کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1318122 | 976657 | 2007 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2α kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization
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موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی معدنی
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چکیده انگلیسی
Heme-regulated eIF2α kinase (HRI) is an important enzyme that modulates protein synthesis during cellular emergency/stress conditions, such as heme deficiency in red cells. It is essential to identify the heme axial ligand(s) and/or binding sites to establish the heme regulation mechanism of HRI. Previous reports suggest that a His residue in the N-terminal region and a Cys residue in the C-terminal region trans to the His are axial ligands of the heme. Moreover, mutational analyses indicate that a residue located in the kinase insertion (KI) domain between Kinase I and Kinase II domains in the C-terminal region is an axial ligand. In the present study, we isolate the KI domain of mouse HRI and employ site-directed mutagenesis to identify the heme axial ligand. The optical absorption spectrum of the Fe(III) hemin-bound wild-type KI displays a broad Soret band at around 373 nm, while that of the Fe(II) heme-bound protein contains a band at 422 nm. Spectral titration studies conducted for both the Fe(III) hemin and Fe(II) heme complexes with KI support a 1:1 stoichiometry of heme iron to protein. Resonance Raman spectra of Fe(III) hemin-bound KI suggest that thiol is the axial ligand in a 5-coordinate high-spin heme complex as a major form. Electron spin resonance (ESR) spectra of Fe(III) hemin-bound KI indicate that the axial ligands are OHâ and Cys. Since Cys385 is the only cysteine in KI, the residue was mutated to Ser, and its spectral characteristics were analyzed. The Soret band position, heme spectral titration behavior and ESR parameters of the Cys385Ser mutant were markedly different from those of wild-type KI. Based on these spectroscopic findings, we conclude that Cys385 is an axial ligand of isolated KI.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Inorganic Biochemistry - Volume 101, Issue 8, August 2007, Pages 1172-1179
Journal: Journal of Inorganic Biochemistry - Volume 101, Issue 8, August 2007, Pages 1172-1179
نویسندگان
Kyoko Hirai, Marketa Martinkova, Jotaro Igarashi, Islam Saiful, Seigo Yamauchi, Samir El-Mashtoly, Teizo Kitagawa, Toru Shimizu,