Identification of Crucial Histidines for Heme Binding in the N-terminal Domain of the Heme-regulated eIF2α Kinase

Abstract

The heme-regulated eukaryotic initiation factor-2␣ (eIF2␣) kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the Nterminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing UV-visible absorption, resonance Raman, EPR and CD spectroscopies, two histidine residues have been identified that are crucial for the binding of heme to the NTD. The UV-visible absorption and resonance Raman spectra of all the histidine to alanine mutants constructed were similar to those of the unmutated NTD. However, the change in the CD spectra of the NTD construct containing mutation of His 78 to Ala (H78A) indicated loss of the specific binding of heme. The EPR spectrum for the ferric H78A mutant was also substantially perturbed. Thus, His 78 is one of the axial ligands for the NTD of HRI. Significant changes in the EPR spectrum of the H123A mutant were also observed, and heme readily dissociated from both the H123A and the H78A NTD mutants, suggesting that His 123 was also an axial heme ligand. However, the CD spectrum for the Soret region of the H123A mutant indicated that this mutant still bound heme specifically. Thus, while both His 78 and His 123 are crucial for stable heme binding, the effects of their mutations on the structure of the NTD differed. His 78 appears to play the primary role in the specific binding of heme to the NTD, acting analogously to the "proximal histidine" ligand of globins, while His 123 appears to act as the "distal" heme ligand.

Elucidation of the Heme Binding Site of Heme-regulated Eukaryotic Initiation Factor 2α Kinase and the Role of the Regulatory Motif in Heme Sensing by Spectroscopic and Catalytic Studies of Mutant Proteins

“…Hg 2ϩ strongly suppresses HRI catalysis with an IC 50 value of 0.6 M, which is restored by NO, supporting the theory that the Cys thiolate is involved catalytic regulation (12). An earlier study shows that two His residues are heme axial ligands of the isolated N-terminal domain of HRI (13), distinct from the His and Cys residues proposed for full-length HRI (7,8). The mechanism proposed for NO-induced catalytic activation of the isolated N-terminal domain is also inconsistent with that for the full-length enzyme (7,8,13).…”

“…8S). protein sensitively reflects the protein structure around the heme binding site, even optical absorption spectra do not manifest any changes upon mutations at the heme binding site (13). To further identify the amino acid(s) that acts as the axial ligand for Fe(III) complex in full-length HRI, we obtained CD spectra of all His mutant proteins.…”

“…The proposed heme axial ligands of the full-length HRI protein are currently a subject of controversy. For the isolated N-terminal domain of HRI, a homology model based on amino acid sequences of the hemoglobin ␣-chain indicates that His-80 is an axial ligand (20), whereas circular dichroism spectra suggest that His-75 and His-120 are axial ligands for the Fe(III) complex of HRI (13). One group proposed the existence of two heme binding sites in the HRI monomer (23).…”

“…Thus, subtle structural changes in heme surroundings would significantly influence the Soret CD spectral bands of the heme proteins even if such changes do not affect other spectroscopies (13), The ratio (RZ value) of the Soret peak absorbance to that at 280 nm of the heme protein is used to measure heme binding to the apoprotein. Because the RZ values of both the H119A and H120A mutants were comparable to that of the wild-type protein, the Fe(III) complex would be adequately incorporated into the mutant proteins.…”

“…An earlier study shows that two His residues are heme axial ligands of the isolated N-terminal domain of HRI (13), distinct from the His and Cys residues proposed for full-length HRI (7,8). The mechanism proposed for NO-induced catalytic activation of the isolated N-terminal domain is also inconsistent with that for the full-length enzyme (7,8,13). Thus, the molecular mechanisms of catalytic activation and inhibition of full-length HRI by heme require clarification, in particular interactions between the heme iron and full-length HRI, the mechanism by which full-length HRI senses the heme iron and the amino acids acting as axial ligands for the heme iron.…”