Mechanism of hydrogen activation by [NiFe] hydrogenases

Abstract

The active site of [NiFe] hydrogenases contains a strictly conserved arginine that suspends a guanidine nitrogen atom <4.5 Å above the nickel and iron atoms. The guanidine headgroup interacts with the side chains of two conserved aspartic acid residues to complete an outer-shell canopy that has thus far proved intractable to investigation by site-directed mutagenesis. Using hydrogenase-1 from Escherichia coli, the strictly conserved residues R509 and D574 have been replaced by lysine (R509K) and asparagine (D574N) and the highly conserved D118 has been replaced by alanine (D118A) or asparagine (D118N/D574N). Each enzyme variant is stable, and their [(RS)2Niμ(SR)2Fe(CO)(CN)2] inner coordination shells are virtually unchanged. The R509K variant had >100-fold lower activity than native enzyme. Conversely, the variants D574N, D118A and D118N/D574N, in which the position of the guanidine headgroup is retained, showed 83%, 26% and 20% activity, respectively. The special kinetic requirement for R509 implicates the suspended guanidine group as the general base in H2 activation by [NiFe] hydrogenases.

Proton Inventory and Dynamics in the Nia-S to Nia-C Transition of a [NiFe] Hydrogenase

“…6,20,66−70 Recently, a conserved arginine residue located above the bridgehead position has been proposed as the initial base. 55 In Pf SHI, this is an unlikely base in the Additionally, the observed pK a of ∼7, relating the ground state Δν CO peak position, photoproduct distribution, and EPT activity, is well below the known pK a range for arginine residues within protein structures. 71 Our results do not preclude the involvement of this arginine in the heterolytic cleavage reaction, however, by a transient frustrated Lewis pair mechanism.…”

Hydrogenase Enzymes and Their Synthetic Models: The Role of Metal Hydrides

“…Indeed, aside from the terminal thiolates, an Arg509 residue proximal to Ni represents another viable H + relay (vide infra). 313 In any case, the situation is much clearer when it comes to electron transport, with a chain of 4Fe–4S, 3Fe–4S, and 4Fe–4S clusters clearly defining a tunneling path to and from the active site.…”

“…Notably, Arg509 is highly conserved, and its basic guanidine group (p K a ≈ 13.8) 332 is only 4.5 Å away from Ni. 313 …”

Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy

“…54 The guanidinium side chain of this arginine residue suspends a nitrogen atom 4.5 Å above the bridging coordination position of the Ni-Fe active site in the wild type enzyme (Figure 6A). Substitution of the arginine residue for lysine generated a variant, R509K, with 100-fold lower H 2 oxidation activity than wild type Hyd-1 (Figure 6B) despite the structure of the inner coordination sphere of the active site being virtually unchanged (Figure 6C).…”

“…35 More transient states have also been probed in time-resolved infrared spectroscopic techniques in which transitions between states of a hydrogenase have been triggered by the release of caged electrons or by photolysis of light-sensitive states of the enzyme. 41,42,60 Attention has also focused on the role of conserved amino acids in the region around the active site in controlling proton transfer, 12,54,61−63 and the correlation between the redox state of the proximal cluster and that of the active site. 36,60 These studies have been aided by the availability of site-directed variants of [NiFe] hydrogenases with particular amino acid exchanges in the region of the active site and the proximal iron sulfur cluster.…”