The Long-Range High-pH Proton Channel Facilitates the Nitrite Reduction in Copper-Containing Nitrite Reductase

Copper-containing nitrite reductase (CuNiR) catalyzes the conversion of nitrite (NO₂ ^-) to nitric oxide (NO), requiring sequential proton delivery through long-range high-pH and primary proton channels. But the proton transfer (PT) and electron transfer (ET) mechanisms during the critical second protonation remain unclear. Here, we investigate these bioenergetic mechanisms using a two-layer ONIOM approach. Results reveal distinct pathways: the high-pH channel features a water-mediated three-stage PT from Glu113 to W_S3, followed by direct PT to Asp98 and proton-coupled β-ET to nitrous acid (HNO₂). In contrast, the primary channel proceeds via water-mediated three-stage PTs from Lys128 to His255 and then a water-mediated two-stage proton-coupled β-ET to HNO₂. Comparative energy analysis shows that the second protonation of NO₂ ^- is the overall rate-limiting step with the long-range high-pH channel serving as the preferred pathway in CuNiR catalysis. These fundamental mechanisms of CuNiR deepen our understanding of the biological denitrification.