Hydrogenase-1 (Hyd-1) from Escherichia coli is a membrane-bound enzyme that catalyses the reversible oxidation of molecular H2. The active site contains one Fe and one Ni atom and several conserved amino acids including an arginine (Arg509), which interacts with two conserved aspartate residues (Asp118 and Asp574) forming an outer shell canopy over the metals. There is also a highly conserved glutamate (Glu28) positioned on the opposite side of the active site to the canopy. The mechanism of hydrogen activation has been dissected by site-directed mutagenesis to identify the catalytic base responsible for splitting molecular hydrogen and possible proton transfer pathways to/from the active site. Previous reported attempts to mutate residues in the canopy were unsuccessful, leading to an assumption of a purely structural role. Recent discoveries, however, suggest a catalytic requirement, for example replacing the arginine with lysine (R509K) leaves the structure virtually unchanged, but catalytic activity falls by more than 100-fold. Variants containing amino acid substitutions at either or both, aspartates retain significant activity. We now propose a new mechanism: heterolytic H2 cleavage is via a mechanism akin to that of a frustrated Lewis pair (FLP), where H2 is polarized by simultaneous binding to the metal(s) (the acid) and a nitrogen from Arg509 (the base).
Hydrogenases are metalloenzymes that catalyse the conversion of molecular H2 to protons and electrons, and the reverse reaction to regenerate H2 (reviewed in ). They are found in the periplasm or cytoplasm of bacteria, archaea and some eukaryotes, either as soluble enzymes or in membrane-bound form, with various functions, including provision of energy by oxidation of H2, balancing the redox potential of the cell, establishing transmembrane (TM) gradients, sensing and signalling. There are three classes, with [NiFe], [FeFe] or [Fe] metal centres respectively, and the chemical mechanism of efficient hydrogen cleavage or generation by these enzymes is of particular interest because, unlike man-made catalysts, they do not require a noble metal such as Pt.
There are three types of [NiFe] hydrogenases: membrane bound, regulatory and soluble. The metal ions are coordinated by two bridging sulfurs from protein cysteine side chains, the Ni by two further ‘terminal’ cysteine sulfurs and the Fe by two inorganic cyanides and one carbonyl ligand. A third bridging site is open for binding of substrate H2 and other small ligands, such as hydroxide (see later). The reaction cycle involves complex proton-coupled electron transfers, with the resulting electrons produced transferred from the metal centre to a string of FeS clusters leading to the protein surface . During the reaction cycle, both Ni and Fe are initially in formal oxidation state II, with Ni passing through Ni(II)–H− (Ni–R, a hydrido species) Ni(III)–H− (Ni–C, also a hydrido species) and probably Ni(I) (Ni–L, in which hydride has migrated as H+) before returning to the starting point II (Ni–SI). Binding of H2 to a metal increases its acidity, facilitating heterolytic cleavage via abstraction of H+ by a nearby base and leaving a bridging H− at the metal centre. [NiFe] hydrogenases are susceptible to aerobic oxidation, forming Ni(III) species that require reductive activation (termed ‘Ready’ and ‘Unready’ states respectively, in order of ease of reductive reactivation). A subclass referred to as ‘O2-tolerant’ [NiFe] hydrogenases are able to sustain H2 oxidation in the presence of O2, owing to their exclusive formation of the Ready state (also known as Ni–B) which is rapidly reactivated .
Hydrogenase structure and activity
Several crystal structures have been determined for bacterial [NiFe] hydrogenases, showing similar folds, which reflect significant sequence conservation and very similar active sites . Figure 1 shows the structure of (Hydrogenase-1; (Hyd-1)), a membrane-bound [NiFe] hydrogenase that is tolerant to O2 exposure . It is a heterotetramer, consisting of two identical small subunits and two identical large subunits. The small subunits anchor the enzyme to the periplasmic side of the cytoplasmic membrane via a C-terminal single-pass TM helix, and contain three FeS clusters which serve to transfer electrons from the active site to an associated membrane-bound Cytochrome b. The large subunits harbour the [NiFe] centre and likely entry channels for H2 and exit pathways for H+.
X-ray crystal structure of E. coli Hyd-1
Structural analysis of various [NiFe] hydrogenases reveal a long channel extending from the surface of the enzyme to the active site that has been proposed to be the entry route for H2. The channel can bind xenon  or krypton  in crystals demonstrating an affinity for gas molecules. Site-directed mutagenesis provides further evidence that H2 enters via this channel, for example double mutant V74I, L122F of Desulfovibrio fructosovorans (Val78 and Leu126 in Escherichia coli) hydrogenase constricts the end of the channel closest to the active site. This does not affect the turnover rate of the variant enzyme, but leads to a 5-fold increase in KM  with respect to H2, suggesting gas molecules have reduced access to the active site. Conversely, increasing the size of gas channels in an oxygen tolerant regulatory hydrogenase from Ralstonia eutropha produces an enzyme that is sensitive to inhibition by oxygen, because oxygen is no longer prevented from reaching the active site .
