Occurrence, classification, and biological function of hydrogenases: an overview.

Investigating and Exploiting the Electrocatalytic Properties of Hydrogenases

ing a hydrogen atom from substrates with relatively weak X−H bonds (Figure 56). Such a situation was found in the catalytic cycle of ACCO, where cleavage of the N−H bond is facilitated by the ferrous ion capable of (partly) reducing the resulting N-based radical to an anion. In the catalytic cycle of HEPD and in the reaction of HppE with an enantiomer of the native substrate, a C−H bond at the carbon hosting a deprotonated alcohol group is severed by the superoxide. In these cases transfer of a hydrogen atom to the superoxide is coupled to one-electron reduction of Fe(III) to Fe(II), which yields aldehyde or ketone products. In a similar way a thioaldehyde is produced during the initial steps of the IPNS catalytic cycle. Figure 53. Reaction catalyzed by NDO. Figure 54. X-ray structure of the non-heme iron cofactor in the NDO active site (PDB 1O7G). Figure 55. Reaction mechanism for the dihydroxylation reaction catalyzed by NDO suggested on the basis of the results of a DFT study. Relative energy values in kilocalories per mole are given beneath the structures shown and above the arrows for the transition states connecting them. Chemical Reviews Review dx.doi.org/10.1021/cr400388t | Chem. Rev. 2014, 114, 3601−3658 3636 The most often encountered reaction of the Fe(III)−O2 species is an electrophilic attack on an electron-rich (co)substrate that yields an Fe(II) intermediate with a peroxide bridge between the ferrous ion and an organic molecule (Figure 57). In all examples shown in the figure the reactive spin state is a quintet with high-spin Fe(III) antiferromagnetically coupled to the superoxide radical. This particular electronic structure allows for a smooth two-electron reaction, leading to a ferrous intermediate featuring a high-spin Fe(II) ion, i.e., also lying on a quintet PES. When the substrates are easily one-electron-oxidized, as, for example, carotenoids or catecholates, already the ternary enzyme−dioxygen−organic substrate complex may contain an organic radical along with the superoxide anion stabilized by coordination to the metal (Figure 58). With proper (antiferromagnetic) alignment of the spins of the unpaired electrons on the two radicals, direct coupling between them proceeds with a straightforward formation of a new C−O bond. With the O2 ligand already protonated, several different reaction scenarios are usually plausible, and one of them, arguably the simplest, involves a transfer of the HOO group from the metal ion to the organic substrate (Figure 59). In the reaction of HEPD, it is the distal, i.e., originally protonated, oxygen atom that attacks the aldehyde group and the proton is transferred to the second (proximal) oxygen atom with the help of a phosphonic group of the substrate. In this reaction HOO acts as a nucleophile. In the case of HGD, the HOO ligand has a partial radical character, and hence, in its attack on the aromatic ring, it behaves as an electrophilic reagent. Moreover, in the HGD and ACO cases, it is the proximal oxygen atom that is directly transferred from the metal ion to the organic radical, i.e., the proton remains on the same oxygen atom throughout the reaction. When a catalytic reaction involves a homolytic O−O bond cleavage, one end of the peroxo group is in contact with the metal ion, and it is reduced to HO− or RO− when the O−O bond breaks (Figure 60). The electron required for the reduction is provided usually by the ferrous ion; in intradiol dioxygenases, which host Fe(III) in the active site, a tyrosinate ligand can serve as a reductant. Heterolytic cleavage of the O−O bond typically yields highvalent iron(IV)−oxo species, and such a reaction requires Fe(II), a deprotonated proximal oxygen atom, and usually also that the distal oxygen atom has a chance to develop a second covalent bond when the O−O gets broken (Figure 61). The bonding partner for the distal oxygen can be a hydrogen (PDHs, IPNS, ACCO) or a (co)substrate’s carbon (αKAOs, ACO, Dke1) atom. Heterolysis to Fe(IV)O usually proceeds on the quintet PES. Finally, heterolytic O−O bond cleavage may proceed without changing the oxidation state of the metal but instead with coupled two-electron oxidation of an organic substrate (Figure Figure 56. Examples of Fe(II)/Fe(III)−O2 reactions involving hydrogen atom abstraction. Only the most relevant fragment of the substrate is shown. Figure 57. Examples of Fe(III)−O2 reactions involving electrophilic attack and two-electron oxidation of an organic (co)substrate. Figure 58. Examples of Fe(II)/Fe(III)−O2 reactions involving radical coupling between the superoxide and an organic radical. Only the most relevant fragment of the substrate is shown. Chemical Reviews Review dx.doi.org/10.1021/cr400388t | Chem. Rev. 2014, 114, 3601−3658 3637 62). In both cases depicted in the figure, a hydroperoxo group is bound to high-spin Fe(III), and when theO−Obond cleaves, the OH group remains on iron and the other oxygen atom forms two covalent bonds with the organic substrate. This kind of heterolytic O−O cleavage requires that the donor orbital of the organic substrate, i.e., the one that provides two electrons for reduction of the peroxo group, has a good overlap with the O−O σ* orbital. 