CO binding study of mouse heme-regulated eIF-2α kinase: kinetics and resonance Raman spectra

Abstract

Heme-regulated eukaryotic initiation factor (eIF)-2α kinase (HRI) regulates the synthesis of globin chains in reticulocytes with heme availability. In the present study, CO binding kinetics to the 6-coordinated Fe(II) heme of the amino-terminal domain of mouse HRI and resonance Raman spectra of the Fe(II)–CO complex are examined to probe the character of the heme environment. The CO association rate constant, k on′, and CO dissociation rate constant, k off, were 0.0029 μM −1s −1 and 0.003 s −1, respectively. These values are very slow compared with those of mouse neuroglobin and sperm whale myoglobin, while the k off value of HRI was close to those of the 6-coordinated hemoglobins from Chlamydomonas and barley (0.0022 and 0.0011 s −1). The dissociation rate constant of an endogenous ligand, which occurs prior to CO association, was 18.3 s −1, which was lower than those (197 and 47 s −1) of the same 6-coordinated hemoglobins. Resonance Raman spectra suggest that the Fe–C–O adopts an almost linear and upright structure and that the bound CO interacts only weakly with nearby amino acid residues.