Formation of an oxygen-bridged adduct between the secondary amine of BU72 and the imidazole ring of His54 would require harsh conditions. Reactive oxygen species (ROS), for instance, can oxidize secondary amines and histidine . But how might these arise? Surprisingly, several potential sources of ROS were present. The BU72-μOR complex was purified and crystallized in HEPES buffer, which generates hydrogen peroxide on exposure to light . HEPES has also been reported to enhance metal-catalyzed generation of other ROS from hydrogen peroxide . A further potential source is the N-terminus, which contains a sequence motif known to generate ROS. The N-terminus used was truncated, leaving glycine as the first residue and histidine as the third . This sequence motif (Gly-Xaa-His) forms redox-active nickel coordination complexes . Moreover, a nickel affinity column was used for purification , and the Gly-Xaa-His motif can capture Ni 2+ ions from these columns . The resulting square planar nickel complexes catalyze the decomposition of hydrogen peroxide to other ROS such as hydroxyl radicals . Thus, the conditions used were sufficient to generate ROS immediately adjacent to His54, potentially oxidizing both the residue itself and BU72.