title: The substrate-bound type 2 copper site of nitrite reductase: the nitrogen hyperfine coupling of nitrite revealed by pulsed EPR.
abstract: 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 (14NO2(-) and 15NO2(-)). The HYSCORE experiments displayed a signal of 15NO2(-) 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 (14N) 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.
authors.affiliation: Department of Molecular Physics, Huygens Laboratory, Leiden University, P.O. Box 9504, The Netherlands.
authors.forename: Maria
authors.initials: M
authors.lastname: Fittipaldi
authors.forename: Hein J
authors.initials: HJ
authors.lastname: Wijma
authors.forename: Martin P
authors.initials: MP
authors.lastname: Verbeet
authors.forename: Gerard W
authors.initials: GW
authors.lastname: Canters
authors.forename: Edgar J J
authors.initials: EJ
authors.lastname: Groenen
authors.forename: Martina
authors.initials: M
authors.lastname: Huber
mesh_terms: D042481:Alcaligenes faecalis / Q000201:enzymology; D001665:Binding Sites; D003300:Copper / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics
chemical_list: D003300:Copper; D009572:Nitrite Reductases