Residues in the Distal Heme Pocket of Neuroglobin: IMPLICATIONS FOR THE MULTIPLE LIGAND BINDING STEPS

Hideaki Sato,Anthony J Wilkinson,Yoshikazu Tomisugi,Tadayuki Uno,Hiroko Tsutsumi,Takashi Hayashi,Yoshinobu Ishikawa,Daisuke Ryu

doi:10.1074/jbc.m311748200

Hideaki Sato, Anthony J Wilkinson + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m311748200

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Abstract

Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wild-type neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His96 by alanine leads to complete loss of heme, indicating that His96 is the proximal ligand. The resonance Raman spectra of M69L and K67T mutants were similar to those of wild-type (WT) neuroglobin in both ferric and ferrous states. By contrast, H64V was six-coordinate high-spin and five-coordinate high-spin in the ferric and ferrous states, respectively, at acidic pH. The spectra were pH-dependent and six-coordinate with the low-spin component dominating at alkaline pH. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states. This implies that His64 is the endogenous ligand and that Lys67 is situated nearby in the distal pocket. In the ferrous H64V and H64V/K67T mutants, the nu(Fe-His) stretching frequency appears at 221 cm(-1), which is similar to that of deoxymyoglobin. In the ferrous CO-bound state, the nu(Fe-CO) stretching frequency was detected at 521 and 494 cm(-1) in WT, M69L, and K67T, while only the 494 cm(-1) component was detected in the H64V and H64V/K67T mutants. Thus, the 521 cm(-1) component is attributed to the presence of polar His64. The CO binding kinetics were biphasic for WT, H64V, and K67T and monophasic for H64V/K67T. Thus, His64 and Lys67 comprise a unique distal heme pocket in neuroglobin.

Highlights

Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods
If massive induction occurs can it be envisaged that the concentration of Ngb would reach levels that are sufficient to sustain an aerobic metabolism during temporary hypoxia
The QuikChange system (Stratagene) was used to introduce mutations into the Ngb coding sequence, which directed the expression of Ngbs with H64V, K67T, M69L, histidine 96 with alanine (H96A), and H64V/K67T mutations

Summary

Introduction

Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states This implies that His is the endogenous ligand and that Lys is situated nearby in the distal pocket. A key feature of Ngb is its six-coordinate heme structure, both in the ferrous and ferric states, with the distal histidine (His64) forming an endogenous ligand [3, 10, 25, 26]. Studies of ligand binding over a wide range of temperatures reveal the presence of multiple, intrinsically heterogeneous distal heme pocket conformations in Ngb-CO [17] This heterogeneity has been attributed to the dissociated His, which is expected to be situated close to the bound gaseous ligand. It has been suggested [17] that other distal residues compete with His for the sixth coordination site

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