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

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

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)10322-10327
Number of pages6
JournalBiochemistry
Volume44
Issue number30
DOIs
Publication statusPublished - 2005 Aug 2
Externally publishedYes

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Myoglobin
Carbon Monoxide
Heme
Atmosphere
Cysteine
Electrons
Dimers
Rate constants
Sperm Whale
Mercaptoethanol
Static Electricity
Disulfides
Polyacrylamide Gel Electrophoresis
Electrostatics
Nitrogen
Iron
Monomers
Air
Oxygen
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Heme reduction by intramolecular electron transfer in cysteine mutant myoglobin under carbon monoxide atmosphere. / Hirota, Shun; Azuma, Kayo; Fukuba, Makoto; Kuroiwa, Shigeki; Funasaki, Noriaki.

In: Biochemistry, Vol. 44, No. 30, 02.08.2005, p. 10322-10327.

Research output: Contribution to journalArticle

Hirota, Shun ; Azuma, Kayo ; Fukuba, Makoto ; Kuroiwa, Shigeki ; Funasaki, Noriaki. / Heme reduction by intramolecular electron transfer in cysteine mutant myoglobin under carbon monoxide atmosphere. In: Biochemistry. 2005 ; Vol. 44, No. 30. pp. 10322-10327.
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