Once hydrogen molecules are split the resultant protons are likely to travel through the enzyme via a Grotthuss-like mechanism, with H+ being transferred between charged or polar groups within hydrogen bonding distance of each other. Replacement of strictly conserved Glu25 of D. fructosovorans (Glu28 in E. coli), positioned next to the [NiFe] cluster, with glutamine produces an enzyme capable of splitting/recombining H2, but incapable of turnover. This suggests Glu28 is an entry point into a proton transfer pathway, since proton transport is a requirement for enzymatic turnover, but not for substrate cleavage . Additionally, structural analysis of Desulfovibrio vulgaris hydrogenase has identified three possible proton transfer routes starting from this conserved glutamate . A second proton transfer pathway was identified by mutagenesis of a highly conserved, histidine-rich motif observed in many membrane-bound hydrogenases . Mutagenesis of His104 from Desulfovibrio gigas hydrogenase (His119 in E. coli) produced an enzyme with a significantly impaired activity and computational analysis identified a possible route for proton transfer independent of Glu28 but including the highly conserved Asp118 (see below) and His119 .
The active site also contains a strictly conserved arginine, Arg509 in Hyd-1, above the metal centre and proposed substrate-binding site, with one N atom only 4.5 Å (1 Å=0.1 nm) from the Ni ion (Figure 2). The very close proximity to the electropositive metal centre and strict sequence conservation implies a functional importance for this arginine, but it has been largely ignored as a candidate for the active site base owing to the typically high pKa of arginine residues. Arg509 also interacts with two highly conserved aspartates, Asp118 and Asp574, to form a conserved canopy above the active site. The conservation of the arginine had been attributed to a role in stabilization of the structure, including a proposed hydrogen bond to one of the Fe cyanide ligands [12,13], although the geometry of this hydrogen bond is not favourable. The functional role of the arginine has not, however, been investigated by site-directed mutagenesis, although there were two unsuccessful attempts to replace the arginine with alternative side chains in enzymes from Thiocapsa roseopersicina  and D. fructosovorans , which did not lead to production of mature enzyme.
Hydrogen oxidation activities of Hyd-1 canopy variants
Proposals for the catalytic mechanism have previously centred on the suggestion that the base might be a terminal cysteine ligand coordinated to the Ni ion, Cys574 in Hyd-1(Figure 2), adjacent to the substrate-binding site [15,16]. A recent 0.89 Å resolution crystal structure for the [NiFe] hydrogenase from D. vulgaris, specifically in the active reduced Ni–R form, gave electron density maps of outstanding quality where many hydrogen atoms were clearly visible . The structure was consistent with the presence of a bound hydride ion bridging the metals, with some difference electron density on the sulfur atom for Cys574, which the authors interpreted as the H+ product of cleavage, although the geometry was unusual.
Structure and activity of E. coli Hyd-1 canopy variants
Recently, a combination of site-directed mutagenesis, electrochemistry and X-ray crystallography has been used to investigate whether the conserved, ‘canopy-forming’ residues in Hyd-1 have a role in activation of H2 . Mutations R509K, D574N, D118A and D118N/D574N (double substitution) were introduced and their hydrogen oxidation activities measured as 0.8%, 83%, 26% and 20% of the H2 oxidation activity of native Hyd-1 (Figure 2). Additionally, protein film electrochemistry (PFE) showed all mutants had similar potentials for the onset of catalytic H2 oxidation, a property mainly dependent on the FeS clusters [18,19]. The characteristic shape of the curve for native enzyme was retained for R509K, whereas the other variants showed negative shifts in the high-potential threshold indicating some stabilization of the ‘resting’ Ni(III)–OH state [20,21]. High resolution X-ray crystal structures showed the mutations had minimal effect on the structure of the protein, and the main chain atoms of the refined atomic models of all variants could be superimposed on those of the native enzyme with root mean square (r.m.s.) deviations in the range 0.12–0.16 Å. The local conformations of the mutated canopy residues, 509, 118 and 574, had even smaller main chain r.m.s. deviations of 0.10–0.12 Å. The cysteine residues coordinating the metal ion were also unaffected by the incorporated amino acid substitutions, although Cys79 showed evidence of partial oxidation to sulfenic acid (Figure 2) as a result of exposure to oxygen during purification . The positions and occupancies of the Ni and Fe ions showed no significant changes between the variants, as judged by Fo–Fo difference electron density maps, which are particularly sensitive to small changes in electron-dense atoms. In all cases, a small ligand was observed bridging the metal ions, and was assigned as a hydroxyl ion .