5.2. Dinuclear Non-Heme Iron Enzymes 5.2.1. Methane Monooxygenase. MMO is an enzyme which inserts one oxygen from O2 into methane to form methanol. The active site of the soluble form of MMO is shown in Figure 63. It contains an iron dimer complex linked by oxygen-derived ligands and has four glutamates and two histidines. Due to the presence of a well-resolved X-ray structure, and the technical importance of the reaction catalyzed, this was actually the first redox-active enzymemechanism that was treated with the cluster model using modern DFT functionals in 1997. Several groups were active at an early stage, and the most essential parts of the reaction mechanism were determined more than a decade ago. A comprehensive review of this development was written by Friesner et al. In short, the active species (compound Q) has a diamond core structure with two bridging oxo groups and is in an Fe2(IV,IV) state, one of themost oxidized species in nature. One of the oxo groups of compound Q activates nethane by an abstraction of one hydrogen atom. The TS is linear in C···H···O to form an Fe2(III,IV) state and amethyl radical. The loss of entropy at the TS is a large part of the barrier. In the second step, the methyl radical recombines with the bridging hydroxide formed in the first step. The TS for this step was first located by Basch et al. This rebound mechanism was criticized by interpretations of radical clock experiments, which appeared to show that there could not be a sufficiently long-lived alkyl radical to be consistent with a two-step mechanism. A concerted mechanism with simultaneous cleavage of the C−H bond and formation of the C−O bond was therefore suggested. Several explanations were suggested to resolve this apparent discrepancy between experiment and theory. In one of them, it was concluded that the radical clock probe molecules Figure 59. Examples of HOO transfer reactions. Only the most relevant fragment of the substrate is shown. Figure 60. Examples of O−O bond homolysis facilitated by oneelectron reduction of the proximal oxygen atom. Only the most relevant fragment of the substrate is shown. Figure 61. Examples of heterolytic O−O bond cleavage yielding iron(IV)−oxo species. Figure 62. Examples of heterolytic O−Obond cleavage proceeding with direct two-electron oxidation of the organic molecule. Chemical Reviews Review dx.doi.org/10.1021/cr400388t | Chem. Rev. 2014, 114, 3601−3658 3638 were chemically so different from methane that different mechanisms were likely. The probes are much easier to ionize and could form cations instead of radicals. Another suggestion was a so-called two-channel mechanism of the dynamics involving a bound radical intermediate. A third possibility could be something analogous to the two-state reactivity mechanism suggested for P450 to explain similar discrepancies in that case, but this has not been tested. In summary, a concerted mechanism, as suggested by the experiments, has never been found in DFT modeling calculations for methane hydroxylation by MMO, at least when reasonable models have been tried, and the suggestion has therefore not survived. In the initial phase of the MMO studies, there were problems converging to proper electronic states. These problems were solved by Friesner et al., who were able to obtain the correct antiferromagnetic coupling of compound Q with two high-spin irons. A state of key importance is also the first intermediate after Q, with an electronic structure characterized as Fe2(III,IV)− O•, discussed further below in connection with mixedMn−Fe dimers. It was found that already at the TS for hydrogen abstraction the iron dimer is in an Fe2(III,IV) state as it is in the product of this step. The bridging oxygen radical would then act as a hydrogen atom abstractor. At the TS, the spins are divided between a bridging oxo ligand and the methyl, while the iron spins stay essentially constant from the Fe2(III,IV)−O state to the product. The O−O bond cleavage to reach compound Q is also a significant step in the catalytic cycle. Again, several groups were involved in studying this step at an early stage. There was essential agreement among these studies on the mechanism. First, a peroxide (compound P) is formed between the two irons in an Fe2(III,III) state. Several different structures of P are nearly degenerate. In the TS for the O−O cleavage, the oxygens are symmetric, but only one of the irons is redox-active. In the final Figure 63. Active site of methane monooxygenase. Figure 64. R1 and R2 proteins in RNR. Chemical Reviews Review dx.doi.org/10.1021/cr400388t | Chem. Rev. 2014, 114, 3601−3658 3639 stage, the other iron also changes its oxidation state to IV, and compound Q is formed. In the study by Friesner et al., 

Quantum chemical studies of mechanisms for metalloenzymes.

