A Dynamic Water Channel Affects O₂ Stability in [FeFe]-Hydrogenases

Oxygen sensitivity remains a major limitation for [FeFe]-hydrogenases, enzymes that catalyze hydrogen production. These enzymes contain a H-cluster cofactor with a [4Fe4S] subunit and a diiron center. Prior research has shown that molecular oxygen can damage the H-cluster, especially the diiron portion. Existing strategies have focused on blocking hydrophobic gas channels to prevent O₂ diffusion. However, it was already known that these approaches have had limited success. This gap motivated an investigation into alternative O₂ diffusion pathways. No prior work had resolved the role of water channels in O₂ transport. Recent studies have suggested that water channels may also contribute to O₂ access to the H-cluster. That uncertainty drove the current work to explore a novel hydrophilic pathway.

This study aimed to identify and block an alternative O₂ diffusion route to improve [FeFe]-hydrogenase stability. The researchers hypothesized that a water channel might serve as a conduit for O₂ to reach the H-cluster. The specific problem addressed was the incomplete understanding of O₂ access routes to the 4Fe_H subcluster. The motivation was to develop more effective strategies for O₂ tolerance in these enzymes. By focusing on a newly identified water channel, the team sought to enhance the enzyme's resistance to O₂-induced damage. The study tested whether mutagenesis could alter the channel's properties to reduce O₂ permeability. The goal was to determine if this approach could lead to improved O₂ stability. The findings could inform future enzyme engineering efforts.

The researchers used molecular dynamics (MD) simulations to identify a novel water channel (W_H) near the H-cluster. They analyzed high-resolution crystal structures to map the channel’s location and composition. Site-directed mutagenesis was applied to amino acids along W_H in proximity to the 4Fe_H subcluster. The mutations targeted residues G302 and S357 to alter the channel’s hydrophilic properties. Protein film electrochemistry experiments were conducted to assess O₂ stability in the modified enzymes. The electrochemical data provided quantitative measures of O₂ tolerance. MD simulations were repeated with the mutated structures to confirm structural changes. The results were compared to wild-type enzyme behavior to evaluate the effectiveness of the mutations.

The study found that variants G302S and S357T showed increased O₂ stability compared to wild-type enzymes. MD simulations revealed a novel water channel (W_H) surrounding the H-cluster. The channel was identified as a possible O₂ diffusion pathway to the 4Fe_H subcluster. Protein film electrochemistry confirmed that the mutations improved O₂ tolerance. The simulations showed enhanced local sieving effects for O₂ near the 4Fe_H in both variants. The results suggest that O₂ diffuses from the 4Fe_H to the 2Fe_H in wild-type enzymes. The mutations appear to block this diffusion route by altering the water channel’s structure. These findings indicate that the water channel plays a critical role in O₂ transport.

The authors propose that the newly identified water channel (W_H) serves as a conduit for O₂ to reach the H-cluster. The study suggests that blocking this channel can improve O₂ stability in [FeFe]-hydrogenases. The results indicate that O₂ diffuses from the 4Fe_H to the 2Fe_H in wild-type enzymes. The mutations tested in this work appear to reduce this diffusion by altering the channel’s properties. The findings reveal a new strategy for improving O₂ tolerance in these enzymes. The authors suggest that focusing on the O₂ diffusion network near the active site is a promising approach. The study supports the idea that water channels can be targeted to enhance enzyme stability. These conclusions are based on the observed effects of the mutations and the supporting MD simulations.