New Insights into the Role of Distal Histidine Flexibility in Ligand Stabilization of Dehaloperoxidase−Hemoglobin from Amphitrite ornata

Abstract

The present work highlights the important role played by the distal histidine in controlling the binding of heme ligands in dehaloperoxidase (DHP) as compared to myoglobin and peroxidases. In DHP the distal histidine is highly mobile and undergoes a conformational change that places it within hydrogen-bonding distance of anionic ligands and water, where strong hydrogen bonding can occur. The detailed resonance Raman (RR) analysis at room temperature shows the presence of an equilibrium between a 5-coordinate and a 6-coordinate (aquo) high-spin form. The equilibrium shifts toward the aquo form at 12 K. These two forms are consistent with the existing X-ray structures where a closed conformation, with His55 positioned in the distal pocket and H-bonded with the distal water molecule (6-coordinate), and an open solvent-exposed conformation, with the His55 displaced from the distal pocket (5-coordinate form), are in equilibrium. Moreover, the comparison between the Raman data at 298 and 12 K and the results obtained by EPR of DHP in the presence of 4-iodophenol highlights the formation of a pure 5-coordinate high-spin form (open conformation). The data reported herein support the role of His55 in facilitating the interaction of substrate and inhibitor in the regulation of enzyme function, as previously suggested. The two conformations of His55 in equilibrium at room temperature provide a level of control that permits the distal histidine to act as both the acid-base catalyst in the peroxidase mechanism and the stabilizing amino acid for exogenous heme-coordinated ligands.

Kinetic Analysis of a Naturally Occurring Bioremediation Enzyme: Dehaloperoxidase-Hemoglobin from Amphitrite ornata

“…The crude DHP A solution was applied to a Ni-NTA agarose column (5 Prime Perfect Pro), washed with washing buffer (50 mM NaH 2 PO 4 300 mM NaCl, 20 mM imidazole, pH 8), and eluted using elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, pH 8). The isolated 6XHis DHP A from the column was oxidized by excess 10 mM K 3 [Fe(CN) 6 ]. Excess K 3 [Fe(CN) 6 ] was removed by gel filtration on a Sephadex G-25 column, which permitted simultaneous buffer exchange into 20 mM KH 2 PO 4 pH 6 buffer.…”

“…The isolated 6XHis DHP A from the column was oxidized by excess 10 mM K 3 [Fe(CN) 6 ]. Excess K 3 [Fe(CN) 6 ] was removed by gel filtration on a Sephadex G-25 column, which permitted simultaneous buffer exchange into 20 mM KH 2 PO 4 pH 6 buffer. The oxidized protein was loaded onto a CM-52 column for further purification.…”

“…Purified 6XHisDHP A was oxidized in the presence of 10 mM K 3 [Fe(CN) 6 ], separated from excess K 3 [Fe(CN) 6 ] by gel filtration on Sephadex G-25, and further purified on CM-52 (as above) prior to each experiment. For kinetic assays the elution buffer was 150 mM KH 2 PO 4 buffer at pH 7.0.…”

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

“…laser (Innova 90/5, Coherent). A detailed description of the room and low-temperature RR experimental procedures has been reported previously [8]. The measurements at 4°C were obtained using a thermostatically controlled water bath.…”

“…EPR spectra were recorded as reported previously [8]. Kinetic measurements were carried out employing a rapid-mixing stopped flow (SX.18MV, Applied Photophysics, Salisbury, UK) with a 1 ms dead time.…”