Characterization of the Heme Environmental Structure of Cytoglobin, a Fourth Globin in Humans

Cytoglobin (Cgb) represents a fourth member of the globin superfamily in mammals, but its function is unknown. Site-directed mutagenesis, in which six histidine residues were replaced with alanine, was carried out, and the results indicate that the imidazoles of His81 (E7) and His113 (F8) bind to th...

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Bibliographic Details
Published in:Biochemistry (Easton) 2003-05, Vol.42 (17), p.5133-5142
Main Authors: Sawai, Hitomi, Kawada, Norifumi, Yoshizato, Katsutoshi, Nakajima, Hiroshi, Aono, Shigetoshi, Shiro, Yoshitsugu
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution
Animals
Circular Dichroism
Globins - chemistry
Globins - genetics
Globins - metabolism
Heme - chemistry
Heme - metabolism
Humans
Lampreys
Molecular Sequence Data
Mutagenesis, Site-Directed
Recombinant Proteins - chemistry
Sequence Alignment
Sequence Homology, Amino Acid
Spectrophotometry
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Summary:Cytoglobin (Cgb) represents a fourth member of the globin superfamily in mammals, but its function is unknown. Site-directed mutagenesis, in which six histidine residues were replaced with alanine, was carried out, and the results indicate that the imidazoles of His81 (E7) and His113 (F8) bind to the heme iron as axial ligands in the hexacoordinate and the low-spin state. The optical absorption, resonance Raman, and IR spectral results are consistent with this conclusion. The redox potential measurements revealed an E‘ of 20 mV (vs NHE) in the ferric/ferrous couple, indicating that the imidazole ligands of His81 and His113 are electronically neutral. On the basis of the νFe - CO and νC - O values in the resonance Raman and infrared spectra of the ferrous−CO complexes of Cgb and its mutants, it was found that CO binds to the ferrous iron after the His81 imidazole is dissociated, and three conformers are present in the resultant CO coordination structure. Two are in closed conformations of the heme pocket, in which the bound CO ligand interacts with the dissociated His81 imidazole, while the third is in an open conformation. The νFe - O 2 in the resonance Raman spectra of oxy Cgb can be observed at 572 cm-1, suggesting a polar heme environment. These structural properties of the heme pocket of Cgb are discussed with respect to its proposed in vivo oxygen storage function.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi027067e