title: Activation of heme-regulated eukaryotic initiation factor 2alpha kinase by nitric oxide is induced by the formation of a five-coordinate NO-heme complex: optical absorption, electron spin resonance, and resonance raman spectral studies.
abstract: Heme-regulated eukaryotic initiation factor 2alpha kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains unclear. In the present study, we utilize in vitro kinase assays, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the Fe(II)-HRI(NO) complex displayed 5-fold greater eukaryotic initiation factor 2alpha kinase activity than the Fe(III)-HRI complex. The Fe(III)-HRI complex exhibited a Soret peak at 418 nm and a rhombic ESR signal with g values of 2.49, 2.28, and 1.87, suggesting coordination with Cys as an axial ligand. Interestingly, optical absorption, ESR, and resonance Raman spectra of the Fe(II)-NO complex were characteristic of five-coordinate NO-heme. Spectral findings on the coordination structure of full-length HRI were distinct from those obtained for the isolated N-terminal heme-binding domain. Specifically, six-coordinate NO-Fe(II)-His was observed but not Cys-Fe(III) coordination. It is suggested that significant conformational change(s) in the protein induced by NO binding to the heme lead to HRI activation. We discuss the role of NO and heme in catalysis by HRI, focusing on heme-based sensor proteins.
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan.
authors.forename: Jotaro
authors.initials: J
authors.lastname: Igarashi
authors.forename: Akira
authors.initials: A
authors.lastname: Sato
authors.forename: Teizo
authors.initials: T
authors.lastname: Kitagawa
authors.forename: Tetsuhiko
authors.initials: T
authors.lastname: Yoshimura
authors.forename: Seigo
authors.initials: S
authors.lastname: Yamauchi
authors.forename: Ikuko
authors.initials: I
authors.lastname: Sagami
authors.forename: Toru
authors.initials: T
authors.lastname: Shimizu
mesh_terms: D000818:Animals; D019281:Dimerization; D004789:Enzyme Activation; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017510:Protein Folding; D013329:Structure-Activity Relationship; D019892:eIF-2 Kinase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009569:Nitric Oxide; D006418:Heme; D019892:eIF-2 Kinase