The side-chain conformations in the variants showed only small differences. The conformation of Lys509 is very similar to Arg509, with small adjustments in side chain torsion angles to maintain the hydrogen bond with Asp118. The position of Nζ, superimposes closely on that of the corresponding Arg509 N, at a similar distance of 4.4 Å above the Ni ion (Figure 2). The smaller side chain allows space for one additional ordered water molecule near the canopy residues . The entire active site structure of R509K shows minimal disturbance from that of the native enzyme, indicating that structural change is not the origin of the considerable reduction in catalytic activity. In D118A, the smaller side chain does not result in additional water molecules, but Arg509 moves slightly closer to the metal ions as a consequence of the loss of the hydrogen bonds to Asp118. The replacement of Asp574 with asparagine results in only a small rotation of the side chain, and one new hydrogen bond between its Nδ2 and Oδ1of Asp118. In the double substitution variant, where both aspartate residues are replaced by asparagine, the side chain of Asn118 rotates away from Arg509 to relieve the unfavourable contact between their terminal NH groups, but retains the hydrogen bond from its Oδ1.
Site-directed mutagenesis in combination with structural and spectroscopic analysis have been invaluable in understanding many aspects of hydrogenase function: including how hydrogen enters to the active site, how protons are transported away after turnover and most recently, a new view as to how activation of H2 facilitates cleavage . The replacement of Arg509 by lysine results in over 100-fold reduction in catalytic activity, despite a lack of structural change, which is remarkable given the significant alterations to the hydrogen bonding network at the active site that the different variants introduce. The significant decrease in hydrogen oxidation activity overturns the previous assumption that the role of Arg509 is simply structural and provides powerful evidence for the direct involvement of the guanidinium group of Arg509 in catalysis. It had recently been suggested that the catalytic base for H2 cleavage was one of the cysteine ligands to the Ni ion (Cys579 in Hyd-1), but a coordinated thiolate-S is expected to be a weak base . On the other hand, the guanidinium group of Arg509 is a very strong base, capable of abstracting a proton if it was itself deprotonated. Arginine side chains normally have a pKa of 12 or more, but enzymes often tune the properties of side chains by creating specific environments and the use of arginine as a catalytic base is not unprecedented. For example it is accepted in the mechanisms of fumarate reductase and L-aspartate oxidase, as well as a proposed role in photosystem II [25–27]. The question that needs to be addressed is how Hyd-1 Arg509 can be deprotonated in order to act as the necessary base. Hyd-1, with other [NiFe] hydrogenases, is a reversible electrocatalyst, so that each step of the catalytic cycle should be reversible according to the appropriate potentials. In addition, a hydrido ligand bound to the reduced metal centre would be an exceptionally strong base , whereas a bound H2 would be a weak acid. The structure of the Ni–R form, with a bound hydride ion was established by the high resolution crystal structure discussed above  and the active site is shown schematically in Figure 3. One N atom of Arg 509 is positioned close to the metal centre with favourable geometry for the strongly basic hydride ligand to deprotonate it and form H2. Consequently, the reverse reaction, heterolytic cleavage of H2 must also be possible. The reaction can be driven in either direction depending on the electrochemical potential, i.e. the supply or withdrawal of electrons. The heterolytic activation of hydrogen by arginine and the metal centre is similar to that of a frustrated Lewis pair (FLP)  where a strong Lewis acid and strong Lewis base are placed in close proximity, but are sterically prevented from reacting with each other. Such an arrangement allows the Lewis acid and base to act cooperatively to activate small molecules such as H2. In the active site of hydrogenases, binding of hydrogen to the metal centre polarizes the molecule and greatly increases its acidity facilitating the abstraction of a proton by the (Lewis) base. It has been proposed that the catalytic base in [NiFe] hydrogenases is Arg509 in Hyd-1 , and the corresponding residue in other homologues. Interestingly, a related mechanism is proposed for [FeFe] hydrogenases, where an unusual bridging ligand places a pendant nitrogen over the substrate-binding site, that is essential for catalysis . In addition, some active synthetic hydrogenase analogues also have a pendant base over a redox-active metal ion . There is now a convergence of [NiFe] and [FeFe] hydrogenases, with the synthetic analogues, towards the FLP mechanism for splitting hydrogen.
Hydrogen activation at the catalytic centre of [NiFe] hydrogenases
The coordinates and structure factors are available from the Protein Data Bank (PDB) with accession codes 4UE3 (R509K), 5A4F (D118A), 5A4I (D574N), 5A4M (deltaTM) and 5ADU (D118N/D574N).
We thank Diamond Light Source for beam time proposal mx9306 and the staff of beamlines i02, i04 and i04-1 for their assistance during data collection.
This work was supported by the U.K. Biological and Biotechnology Sciences Research Council [grant numbers BB/I022309-1, BB/L009722/1 (to F.A.A.) and BB/L008521/1 (to F.S.)]; the U.K. Medical Research Council [grant number G0901251 (to S.E.V.P. and S.B.C.)]; the Global Innovation Initiative (to E.J.B.); the U.K. Engineering and Physical Sciences Research Council [grant number EP/H019480/1]; and the Royal Society Wolfson Research Merit Award (to F.A.A.).
Membrane Proteins From A to Z: Held at University of Leeds, U.K., 16–17 December 2015