Heme reduction by intramolecular electron transfer in cysteine mutant myoglobin under carbon monoxide atmosphere

Shun Hirota, Kayo Azuma, Makoto Fukuba, Shigeki Kuroiwa, Noriaki Funasaki

研究成果: Article査読

14 被引用数 (Scopus)

抄録

Human myoglobin (Mb) possesses a unique cysteine (Cys110), whereas other mammalian Mbs do not. To investigate the effect of a cysteine residue on Mb, we introduced cysteine to various sites on the surface of sperm whale Mb (K56C, V66C, K96C, K102C, A125C, and A144C) by mutation. The cysteines were inserted near the end of α-helices, except for V66C, where the cysteine was introduced in the middle of an α-helix. Reduction of the heme was observed for each mutant metMb by incubation at 37°C under carbon monoxide atmosphere, which was much faster than reduction of wild-type metMb under the same condition. Heme reduction did not occur significantly under nitrogen or oxygen atmospheres. The rate constant for heme reduction increased for higher mutant Mb concentration, whereas it did not change significantly when the CO concentration was reduced from 100% CO to 50% CO with 50% O2. The similarity in the rate constants with different CO concentrations indicates that CO stabilizes the reduced heme by coordination to the heme iron. SDS-PAGE analysis showed that mutant Mb dimers were formed by incubation under CO atmosphere but not under air. These dimers were converted back to Mb monomers by an addition of 2-mercaptoethanol, which showed formation of a Mb dimer through a disulfide bond. The rate constant decreased in general as the heme-cysteine distance was increased, although V66C Mb exhibited a very small rate constant. Since V66 is placed in the middle of an α-helix, steric hindrance would occur and prevent formation of a dimer when the cysteine residues of two different V66C Mb molecules interact with each other. The rate constants also decreased for K56C and A144C Mbs presumably because of the electrostatic repulsion during dimer formation, since they are relatively charged around the inserted cysteine.

本文言語English
ページ(範囲)10322-10327
ページ数6
ジャーナルBiochemistry
44
30
DOI
出版ステータスPublished - 2005 8 2
外部発表はい

ASJC Scopus subject areas

  • Biochemistry

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