ABSTRACT
Copper-containing nitrite reductases (CuNIRs) are multifunctional enzymes that catalyse the one-electron reduction of nitrite (NO2-) to nitric oxide (NO) and the two-electron reduction of dioxygen (O2) to hydrogen peroxide (H2O2). In contrast to the mechanism of nitrite reduction, that of dioxygen reduction is poorly understood. Here, results from anaerobic synchrotron-radiation crystallography (SRX) and aerobic in-house radiation crystallography (iHRX) with a CuNIR from the thermophile Geobacillus thermodenitrificans (GtNIR) support the hypothesis that the dioxygen present in an aerobically manipulated crystal can bind to the catalytic type 2 copper (T2Cu) site of GtNIR during SRX experiments. The anaerobic SRX structure showed a dual conformation of one water molecule as an axial ligand in the T2Cu site, while previous aerobic SRX GtNIR structures were refined as diatomic molecule-bound states. Moreover, an SRX structure of the C135A mutant of GtNIR with peroxide bound to the T2Cu atom was determined. The peroxide molecule was mainly observed in a side-on binding manner, with a possible minor end-on conformation. The structures provide insights into dioxygen chemistry in CuNIRs and hence help to unmask the other face of CuNIRs.
