Laser‐flash photolysis indicates that internal electron transfer is triggered by proton uptake by Alcaligenes xylosoxidans copper‐dependent nitrite reductase

Enzyme‐catalysed electron transfer reactions are often controlled by protein motions and coupled to chemical change such as proton transfer. We have investigated the nature of this control in the blue copper‐dependent nitrite reductase from Alcaligenes xylosoxidans ( Ax NiR). Inter‐Cu electron transfer from the T1Cu site to the T2Cu catalytic site in Ax NiR occurs via a proton‐coupled electron transfer mechanism. Here we have studied the kinetics of both electron and proton transfer independently using laser‐flash photolysis for native Ax NiR and its proton‐channel mutant N90S. In native Ax NiR, both inter‐Cu electron transfer and proton transfer exhibit similar rates, and show an unusual dependence on the nitrite concentration. An initial decrease in the observed rates at low nitrite concentrations is followed by an increase in the observed rates at high nitrite concentrations (> 5 m m ). In N90S, in which the T1Cu reduction potential is elevated by 60 mV, no inter‐Cu electron transfer or proton transfer was observed in the absence of nitrite. Only in the presence of nitrite were both processes detected, with similar [nitrite] dependence, but the nitrite dependence was different compared with native enzyme. The substrate dependence in N90S was similar to that observed in steady‐state assays, suggesting that this substitution resulted in proton‐coupled electron transfer becoming rate‐limiting. A pH perturbation experiment with native Ax NiR revealed that protonation triggers inter‐Cu electron transfer and generation of NO. Our results show a strong coupling of inter‐Cu electron transfer and proton transfer for both native Ax NiR and N90S, and provide novel insights into the controlled delivery of electrons and protons to the substrate‐utilization T2Cu active site of Ax NiR.