Alteration of Sperm Whale Myoglobin Heme Axial Ligation by Site-Directed Mutagenesis

Karen D. Egeberg, Barry A. Springer, Susan A. Martinis, Stephen G. Sligar, Dimitrios Morikis, Paul M. Champion

Research output: Contribution to journalArticle

Abstract

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E 11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(βE7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(αF8)Tyr] and Hb M Hyde Park [His(βF8)Tyr], Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(βE11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact. The results presented here demonstrate the power of site-directed mutagenesis as a tool for altering the electronic structure of metal centers and aiding in the assignment of complicated vibrational spectra.

Original languageEnglish (US)
Pages (from-to)9783-9791
Number of pages9
JournalBiochemistry
Volume29
Issue number42
DOIs
StatePublished - Oct 1 1990

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Sperm Whale
Mutagenesis
Myoglobin
Site-Directed Mutagenesis
Heme
Ligation
Iron
Histidine
Tyrosine
Paramagnetic resonance
Glutamic Acid
Hemoglobin M
Substitution reactions
Synthetic Genes
Valine
Vibrational spectra
Mutant Proteins
Light absorption
Electronic structure

ASJC Scopus subject areas

  • Biochemistry

Cite this

Alteration of Sperm Whale Myoglobin Heme Axial Ligation by Site-Directed Mutagenesis. / Egeberg, Karen D.; Springer, Barry A.; Martinis, Susan A.; Sligar, Stephen G.; Morikis, Dimitrios; Champion, Paul M.

In: Biochemistry, Vol. 29, No. 42, 01.10.1990, p. 9783-9791.

Research output: Contribution to journalArticle

Egeberg, Karen D. ; Springer, Barry A. ; Martinis, Susan A. ; Sligar, Stephen G. ; Morikis, Dimitrios ; Champion, Paul M. / Alteration of Sperm Whale Myoglobin Heme Axial Ligation by Site-Directed Mutagenesis. In: Biochemistry. 1990 ; Vol. 29, No. 42. pp. 9783-9791.
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abstract = "Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E 11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(βE7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(αF8)Tyr] and Hb M Hyde Park [His(βF8)Tyr], Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(βE11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact. The results presented here demonstrate the power of site-directed mutagenesis as a tool for altering the electronic structure of metal centers and aiding in the assignment of complicated vibrational spectra.",
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AU - Egeberg, Karen D.

AU - Springer, Barry A.

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AU - Morikis, Dimitrios

AU - Champion, Paul M.

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N2 - Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E 11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(βE7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(αF8)Tyr] and Hb M Hyde Park [His(βF8)Tyr], Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(βE11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact. The results presented here demonstrate the power of site-directed mutagenesis as a tool for altering the electronic structure of metal centers and aiding in the assignment of complicated vibrational spectra.

AB - Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E 11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(βE7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(αF8)Tyr] and Hb M Hyde Park [His(βF8)Tyr], Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(βE11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact. The results presented here demonstrate the power of site-directed mutagenesis as a tool for altering the electronic structure of metal centers and aiding in the assignment of complicated vibrational spectra.

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