Corrigendum to "Transport limited adsorption experiments give a new lower estimate of the turnover frequency of hydrogenase 1" BBA Advances 3 (2023) 100090.

[This corrects the article DOI: 10.1016/j.bbadva.

Outer-sphere effects on the O sensitivity, catalytic bias and catalytic reversibility of hydrogenases.

The comparison of homologous metalloenzymes, in which the same inorganic active site is surrounded by a variable protein matrix, has demonstrated that residues that are remote from the active site may have a great influence on catalytic properties. In this review, we summarise recent findings on the diverse molecular mechanisms by which the protein matrix may define the oxygen tolerance, catalytic directionality and catalytic reversibility of hydrogenases, enzymes that catalyse the oxidation and evolution of H. These mechanisms involve residues in the second coordination sphere of the active site metal ion, more distant residues affecting protein flexibility through their side chains, residues lining the gas channel and even accessory subunits.

Combining a Commercial Mixer with a Wall-Tube Electrode Allows the Arbitrary Control of Concentrations in Protein Film Electrochemistry.

Protein film electrochemistry is a technique in which an enzyme is immobilized on an electrode in a configuration that allows following the changes in turnover frequency as a response to changes in the experimental conditions. Insights into the reactivity of the enzyme can be obtained by quantitatively modeling such responses. As a consequence, the more the technique allows flexibility in changing conditions, the more useful it becomes.

Kinetic Modeling of the Reversible or Irreversible Electrochemical Responses of FeFe-Hydrogenases.

The enzyme FeFe-hydrogenase catalyzes H evolution and oxidation at an active site that consists of a [4Fe-4S] cluster bridged to a [Fe(CO)(CN)(azadithiolate)] subsite. Previous investigations of its mechanism were mostly conducted on a few "prototypical" FeFe-hydrogenases, such as that from (Cr HydA1), but atypical hydrogenases have recently been characterized in an effort to explore the diversity of this class of enzymes. We aim at understanding why prototypical hydrogenases are active in either direction of the reaction in response to a small deviation from equilibrium, whereas the homologous enzyme from (Tam HydS) shows activity only under conditions of very high driving force, a behavior that was referred to as "irreversible catalysis".

An allosteric redox switch involved in oxygen protection in a CO reductase.

Metal-dependent formate dehydrogenases reduce CO with high efficiency and selectivity, but are usually very oxygen sensitive. An exception is Desulfovibrio vulgaris W/Sec-FdhAB, which can be handled aerobically, but the basis for this oxygen tolerance was unknown. Here we show that FdhAB activity is controlled by a redox switch based on an allosteric disulfide bond.

Kinetic modeling of 2e/1H and 2e/2H bidirectional catalytic cycles.

When a redox enzyme or synthetic catalyst is interfaced with an electrode, the electrochemical response depends on the details of the catalytic cycle. Here we focus on the steady-state catalytic waveshape of enzymes such as formate dehydrogenase (2e/1H), hydrogenases (2e/2H) and other bidirectional molecular catalysts that can be adsorbed on, and undergo direct electron transfer with an electrode. We seek to examine the relations between the dependence on pH of the waveshape, the sequence of events in the catalytic cycle, and the properties of the catalytic intermediates (their reduction potentials and pK's).

A Chimeric NiFe Hydrogenase Heterodimer to Assess the Role of the Electron Transfer Chain in Tuning the Enzyme's Catalytic Bias and Oxygen Tolerance.

The observation that some homologous enzymes have the same active site but very different catalytic properties demonstrates the importance of long-range effects in enzyme catalysis, but these effects are often difficult to rationalize. The NiFe hydrogenases 1 and 2 (Hyd 1 and Hyd 2) from both consist of a large catalytic subunit that embeds the same dinuclear active site and a small electron-transfer subunit with a chain of three FeS clusters. Hyd 1 is mostly active in H oxidation and resistant to inhibitors, whereas Hyd 2 also catalyzes H production and is strongly inhibited by O and CO.

Transport limited adsorption experiments give a new lower estimate of the turnover frequency of hydrogenase 1.

Protein Film Electrochemistry is a technique in which a redox enzyme is directly wired to an electrode, which substitutes for the natural redox partner. In this technique, the electrical current flowing through the electrode is proportional to the catalytic activity of the enzyme. However, in most cases, the amount of enzyme molecules contributing to the current is unknown and the absolute turnover frequency cannot be determined.

Assessing the Extent of Potential Inversion by Cyclic Voltammetry: Theory, Pitfalls, and Application to a Nickel Complex with Redox-Active Iminosemiquinone Ligands.

Potential inversion refers to the situation where a protein cofactor or a synthetic molecule can be oxidized or reduced twice in a cooperative manner; that is, the second electron transfer is easier than the first. This property is very important regarding the catalytic mechanism of enzymes that bifurcate electrons and the properties of bidirectional redox molecular catalysts that function in either direction of the reaction with no overpotential. Cyclic voltammetry is the most common technique for characterizing the thermodynamics and kinetics of electron transfer to or from these molecules.

Increasing the O Resistance of the [FeFe]-Hydrogenase CbA5H through Enhanced Protein Flexibility.

The high turnover rates of [FeFe]-hydrogenases under mild conditions and at low overpotentials provide a natural blueprint for the design of hydrogen catalysts. However, the unique active site (H-cluster) degrades upon contact with oxygen. The [FeFe]-hydrogenase from (CbA5H) is characterized by the flexibility of its protein structure, which allows a conserved cysteine to coordinate to the active site under oxidative conditions.

Electrochemical Kinetics Support a Second Coordination Sphere Mechanism in Metal-Based Formate Dehydrogenase.

