Coordination-Sphere Perturbations Partition Catalytic Bias Control in a Thermostable [FeFe]-Hydrogenase

Technical Summary: Coordination-Sphere Perturbations Partition Catalytic Bias Control in a Thermostable [FeFe]-Hydrogenase

Problem Statement
While [FeFe]-hydrogenases are renowned for their high turnover frequencies in reversible hydrogen interconversion, the molecular mechanisms governing their "catalytic bias" (the preference for hydrogen oxidation versus proton reduction) remain incompletely understood. Existing models largely attribute catalytic bias to the thermodynamic gating of the distal iron-sulfur (FeS) cluster, specifically its redox potential relative to the $H_2/H^+$ couple. However, these macroscopic models often overlook the microscopic contributions of the first- and second-coordination spheres of the FeS clusters, the specific roles of individual ligands, and the integrated behavior of the entire electron-transfer network. There is a need to determine whether local structural perturbations, independent of large thermodynamic shifts, can fine-tune catalytic directionality through kinetic and electrostatic mechanisms.

Methodology
The study utilized a thermostable [FeFe]-hydrogenase module (TkHydA2) derived from the hydrogen-dependent carbon dioxide reductase (HDCR) of Thermoanaerobacter kivui as the primary model system. This enzyme was selected for its complete active-site architecture, including the H-cluster, a proximal [4Fe-4S]H cluster, and two additional F-clusters, offering a simplified yet complete electron-transfer chain.

The researchers employed a systematic rational design approach involving site-directed mutagenesis of:

1. Distal FeS Cluster ($FeS_{en/ex}$): Mutations in the second-coordination sphere (specifically R20) and the first-coordination sphere (cysteine ligands C15, C18, C21, C55).
2. Proximal [4Fe-4S]H Cluster: Mutations in the first-coordination sphere (C383) and second-coordination sphere (T382).

Comparative analyses were conducted against the homologous Acetobacterium woodii hydrogenase (AwHydA2). The experimental toolkit included:

• Protein Film Electrochemistry (PFE): Cyclic voltammetry (CV) and rotating disk electrode measurements to assess catalytic bias, overpotential, and turnover rates across varying pH and temperatures.
• EPR Spectroscopy: To probe the electronic structure, spin states, and ligand-field perturbations of the FeS clusters in oxidized and reduced states.
• Electrostatic Potential Mapping: Computational modeling (APBS) to visualize changes in the electrostatic landscape.
• Kinetic Modeling: Application of the Eyring equation and a unified electrocatalytic model (Armstrong et al.) to deconvolute activation enthalpies ($\Delta H^\ddagger$) for interfacial electron transfer versus intramolecular catalytic steps.

Key Results
The study revealed that catalytic bias is governed by a spatially partitioned, hierarchical control system rather than a single thermodynamic determinant:

1. Distal $FeS_{en/ex}$ as a Reductive-Bias Module:

   • Second-Coordination Sphere (R20): The R20E mutation (introducing a negative charge) shifted the bias toward proton reduction by suppressing hydrogen oxidation. This was driven by local electrostatic repulsion and kinetic modulation of electron transfer rates, not a shift in the cluster's redox potential. The R20 residue acts as an electrostatic switch; its positive charge in the wild type facilitates electron injection.
   • First-Coordination Sphere (C55H): Substitution of a coordinating cysteine with histidine (C55H) also shifted bias toward proton reduction but fundamentally altered the reaction kinetics. Unlike [NiFe]-hydrogenases where Cys→His shifts potentials positively, in TkHydA2, this mutation created a kinetic bottleneck at the interface, converting the voltammetric response from sigmoidal (fast/reversible) to linear (slow/quasi-reversible) without significantly changing the apparent equilibrium potential.

2. Proximal [4Fe-4S]H as an Oxidative-Bias Module:

   • First-Coordination Sphere (C383H): Mutating the proximal cluster ligand C383 to histidine induced a positive shift in the apparent equilibrium potential (~50 mV) and strongly favored hydrogen oxidation. EPR and modeling indicated this resulted from the histidine imidazole ring withdrawing electron density, stabilizing the reduced state, and raising the potential of the [4Fe-4S]H cluster. This created a thermodynamic barrier for electron injection from the electrode, effectively retuning the entire network toward oxidation.
   • Second-Coordination Sphere (T382A, C383A): Mutations in the immediate vicinity of the proximal cluster (T382A) enhanced oxidative bias without altering the overpotential. These changes were attributed to electrostatic reorganization and the modulation of proton-coupled electron transfer pathways, acting as "fine-tuners" that preserve enzyme stability while shifting directionality.

3. Kinetic vs. Thermodynamic Control:
   Kinetic analysis demonstrated that bias shifts in these mutants were primarily driven by changes in activation enthalpies for interfacial electron transfer and intramolecular catalytic steps, rather than large shifts in the redox potentials of the distal clusters. The distal cluster primarily regulates interfacial electron transfer rates, while the proximal cluster influences the thermodynamic landscape of the electron relay.

Significance and Claims
The authors claim that this work establishes a "distributed regulation model" for [FeFe]-hydrogenase catalytic bias. The study challenges the prevailing view that bias is solely determined by the redox potential of the distal electron entry/exit site. Instead, it demonstrates that:

• Catalytic bias emerges from the cooperative regulation of multiple components within the electron-transfer network.
• The distal and proximal FeS clusters function as distinct regulatory modules (reductive-bias vs. oxidative-bias).
• Local coordination-sphere perturbations can partition control between kinetic gating (first-coordination sphere) and electrostatic tuning (second-coordination sphere).
• This framework provides a refined basis for the rational design of hydrogenases with tailored catalytic properties, moving beyond simple thermodynamic tuning to include kinetic and electrostatic engineering of the entire electron-transfer chain.

The paper concludes that understanding these spatially partitioned mechanisms is essential for advancing the bioenergetic understanding of hydrogenases and for developing optimized biocatalysts for biotechnological hydrogen production.