The interaction of hydroxylamine (HA) with Arthromyces ramosus peroxidase (ARP) was investigated by kinetic, spectroscopic, and x-ray crystallographic techniques. HA inhibited the reaction of native ARP with H2O2 in a competitive manner. Electron absorption and resonance Raman spectroscopic studies indicated that pentacoordinate high spin species of native ARP are converted to hexacoordinate low spin species upon the addition of HA, strongly suggesting the occurrence of a direct interaction of HA with ARP heine iron. Kinetic analysis exhibited that the apparent dissociation constant is 6.2 mM at pH 7.0 and that only one HA molecule likely binds to the vicinity of the heme. pH dependence of HA binding suggested that the nitrogen atom of HA could be involved in the interaction with the heine iron. X-ray crystallographic analysis of ARP in complex with HA at 2.0 Å resolution revealed that the electron density ascribed to HA is located in the distal pocket between the heine iron and the distal His56. HA seems to directly interact with the heine iron but is too far away to interact with Arg52. In HA, it is likely that the nitrogen atom is coordinated to the heine iron and that hydroxyl group is hydrogen bonded to the distal His56.
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