Abstract
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 H(2)O(2) 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 heme 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 heme iron. X-ray crystallographic analysis of ARP in complex with HA at 2.0 A resolution revealed that the electron density ascribed to HA is located in the distal pocket between the heme iron and the distal His(56). HA seems to directly interact with the heme iron but is too far away to interact with Arg(52). In HA, it is likely that the nitrogen atom is coordinated to the heme iron and that hydroxyl group is hydrogen bonded to the distal His(56).
Highlights
The Soret peak of HA complex was not shifted in the pH range of 5– 8 and was stable for several hours
We reported that the binding of ammonia to the heme iron of ARP (32), and the direct interaction of HA is not surprising
Substrate Binding Site of ARP—The crystal structure of ARP-BHA complex reported by Itakura et al (20) suggests that one of the major factors controlling the binding mode of BHA with ARP is the hydrogen bonding network caused by the side chain functional groups and distal amino acid residues
Summary
In addition to cytochrome c peroxidase, whose structure has been known for over 10 years (1), the structures of lignin peroxidase (LiP)[1] (2, 3), Arthromyces ramosus peroxidase (ARP) (4), manganese peroxidase (5), peanut peroxidase (6), chloroperoxidase, (7), barley peroxidase 1 (8), ascorbate peroxidase (9), and horseradish peroxidase (HRP) (10) are available From the inhibitory damage to the heme of HRP, it has been suggested that the binding sites of the aromatic substrates are located near the heme in the vicinity of the ␦-meso carbon and 8-methyl group of the heme (15, 16). This region is exposed to solvent in many peroxidases. For HRP, the binding mode of the naturally occurring substrate, ferulic acid, to the
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