Regulatory Mechanisms and Synergistic Enhancement of the Diiron YtfE Protein in Nitric Oxide Reduction.

The diiron-containing YtfE protein in Escherichia coli is pivotal in counteracting nitrosative stress, a critical barrier to bacterial viability. This study delves into the biochemical complexity governing YtfE's conversion of nitric oxide (NO) to nitrous oxide, a key process for alleviating nitrosative stress. Through site-directed mutagenesis, we explored YtfE's molecular structure, with a particular focus on two internal transport tunnels important for its activity. Our findings illuminate Tunnel 1 as the primary conduit for substrate transport, regulated by conformational shifts within the N-terminal domain that enable substrate access to the diiron core in the C-terminal domain. Tunnel 2 emerges as a secondary, supportive route, activated when Tunnel 1 is compromised. This result challenges a previous model of distinct tunnels for substrate entry and product exit, suggesting both tunnels are capable of transporting substrates and products. Our engineering efforts enhanced the role of Tunnel 2, enabling a synergistic operation with Tunnel 1 and tripling YtfE's enzymatic activity compared to its wild-type form. This research not only deepens our understanding of YtfE's regulatory mechanism for NO reduction but also introduces a strategy to amplify its enzymatic efficiency. The outcomes offer new ravenues for modulating bacterial stress responses.