Activation and Inactivation of Hydrogenase Function and the Catalytic Cycle: Spectroelectrochemical Studies

[NiFe] and [FeFe] hydrogenases studied by advanced magnetic resonance techniques.

The Ni-Fe site in the active membrane-bound [NiFe]-hydrogenase from Allochromatium Vinosum can exist in three different redox states. In the most oxidized state (Nia-S) the nickel is divalent. The most reduced state (Nia-SR) likewise has Ni2+, while the intermediate state (Nia-C*) has Ni3+. The transitions between these states have been studied by stopped-flow Fourier transform infrared spectroscopy. It is inferred from the data that the Nia-S f Nia-C* and Nia-C* f Nia-SR transitions induced by dihydrogen require one of the [4Fe-4S] clusters to be oxidized. Enzyme in the Nia-S state with all of the iron-sulfur clusters reduced reacts with dihydrogen to form the Nia-SR state in milliseconds. By contrast, when one of the cubane clusters is oxidized, the Nia-S state reacts with dihydrogen to form the Nia-C* state with all of the iron-sulfur clusters reduced. The competition between dihydrogen and carbon monoxide for binding to the active site was dependent on the redox state of the nickel ion. Formation of the Nia-SâCO state (Ni2+) by reacting CO with enzyme in the Nia-SR and Nia-S states (Ni2+) is considerably faster than its formation from enzyme in the Nia-C* (Ni3+) state. Excess oxygen converted hydrogen-reduced enzyme to the inactive Nir* state within 158 ms, suggesting a direct reaction at the Ni-Fe site. With lower O2 concentrations the formation of intermediate states was observed. The results are discussed in the light of the present knowledge of the structure and mechanism of action of the A. Vinosum enzyme.

Reactions of H2, CO, and O2 with active [NiFe]-hydrogenase from Allochromatium vinosum. A stopped-flow infrared study.

The reaction between hydrogen and the [NiFe]-hydrogenase from Allochromatium Vinosum in its inactive form has been studied by stopped-flow infrared spectroscopy. The data, for the first time, clearly show that at room temperature enzyme in the unready state, either oxidized or reduced, does not react with hydrogen. Enzyme in the ready state reacts with hydrogen after a lag phase of about six seconds, whereby a specific reduction of the enzyme occurs. The lag phase and the rate of reduction of the ready enzyme are neither dependent on the enzyme concentration nor on the substrate concentration, i.e., substoichiometric and 8-fold excess amounts of H2 reduce the ready enzyme at the same rate. Oxygen delays this reaction but does not prevent it. The infrared changes lead us to suggest that the hydroxyl group, bridging between the Ni and the Fe atom in the active site, becomes protonated during this reduction. At physiological temperatures, this property of the inactive ready enzyme enables a full development of activity by substoichiometric H2 concentrations.

Hydrogen-induced activation of the [NiFe]-hydrogenase from Allochromatium vinosum as studied by stopped-flow infrared spectroscopy.

The purified membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri was studied with electron paramagnetic resonance (EPR) focusing on the properties of the iron-sulphur clusters. The EPR spectra showed signals from three different [4Fe-4S] clusters. Two of the clusters could be reduced under 101 kPa of H2, whereas the third cluster was only partially reduced. Magnetic interaction of one of the clusters with an unpaired electron localized on the Ni-Fe site indicated that this was the proximal cluster as found in all [NiFe]-hydrogenases. Hence, this cluster was assigned to be located in the EchC subunit. The other two clusters could therefore be assigned to be bound to the EchF subunit, which has two conserved four-Cys motifs for the binding of a [4Fe-4S] cluster. Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential. The possible relevance of these properties for the function of this proton-pumping [NiFe]-hydrogenase is discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1033.2002.03328.x

Sergei Kurkin

Papers

Reactions of H2, CO, and O2 with active [NiFe]-hydrogenase from Allochromatium vinosum. A stopped-flow infrared study.

Hydrogen-induced activation of the [NiFe]-hydrogenase from Allochromatium vinosum as studied by stopped-flow infrared spectroscopy.

The membrane-bound [NiFe]-hydrogenase (Ech) from Methanosarcina barkeri: unusual properties of the iron-sulphur clusters