proceedings of biological hydrogen turnover inspired the design of novel catalytic compounds for H2 generation. The bidirectional “O2-sensitive” [NiFe]-hydrogenase from Escherichia coli HYD-2 has recently been crystallized; however, a systematic infrared characterization in the presence of natural reactants is not available yet. In this study, we analyze HYD-2 from E. coli by in situ attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR) under quantitative gas control. We provide an experimental assignment of all catalytically relevant redox intermediates alongside the O2- and CO-inhibited cofactor species. Furthermore, the reactivity and mutual competition between H2, O2, and CO was probed in real time, which lays the foundation for a comparison with other enzymes, e.g., “O2-tolerant” [NiFe]-hydrogenases. Surprisingly, only Ni-B was observed in the presence of O2 with no indications for the “unready” Ni-A state. The presented work proves the capabilities of in situ ATR FTIR spectroscopy as an efficient and powerful technique for the analysis of biological macromolecules and enzymatic small molecule catalysis

Cyanide Binding to [FeFe]-Hydrogenase Stabilizes the Alternative Configuration of the Proton Transfer Pathway

Hydrogenases are H2 converting enzymes that harbor catalytic cofactors in which iron (Fe) ions are coordinated by biologically unusual carbon monoxide (CO) and cyanide (CN−) ligands. Extrinsic CO and CN−, however, inhibit hydrogenases. The mechanism by which CN− binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CN−-treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39 Å allowed us to distinguish intrinsic CN− and CO ligands and to show that extrinsic CN− binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CN− treated crystals show conformational changes of conserved residues within the proton transfer pathway which could allow a direct proton transfer between E279 and S319. This configuration has been proposed to be vital for efficient proton transfer, but has never been observed structurally

[NiFe]-hydrogenase maturation in vitro: analysis of the roles of the HybG and HypD accessory proteins

[NiFe]-hydrogenases (Hyd) bind a nickel-iron-based cofactor. The Fe ion of the cofactor is bound by two cyanide ligands and a single carbon monoxide ligand. Minimally six accessory proteins (HypA–HypF) are necessary for NiFe(CN)2CO cofactor biosynthesis in Escherichia coli. It has been shown that the anaerobically purified HypC–HypD–HypE scaffold complex carries the Fe(CN)2CO moiety of this cofactor. In the present study, we have purified the HybG–HypDE complex and used it to successfully reconstitute in vitro active Hyd from E. coli. HybG is a homologue of HypC that is specifically required for the maturation of Hyd-2 and also functions in the maturation of Hyd-1 of E. coli. Maturation of active Hyd-1 and Hyd-2 could be demonstrated in extracts derived from HybG- and HypD-deficient E. coli strains by adding anaerobically purified HybG–HypDE complex. In vitro maturation was dependent on ATP, carbamoylphosphate, nickel and reducing conditions. Hydrogenase maturation was prevented when the purified HybG–HypDE complex used in the maturation assay lacked a bound Fe(CN)2CO moiety. These findings demonstrate that it is possible to isolate incompletely processed intermediates on the maturation pathway and to use these to activate apo-forms of [NiFe]-hydrogenase large subunits

Quantification of Local Electric Field Changes at the Active Site of Cytochrome c Oxidase by Fourier Transform Infrared Spectroelectrochemical Titrations

Cytochrome c oxidase (CcO) is a transmembrane protein complex that reduces molecular oxygen to water while translocating protons across the mitochondrial membrane. Changes in the redox states of its cofactors trigger both O2 reduction and vectorial proton transfer, which includes a proton-loading site, yet unidentified. In this work, we exploited carbon monoxide (CO) as a vibrational Stark effect (VSE) probe at the binuclear center of CcO from Rhodobacter sphaeroides. The CO stretching frequency was monitored as a function of the electrical potential, using Fourier transform infrared (FTIR) absorption spectroelectrochemistry. We observed three different redox states (R4CO, R2CO, and O), determined their midpoint potential, and compared the resulting electric field to electrostatic calculations. A change in the local electric field strength of +2.9 MV/cm was derived, which was induced by the redox transition from R4CO to R2CO. We performed potential jump experiments to accumulate the R2CO and R4CO species and studied the FTIR difference spectra in the protein fingerprint region. The comparison of the experimental and computational results reveals that the key glutamic acid residue E286 is protonated in the observed states, and that its hydrogen-bonding environment is disturbed upon the redox transition of heme a3. Our experiments also suggest propionate A of heme a3 changing its protonation state in concert with the redox state of a second cofactor, heme a. This supports the role of propionic acid side chains as part of the proton-loading site

The Geometry of the Catalytic Active Site in [FeFe]-hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

[FeFe]-hydrogenases are efficient metalloenzymes that catalyze the oxidation and evolution of molecular hydrogen, H2. They serve as a blueprint for the design of synthetic H2-forming catalysts. [FeFe]-hydrogenases harbor a six-iron cofactor that comprises a [4Fe-4S] cluster and a unique diiron site with cyanide, carbonyl, and hydride ligands. To address the ligand dynamics in catalytic turnover and upon carbon monoxide (CO) inhibition, we replaced the native aminodithiolate group of the diiron site by synthetic dithiolates, inserted into wild-type and amino acid variants of the [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. The reactivity with H2 and CO was characterized using in situ and transient infrared spectroscopy, protein crystallography, quantum chemical calculations, and kinetic simulations. All cofactor variants adopted characteristic populations of reduced species in the presence of H2 and showed significant changes in CO inhibition and reactivation kinetics. Differences were attributed to varying interactions between polar ligands and the dithiolate head group and/or the environment of the cofactor (i.e., amino acid residues and water molecules). The presented results show how catalytically relevant intermediates are stabilized by inner-sphere hydrogen bonding suggesting that the role of the aminodithiolate group must not be restricted to proton transfer. These concepts may inspire the design of improved enzymes and biomimetic H2-forming catalysts

Isolation of a HypC–HypD complex carrying diatomic CO and CN− ligands

The HypC and HypD maturases are required for the biosynthesis of the Fe(CN)2CO cofactor in the large subunit of [NiFe]-hydrogenases. Using infrared spectroscopy we demonstrate that an anaerobically purified, Strep-tagged HypCD complex from Escherichia coli exhibits absorption bands characteristic of diatomic CO and CN− ligands as well as CO2. Metal and sulphide analyses revealed that along with the [4Fe–4S]2+ cluster in HypD, the complex has two additional oxygen-labile Fe ions. We prove that HypD cysteine 41 is required for the coordination of all three ligands. These findings suggest that the HypCD complex carries minimally the Fe(CN)2CO cofactor