EPR AND ELECTRON-NUCLEAR DOUBLE-RESONANCE (ENDOR) STUDIES SHOW NITRITE BINDING TO THE TYPE-2 COPPER CENTERS OF THE DISSIMILATORY NITRITE REDUCTASE OF ALCALIGENES XYLOSOXIDANS (NCIMB-11015)

EPR and H-1,N-14,N-15 ENDOR spectra are described for the type 1 and t ype 2 Cu(II) centers of dissimilatory nitrite reductase (NiR) from Alc aligenes xylosoxidans. The study was carried out on preparations of Ni R containing both type 1 and type 2 Cu sites, and also on preparations of lower activity which contained essentially only type 1 Cu centers. This has enabled ENDOR studies of type 1 and type 2 sites to be carri ed out largely independently of each other, by appropriate choice of t he excitation field. Spectra were recorded both in the absence and pre sence of nitrite, allowing a clear determination of which of the two t ypes of Cu center constitutes the substrate binding site. The EPR resu lts show large changes in the type 2 site g(parallel to) (which decrea ses by 0.065) and (Cu)A(parallel to) (which increases by 2.0 mT) while the type 1 site EPR is not affected. In addition, both H-1 and N-14 E NDOR of the type 2 Cu site undergo considerable changes on addition of nitrite whereas the type 1 Cu site ENDOR is unaffected. Our results c learly demonstrate that nitrite binds to the type 2 copper and that th is process significantly perturbs the ligation of this copper by the p rotein histidine residues. No N-15 ENDOR resonances were observed from N-15 nitrite. The accessibility of the copper sites to solvent has be en studied using (H2O)-H-2. The results indicate that nitrite binds to the type 2 Cu by displacing a proton, probably on a water molecule bo und to the copper atom.