Importance of the Active Site “Canopy” Residues in an O₂-Tolerant [NiFe]-Hydrogenase

Abstract

The active site of Hyd-1, an oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Escherichia coli, contains four highly conserved residues that form a "canopy" above the bimetallic center, closest to the site at which exogenous agents CO and O 2 interact, substrate H 2 binds, and a hydrido intermediate is stabilized. Genetic modification of the Hyd-1 canopy has allowed the first systematic and detailed kinetic and structural investigation of the influence of the immediate outer coordination shell on H 2 activation. The central canopy residue, arginine 509, suspends a guanidine/guanidinium side chain at close range above the open coordination site lying between the Ni and Fe atoms (N−metal distance of 4.4 Å): its replacement with lysine lowers the H 2 oxidation rate by nearly 2 orders of magnitude and markedly decreases the H 2 /D 2 kinetic isotope effect. Importantly, this collapse in rate constant can now be ascribed to a very unfavorable activation entropy (easily overriding the more favorable activation enthalpy of the R509K variant). The second most important canopy residue for H 2 oxidation is aspartate 118, which forms a salt bridge to the arginine 509 headgroup: its mutation to alanine greatly decreases the H 2 oxidation efficiency, observed as a 10-fold increase in the potential-dependent Michaelis constant. Mutations of aspartate 574 (also salt-bridged to R509) to asparagine and proline 508 to alanine have much smaller effects on kinetic properties. None of the mutations significantly increase sensitivity to CO, but neutralizing the expected negative charges from D118 and D574 decreases O 2 tolerance by stabilizing the oxidized resting Ni III −OH state ("Ni-B"). An extensive model of the catalytic importance of residues close to the active site now emerges, whereby a conserved gas channel culminates in the arginine headgroup suspended above the Ni and Fe.

Frustration across the periodic table: heterolytic cleavage of dihydrogen by metal complexes

“…[41][42][43][44]. Work by Armstrong and co-workers [37] suggests that a basic residue in close proximity to the bimetallic active site of the [NiFe]-hydrogenase could serve as a base for H 2 heterolysis, which would more closely resemble the proposed mechanism for the…”

“…(Online version in colour.) (e) Biologically inspired systems (i) Models of [FeFe]-hydrogenase The [FeFe]-hydrogenase (figure 2) reversibly and catalytically cleaves H 2 , and hydrogenases have been recognized as exhibiting FLP reactivity[36,37]. The remarkable identification of organometallic compounds in nature[38][39][40] spawned research by many groups worldwide to…”

The structure of hydrogenase-2 from Escherichia coli: implications for H2-driven proton pumping

“…The main compartment, equipped with gas inlet and outlet ports and typically accommodating 2 ml of buffer-electrolyte solution, housed the pyrolytic graphite ‘edge’ (PGE) working electrode (which could be rotated at high speed) and a Pt counter electrode. For each experiment, the PGE (geometric surface area 0.03 cm 2 ) was sanded (Hookit P400 sand paper), and hydrogenase solution (0.5–2 µl at 1–5 mg/ml) was applied by repeated ‘add/withdraw’ pipetting for 30 s as previously described [ 25 ]. The electrode was then rinsed with ultrapure H 2 O (Millipore, 18 MΩ cm) to remove unabsorbed protein and attached to the electrode rotator motor.…”

“…Solution assays for determining the rate of H 2 oxidation were carried out following an established procedure [ 25 ]. A micro-Eppendorf tube of the enzyme was placed under a 100% H 2 environment for 12–18 h to ensure full reductive activation of the enzyme.…”

O2 sensitivity and H2 production activity of hydrogenases—A review

“…To uncover the molecular mechanism of O 2 sensitivity, efforts have been made through experimental and theoretical exploration on hydrogenases in the past over 30 years (Abou Hamdan et al, ). Comprehensive genetic, biochemical, electrochemical, spectroscopic and crystallographic investigations on hydrogenases from different organisms have revealed the role of active site, the involvement of chaperones, water movement via hydrophilic cavities, the influence of gas diffusion, proton transfer pathways, dependence on quaternary structure, and so on (Brooke et al, ; Fritsch et al, ; Pandelia et al, ; Wulff, Thomas, Sargent, & Armstrong, ). To better understand the mechanism of O 2 diffusion, molecular dynamic simulation, density functional theory calculations, volumetric solvent accessibility maps, and other computational analyses have been conducted.…”