The Substrate-Bound Type 2 Copper Site of Nitrite Reductase: The Nitrogen Hyperfine Coupling of Nitrite Revealed by Pulsed EPR†

A pulsed electron paramagnetic resonance study has been performed on the type 2 copper site of nitrite reductase (NiR) from Alcaligenes faecalis. The H145A mutant, in which histidine 145 is replaced by alanine, was studied by ESEEM and HYSCORE experiments at 9 GHz on frozen solutions. This mutant contains a reduced type 1 copper site which allowed a selective investigation of the type 2 site of H145A and of its nitrite-bound form H145A (NO2-). The experiments yielded hyperfine and quadrupole parameters of the remote nitrogens of two of the histidines in the type 2 copper site of the protein and revealed the changes of these values induced by substrate binding ((NO2-)-N-14 and (NO2-)-N-15). The HYSCORE experiments displayed a signal of (NO2-)-N-15 bound to H145A, from which hyperfine parameters of the nitrite nitrogen were estimated. The small isotropic hyperfine coupling, 0.36 MHz, of the nitrite nitrogen (N-14) suggests that the substrate binds in an axial position to the copper in the type 2 site and that the molecular orbital containing the unpaired electron extends onto the substrate. This and other changes in the EPR parameters occurring after nitrite binding suggest a change in electronic structure of the site, which most likely prepares the site for the catalytic reaction. We propose that this change is essential for the reaction to occur.