Oxygen sensitivity of [FeFe]-hydrogenase: a comparative study of active site mimics inside outside the enzyme.

[FeFe]-hydrogenase is nature's most efficient proton reducing and H-oxidizing enzyme. However, biotechnological applications are hampered by the O sensitivity of this metalloenzyme, and the mechanism of aerobic deactivation is not well understood. Here, we explore the oxygen sensitivity of four mimics of the organometallic active site cofactor of [FeFe]-hydrogenase, [Fe(adt)(CO)(CN)] and [Fe(pdt)(CO)(CN)] ( = 1, 2) as well as the corresponding cofactor variants of the enzyme by means of infrared, Mössbauer, and NMR spectroscopy.

Infrared spectroscopy reveals metal-independent carbonic anhydrase activity in crotonyl-CoA carboxylase/reductase.

The conversion of CO by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many biological processes. However, methods following the microscopic details of CO conversion at the active site are limited. Here, we used infrared spectroscopy to study the interaction of CO, water, bicarbonate, and other reactants with β-carbonic anhydrase from (CA) and crotonyl-CoA carboxylase/reductase from (Ccr), two of the fastest CO-converting enzymes in nature.

The catalytic reaction of cytochrome c oxidase probed by in situ gas titrations and FTIR difference spectroscopy.

Cytochrome c oxidase (CcO) is a transmembrane heme‑copper metalloenzyme that catalyzes the reduction of O to HO at the reducing end of the respiratory electron transport chain. To understand this reaction, we followed the conversion of CcO from Rhodobacter sphaeroides between several active-ready and carbon monoxide-inhibited states via attenuated total reflection Fourier-transform infrared (ATR FTIR) difference spectroscopy. Utilizing a novel gas titration setup, we prepared the mixed-valence, CO-inhibited RCO state as well as the fully-reduced R and RCO states and induced the "active ready" oxidized state O.

A personal account on 25 years of scientific literature on [FeFe]-hydrogenase.

[FeFe]-hydrogenases are gas-processing metalloenzymes that catalyze H oxidation and proton reduction (H release) in microorganisms. Their high turnover frequencies and lack of electrical overpotential in the hydrogen conversion reaction has inspired generations of biologists, chemists, and physicists to explore the inner workings of [FeFe]-hydrogenase. Here, we revisit 25 years of scientific literature on [FeFe]-hydrogenase and propose a personal account on 'must-read' research papers and review article that will allow interested scientists to follow the recent discussions on catalytic mechanism, O sensitivity, and the in vivo synthesis of the active site cofactor with its biologically uncommon ligands carbon monoxide and cyanide.

Molecular Basis of the Electron Bifurcation Mechanism in the [FeFe]-Hydrogenase Complex HydABC.

Electron bifurcation is a fundamental energy coupling mechanism widespread in microorganisms that thrive under anoxic conditions. These organisms employ hydrogen to reduce CO, but the molecular mechanisms have remained enigmatic. The key enzyme responsible for powering these thermodynamically challenging reactions is the electron-bifurcating [FeFe]-hydrogenase HydABC that reduces low-potential ferredoxins (Fd) by oxidizing hydrogen gas (H).

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

Hydrogenases are H 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.

An in vitro reconstitution system to monitor iron transfer to the active site during the maturation of [NiFe]-hydrogenase.

[NiFe]-hydrogenases (Hyds) comprise a small and a large subunit. The latter harbors the biologically unique [NiFe](CN)CO active-site cofactor. The maturation process includes the assembly of the [Fe](CN)CO cofactor precursor, nickel binding, endoproteolytic cleavage of the large subunit, and dimerization with the small subunit to yield active enzyme.

Second and Outer Coordination Sphere Effects in Nitrogenase, Hydrogenase, Formate Dehydrogenase, and CO Dehydrogenase.

Gases like H, N, CO, and CO are increasingly recognized as critical feedstock in "green" energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N, CO, and CO and the production of H require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur.

Trapping an Oxidized and Protonated Intermediate of the [FeFe]-Hydrogenase Cofactor under Mildly Reducing Conditions.

The H-cluster is the catalytic cofactor of [FeFe]-hydrogenase, a metalloenzyme that catalyzes the formation of dihydrogen (H). The catalytic diiron site of the H-cluster carries two cyanide and three carbon monoxide ligands, making it an excellent target for IR spectroscopy. In previous work, we identified an oxidized and protonated H-cluster species, whose IR signature differs from that of the oxidized resting state (Hox) by a small but distinct shift to higher frequencies.

Electron inventory of the iron-sulfur scaffold complex HypCD essential in [NiFe]-hydrogenase cofactor assembly.

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN- and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E.

Characterization of a putative sensory [FeFe]-hydrogenase provides new insight into the role of the active site architecture.

[FeFe]-hydrogenases are known for their high rates of hydrogen turnover, and are intensively studied in the context of biotechnological applications. Evolution has generated a plethora of different subclasses with widely different characteristics. The M2e subclass is phylogenetically distinct from previously characterized members of this enzyme family and its biological role is unknown.

Two ligand-binding sites in CO-reducing V nitrogenase reveal a general mechanistic principle.

Besides its role in biological nitrogen fixation, vanadium-containing nitrogenase also reduces carbon monoxide (CO) to hydrocarbons, in analogy to the industrial Fischer-Tropsch process. The protein yields 93% of ethylene (CH), implying a C-C coupling step that mandates the simultaneous binding of two CO at the active site FeV cofactor. Spectroscopic data indicated multiple CO binding events, but structural analyses of Mo and V nitrogenase only confirmed a single site.

