Spectroscopic, electrochemical, and ligand binding properties of the horse heart metmyoglobin His64-Tyr variant

Hai Lun Tang, Britton Chance, A. Grant Mauk, Linda S Powers, Konda S. Reddy, Michael Smith

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The distal histidine (E7) of horse heart myoglobin (Mb) has been replaced by tyrosine using site-specific mutagenesis. The resulting green Mb variant (His64-Tyr) was expressed in Escherichia coli JM101, isolated and purified to homogeneity. Spectrophotometric pH titrations of the variant exhibit a change in spectrum that occurs with a pKa of 4.7 (25°C). The midpoint reduction potential of the variant is 20 mV (vs. SHE at pH 7, 25°c). Cyanide and azide binding measurements indicate that the oxidized variant binds these anionic ligands with much greater affinity at pH 4.0 than at neutral pH. Extended X-ray absorption fine structure (EXAFS) spectroscopy establishes that the variant is six coordinate at pH 7.0 and pH 4.2. Higher shell contributions to the iron EXAFS observed at pH 7.0 are attributed to tyrosine. These contributions are absent at pH 4.2. Thus, the sixth heme iron ligand of the oxidized variant Mb at pH 7.0 is attributed to oxygen from the hydroxyl group of tyrosine and the sixth ligand present at pH 4.2 is attributed to the oxygen atom of a coordinated water. The EXAFS spectra, electronic absorption spectra, and ligand binding properties of the His64-Tyr Mb variant are consistent with the binding of Tyr-64 as the sixth heme iron ligand between pH 5 and 12 and with the replacement of Tyr-64 by a water molecule at low pH with a pKa of 4.7.

Original languageEnglish (US)
Pages (from-to)90-96
Number of pages7
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1206
Issue number1
DOIs
StatePublished - May 18 1994
Externally publishedYes

Fingerprint

Metmyoglobin
Myoglobin
Horses
Ligands
Tyrosine
Iron
X ray absorption
Heme
Extended X ray absorption fine structure spectroscopy
Oxygen
Mutagenesis
Azides
Water
Cyanides
Titration
Histidine
Hydroxyl Radical
Escherichia coli
X-Rays
Absorption spectra

Keywords

  • (Horse heart)
  • EXAFS
  • Heme iron ligand
  • Myoglobin
  • Site-specific mutagenesis
  • Tyrosine-64

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

Cite this

Spectroscopic, electrochemical, and ligand binding properties of the horse heart metmyoglobin His64-Tyr variant. / Tang, Hai Lun; Chance, Britton; Mauk, A. Grant; Powers, Linda S; Reddy, Konda S.; Smith, Michael.

In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 1206, No. 1, 18.05.1994, p. 90-96.

Research output: Contribution to journalArticle

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abstract = "The distal histidine (E7) of horse heart myoglobin (Mb) has been replaced by tyrosine using site-specific mutagenesis. The resulting green Mb variant (His64-Tyr) was expressed in Escherichia coli JM101, isolated and purified to homogeneity. Spectrophotometric pH titrations of the variant exhibit a change in spectrum that occurs with a pKa of 4.7 (25°C). The midpoint reduction potential of the variant is 20 mV (vs. SHE at pH 7, 25°c). Cyanide and azide binding measurements indicate that the oxidized variant binds these anionic ligands with much greater affinity at pH 4.0 than at neutral pH. Extended X-ray absorption fine structure (EXAFS) spectroscopy establishes that the variant is six coordinate at pH 7.0 and pH 4.2. Higher shell contributions to the iron EXAFS observed at pH 7.0 are attributed to tyrosine. These contributions are absent at pH 4.2. Thus, the sixth heme iron ligand of the oxidized variant Mb at pH 7.0 is attributed to oxygen from the hydroxyl group of tyrosine and the sixth ligand present at pH 4.2 is attributed to the oxygen atom of a coordinated water. The EXAFS spectra, electronic absorption spectra, and ligand binding properties of the His64-Tyr Mb variant are consistent with the binding of Tyr-64 as the sixth heme iron ligand between pH 5 and 12 and with the replacement of Tyr-64 by a water molecule at low pH with a pKa of 4.7.",
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N2 - The distal histidine (E7) of horse heart myoglobin (Mb) has been replaced by tyrosine using site-specific mutagenesis. The resulting green Mb variant (His64-Tyr) was expressed in Escherichia coli JM101, isolated and purified to homogeneity. Spectrophotometric pH titrations of the variant exhibit a change in spectrum that occurs with a pKa of 4.7 (25°C). The midpoint reduction potential of the variant is 20 mV (vs. SHE at pH 7, 25°c). Cyanide and azide binding measurements indicate that the oxidized variant binds these anionic ligands with much greater affinity at pH 4.0 than at neutral pH. Extended X-ray absorption fine structure (EXAFS) spectroscopy establishes that the variant is six coordinate at pH 7.0 and pH 4.2. Higher shell contributions to the iron EXAFS observed at pH 7.0 are attributed to tyrosine. These contributions are absent at pH 4.2. Thus, the sixth heme iron ligand of the oxidized variant Mb at pH 7.0 is attributed to oxygen from the hydroxyl group of tyrosine and the sixth ligand present at pH 4.2 is attributed to the oxygen atom of a coordinated water. The EXAFS spectra, electronic absorption spectra, and ligand binding properties of the His64-Tyr Mb variant are consistent with the binding of Tyr-64 as the sixth heme iron ligand between pH 5 and 12 and with the replacement of Tyr-64 by a water molecule at low pH with a pKa of 4.7.

AB - The distal histidine (E7) of horse heart myoglobin (Mb) has been replaced by tyrosine using site-specific mutagenesis. The resulting green Mb variant (His64-Tyr) was expressed in Escherichia coli JM101, isolated and purified to homogeneity. Spectrophotometric pH titrations of the variant exhibit a change in spectrum that occurs with a pKa of 4.7 (25°C). The midpoint reduction potential of the variant is 20 mV (vs. SHE at pH 7, 25°c). Cyanide and azide binding measurements indicate that the oxidized variant binds these anionic ligands with much greater affinity at pH 4.0 than at neutral pH. Extended X-ray absorption fine structure (EXAFS) spectroscopy establishes that the variant is six coordinate at pH 7.0 and pH 4.2. Higher shell contributions to the iron EXAFS observed at pH 7.0 are attributed to tyrosine. These contributions are absent at pH 4.2. Thus, the sixth heme iron ligand of the oxidized variant Mb at pH 7.0 is attributed to oxygen from the hydroxyl group of tyrosine and the sixth ligand present at pH 4.2 is attributed to the oxygen atom of a coordinated water. The EXAFS spectra, electronic absorption spectra, and ligand binding properties of the His64-Tyr Mb variant are consistent with the binding of Tyr-64 as the sixth heme iron ligand between pH 5 and 12 and with the replacement of Tyr-64 by a water molecule at low pH with a pKa of 4.7.

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