Metal-based formate dehydrogenases are molybdenum or tungsten-dependent enzymes that catalyze the interconversion between formate and CO . According to the current consensus, the metal ion of the catalytic center in its active form is coordinated by 6 S (or 5 S and 1 Se) atoms, leaving no free coordination sites to which formate could bind to the metal. Some authors have proposed that one of the active site ligands decoordinates during turnover to allow formate binding.

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.

Reversible or Irreversible Catalysis of H/H Conversion by FeFe Hydrogenases.

Studies of molecular catalysts traditionally aim at understanding how a certain mechanism allows the reaction to be fast. A distinct question, which has only recently received attention in the case of bidirectional molecular catalysts, is how much thermodynamic driving force is required to achieve fast catalysis in either direction of the reaction. "Reversible" catalysts are bidirectional catalysts that work either way in response to even a small departure from equilibrium and thus do not waste input free energy as heat; conversely, "irreversible" catalysts require a large driving force to proceed at an appreciable rate [Fourmond et al.

Electrochemical Studies of CO -Reducing Metalloenzymes.

Only two enzymes are capable of directly reducing CO : CO dehydrogenase, which produces CO at a [NiFe S ] active site, and formate dehydrogenase, which produces formate at a mononuclear W or Mo active site. Both metalloenzymes are very rapid, energy-efficient and specific in terms of product. They have been connected to electrodes with two different objectives.

Reversible catalysis.

We describe as 'reversible' a bidirectional catalyst that allows a reaction to proceed at a significant rate in response to even a small departure from equilibrium, resulting in fast and energy-efficient chemical transformation. Examining the relation between reaction rate and thermodynamic driving force is the basis of electrochemical investigations of redox reactions, which can be catalysed by metallic surfaces and biological or synthetic molecular catalysts. This relation has also been discussed in the context of biological energy transduction, regarding the function of biological molecular machines that harness chemical reactions to do mechanical work.

Reversible H Oxidation and Evolution by Hydrogenase Embedded in a Redox Polymer Film.

Efficient electrocatalytic energy conversion requires the devices to function reversibly, deliver a significant current at minimal overpotential. Redox-active films can effectively embed and stabilise molecular electrocatalysts, but mediated electron transfer through the film typically makes the catalytic response irreversible. Here, we describe a redox-active film for bidirectional (oxidation or reduction) and reversible hydrogen conversion, consisting of [FeFe] hydrogenase embedded in a low-potential, 2,2'-viologen modified hydrogel.

Formate Dehydrogenases Reduce CO Rather than HCO : An Electrochemical Demonstration.

Mo/W formate dehydrogenases catalyze the reversible reduction of CO species to formate. It is thought that the substrate is CO and not a hydrated species like HCO , but there is still no indisputable evidence for this, in spite of the extreme importance of the nature of the substrate for mechanistic studies. We devised a simple electrochemical method to definitively demonstrate that the substrate of formate dehydrogenases is indeed CO .

A safety cap protects hydrogenase from oxygen attack.

[FeFe]-hydrogenases are efficient H-catalysts, yet upon contact with dioxygen their catalytic cofactor (H-cluster) is irreversibly inactivated. Here, we combine X-ray crystallography, rational protein design, direct electrochemistry, and Fourier-transform infrared spectroscopy to describe a protein morphing mechanism that controls the reversible transition between the catalytic H-state and the inactive but oxygen-resistant H-state in [FeFe]-hydrogenase CbA5H of Clostridium beijerinckii. The X-ray structure of air-exposed CbA5H reveals that a conserved cysteine residue in the local environment of the active site (H-cluster) directly coordinates the substrate-binding site, providing a safety cap that prevents O-binding and consequently, cofactor degradation.

Electrochemical Characterization of a Complex FeFe Hydrogenase, the Electron-Bifurcating Hnd From .

Hnd, an FeFe hydrogenase from , is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H to both the exergonic reduction of NAD and the endergonic reduction of a ferredoxin. We previously showed that Hnd retains activity even when purified aerobically unlike other electron-bifurcating hydrogenases.

Artificial maturation of [FeFe] hydrogenase in a redox polymer film.

We demonstrate that the insertion of the dinuclear active site of [FeFe] hydrogenase into the apo-enzyme can occur when the enzyme is embedded in a film of redox polymer, under conditions of mediated electron transfer. The maturation can be monitored by electrochemistry, and is as fast as under conditions of direct electron transfer. This new approach further clears the way to the implementation of hydrogenases in large scale real life processes.

The Solvent-Exposed Fe-S D-Cluster Contributes to Oxygen-Resistance in Ni-Fe Carbon Monoxide Dehydrogenase.

Ni-Fe CO-dehydrogenases (CODHs) catalyze the conversion between CO and CO using a chain of Fe-S clusters to mediate long-range electron transfer. One of these clusters, the D-cluster, is surface-exposed and serves to transfer electrons between CODH and external redox partners. These enzymes tend to be extremely O-sensitive and are always manipulated under strictly anaerobic conditions.

The two CO-dehydrogenases of Thermococcus sp. AM4.

Ni-containing CO-dehydrogenases (CODHs) allow some microorganisms to couple ATP synthesis to CO oxidation, or to use either CO or CO as a source of carbon. The recent detailed characterizations of some of them have evidenced a great diversity in terms of catalytic properties and resistance to O. In an effort to increase the number of available CODHs, we have heterologously produced in Desulfovibrio fructosovorans, purified and characterized the two CooS-type CODHs (CooS1 and CooS2) from the hyperthermophilic archaeon Thermococcus sp.

Author Correction: The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.