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

Cytochrome oxidase (CO) 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 O 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 CO from .

Following Electroenzymatic Hydrogen Production by Rotating Ring-Disk Electrochemistry and Mass Spectrometry*.

Gas-processing metalloenzymes are of interest to future bio- and bioinspired technologies. Of particular importance are hydrogenases and nitrogenases, which both produce molecular hydrogen (H ) from proton (H ) reduction. Herein, we report on the use of rotating ring-disk electrochemistry (RRDE) and mass spectrometry (MS) to follow the production of H and isotopes produced from deuteron (D ) reduction (HD and D ) using the [FeFe]-hydrogenase from Clostridium pasteurianum, a model hydrogen-evolving metalloenzyme.

Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase.

Hydrogenases are bidirectional redox enzymes that catalyze hydrogen turnover in archaea, bacteria, and algae. While all types of hydrogenase show H2 oxidation activity, [FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands.

Proton Transfer Mechanisms in Bimetallic Hydrogenases.

Hydrogenases are metalloenzymes that catalyze proton reduction and H oxidation with outstanding efficiency. They are model systems for bioinorganic chemistry, including low-valent transition metals, hydride chemistry, and proton-coupled electron transfer. In this Account, we describe how photochemistry and infrared difference spectroscopy can be used to identify the dynamic hydrogen-bonding changes that facilitate proton transfer in [NiFe]- and [FeFe]-hydrogenase.

Temperature Dependence of Structural Dynamics at the Catalytic Cofactor of [FeFe]-hydrogenase.

[FeFe]-hydrogenases are nature's blueprint for efficient hydrogen turnover. Understanding their enzymatic mechanism may improve technological H fuel generation. The active-site cofactor (H-cluster) consists of a [4Fe-4S] cluster ([4Fe]), cysteine-linked to a diiron site ([2Fe]) carrying an azadithiolate (adt) group, terminal cyanide and carbon monoxide ligands, and a bridging carbon monoxide (μCO) in the oxidized protein ().

[FeFe]-hydrogenase maturation: H-cluster assembly intermediates tracked by electron paramagnetic resonance, infrared, and X-ray absorption spectroscopy.

[FeFe]-hydrogenase enzymes employ a unique organometallic cofactor for efficient and reversible hydrogen conversion. This so-called H-cluster consists of a [4Fe-4S] cubane cysteine linked to a diiron complex coordinated by carbon monoxide and cyanide ligands and an azadithiolate ligand (adt = NH(CHS))·[FeFe]-hydrogenase apo-protein binding only the [4Fe-4S] sub-complex can be fully activated in vitro by the addition of a synthetic diiron site precursor complex ([2Fe]). Elucidation of the mechanism of cofactor assembly will aid in the design of improved hydrogen processing synthetic catalysts.

Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase.

Hydrogenases are among the fastest H evolving catalysts known to date and have been extensively studied under conditions. Here, we report the first mechanistic investigation of an [FeFe]-hydrogenase under whole-cell conditions. Functional [FeFe]-hydrogenase from the green alga is generated in genetically modified cells by addition of a synthetic cofactor to the growth medium.

Discovery of novel [FeFe]-hydrogenases for biocatalytic H-production.

A new screening method for [FeFe]-hydrogenases is described, circumventing the need for specialized expression conditions as well as protein purification for initial characterization. [FeFe]-hydrogenases catalyze the formation and oxidation of molecular hydrogen at rates exceeding 10 s, making them highly promising for biotechnological applications. However, the discovery of novel [FeFe]-hydrogenases is slow due to their oxygen sensitivity and dependency on a structurally unique cofactor, complicating protein expression and purification.

How [FeFe]-Hydrogenase Facilitates Bidirectional Proton Transfer.

Hydrogenases are metalloenzymes that catalyze the conversion of protons and molecular hydrogen, H. [FeFe]-hydrogenases show particularly high rates of hydrogen turnover and have inspired numerous compounds for biomimetic H production. Two decades of research on the active site cofactor of [FeFe]-hydrogenases have put forward multiple models of the catalytic proceedings.

Differential Protonation at the Catalytic Six-Iron Cofactor of [FeFe]-Hydrogenases Revealed by Fe Nuclear Resonance X-ray Scattering and Quantum Mechanics/Molecular Mechanics Analyses.

[FeFe]-hydrogenases are efficient biological hydrogen conversion catalysts and blueprints for technological fuel production. The relations between substrate interactions and electron/proton transfer events at their unique six-iron cofactor (H-cluster) need to be elucidated. The H-cluster comprises a four-iron cluster, [4Fe4S], linked to a diiron complex, [FeFe].

Crystallographic and spectroscopic assignment of the proton transfer pathway in [FeFe]-hydrogenases.

The unmatched catalytic turnover rates of [FeFe]-hydrogenases require an exceptionally efficient proton-transfer (PT) pathway to shuttle protons as substrates or products between bulk water and catalytic center. For clostridial [FeFe]-hydrogenase CpI such a pathway has been proposed and analyzed, but mainly on a theoretical basis. Here, eleven enzyme variants of two different [FeFe]-hydrogenases (CpI and HydA1) with substitutions in the presumptive PT-pathway are examined kinetically, spectroscopically, and crystallographically to provide solid experimental proof for its role in hydrogen-turnover.