文摘
A pulsed electron paramagnetic resonance study has been performed on the type 2 copper siteof nitrite reductase (NiR) from Alcaligenes faecalis. The H145A mutant, in which histidine 145 is replacedby alanine, was studied by ESEEM and HYSCORE experiments at 9 GHz on frozen solutions. Thismutant contains a reduced type 1 copper site which allowed a selective investigation of the type 2 site ofH145A and of its nitrite-bound form H145A (NO2-). The experiments yielded hyperfine and quadrupoleparameters of the remote nitrogens of two of the histidines in the type 2 copper site of the protein andrevealed the changes of these values induced by substrate binding (14NO2- and 15NO2-). The HYSCOREexperiments displayed a signal of 15NO2- bound to H145A, from which hyperfine parameters of thenitrite nitrogen were estimated. The small isotropic hyperfine coupling, 0.36 MHz, of the nitrite nitrogen(14N) suggests that the substrate binds in an axial position to the copper in the type 2 site and that themolecular orbital containing the unpaired electron extends onto the substrate. This and other changes inthe 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 forthe reaction to occur.