Direct Binding of Hydroxylamine to the Heme Iron ofArthromyces ramosus Peroxidase

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 Å 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

Influence of the Unusual Covalent Adduct on the Kinetics and Formation of Radical Intermediates in Synechocystis Catalase Peroxidase

“…The spectrum of the intermediate, which was observed upon incubation of both Arg 439 variants with excess hydrogen peroxide (bands at 414, 542, and 572 nm; Fig. 4, C and D), was very similar to that of the hexacoordinated low-spin species obtained upon mixing of hydroxylamine with Arthromyces ramosus peroxidase (29). This was interpreted as a structural model for compound 0, normally difficult to observe because its formation is followed by the rapid heterolytic cleavage of hydrogen peroxide.…”

Hydroxylamines and Oximes: Biological Properties and Potential Uses as Therapeutic Agents

“…This is achieved by the unique structure of the HAO protein that permits electron transfer between the P460 and other heme moieties of the enzyme 33 . The crystallographic analysis of a fungal peroxidase complex with HA provided evidence that HA interacts directly with the heme via coordination of the nitrogen atom to the heme iron 65,66 . The binding mode of HA was suggested by the authors to simulate the binding of the natural substrate H 2 O 2 to peroxidase, which is consistent with the observation that HA is a competitive inhibitor of hydrogen peroxide 65 .…”

“…Sarnoff suggested that, in addition to its role in the induction of formation of methemoglobin, HA may also act as a respiratory stimulant in cyanide poisoning, but offered no mechanism to explain how this might occur 84 . Since HA was demonstrated to bind directly to the iron atom of a peroxidase, as does cyanide 65,66 , it may compete out cyanide from cytochrome oxidase, and thus contribute to its overall antidotal activity.…”

Hydroxylamine and hydrazine bind directly to the heme iron of the heme–heme oxygenase-1 complex

“…The pH dependency of the K D;app for both amines was analyzed by the following equations [13] on the basis of the assumption that only the neutral form of amines is competent to bind to the heme in HO. For example, the NH 2 OH form of HA is assumed to bind to the heme,…”

“…These two amines, both of which serve as reductants for the aerobic HO reaction, might provide insight into the nature of the hydroperoxide species if stable adducts could be formed between the amines and the heme bound to HO. HA does indeed bind to the heme iron of Arthromyces ramosus peroxidase [13]. In this study, we investigated the anaerobic binding of HA and HZ to heme in a complex with recombinant rat HO-1 (rHO-1) in which the C-terminal membrane-spanning region had been truncated.…”