The proximal ligand variant His93Tyr of horse heart myoglobin.
Abstract: The spectroscopic and structural properties of the His93Tyr variant of horse heart myoglobin have been studied to assess the effects of replacing the proximal His residue of this protein with a tyrosyl residue as occurs in catalases from various sources. The variant in the ferric form exhibits electronic spectra that are independent of pH between pH 7 and 10, and it exhibits changes in absorption maxima and intensity that are consistent with a five-coordinate heme iron center at the active site. The EPR spectrum of the variant is that of a high-spin, rhombic system similar to that reported for bovine liver catalase. The 1D 1H-NMR spectrum of the variant confirms the five-coordinate nature of the heme iron center and exhibits a broad resonance at 112.5 ppm that is attributable to the meta protons of the phenolate ligand. This result indicates that the new Tyr ligand flips at a significant rate in this protein. The thermal stability of the Fe(III) derivative is unchanged from that of the wild-type protein (pH 8) while the midpoint reduction potential [-208 mV vs SHE (pH 8.0, 25 degrees C)] is about 250 mV lower. The three-dimensional structure of the variant determined by X-ray diffraction analysis confirms the five-coordinate nature of the heme iron center and establishes that the introduction of a proximal Tyr ligand is accommodated by a shift of the F helix (residues 88-99) in which this residue resides away from the heme pocket. Additional effects of this change are small shifts in the positions of Leu29, a heme propionate, and a heme vinyl group that are accompanied by altered hydrogen bonding interactions with the heme prosthetic group.(ABSTRACT TRUNCATED AT 250 WORDS)
Publication Date: 1995-02-14 PubMed ID: 7849057DOI: 10.1021/bi00006a021Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The study examined the impact of replacing the His residue of horse heart myoglobin with a tyrosyl residue, as observed in catalases, on its spectroscopic and structural properties. Findings point to a consistent five-coordinate heme iron center and significantly lower midpoint reduction potential, while the thermal stability remained unchanged from the wild-type protein.
Research Objective
- The main goal of this research is to evaluate the effects of substituting the proximal His residue in horse heart myoglobin with a tyrosyl residue, emulating the characteristic seen in catalases – a group of enzymes that break down harmful hydrogen peroxide into water and oxygen.
Scientific Approach
- The researchers analyzed the electronic spectra of the variant in its ferric form (iron ion contained) within a pH level balance between 7 and 10.
- Changes in absorption maxima and intensity of light were examined to verify a consistent five-coordinate heme iron center at the active site – a key area in the molecule where reactions consistently take place.
- The EPR (Electron Paramagnetic Resonance) spectrum of the variant was compared to that of bovine liver catalase to determine similarities.
- They validated the five-coordinate nature of the heme iron center using the 1D 1H-NMR (Nuclear Magnetic Resonance) spectrum of the variant and observed the behavior of the newly introduced Tyr ligand.
- Researchers also examined changes in the thermal stability and midpoint reduction potential, a measure of the tendency of a chemical species to acquire electrons and thereby be reduced, of the Fe(III) derivative.
- Finally, the three-dimensional structure of the protein was analyzed.
Research Findings
- The findings confirmed the five-coordinate nature of the heme iron center, showing changes consistent with this structure.
- Phenolate ligand behavior suggests a significant rate of flipping or rotation.
- The thermal stability of the Fe(III) derivative is found to be consistent with that of the wild-type (non-modified) protein.
- The midpoint reduction potential was approximately 250 mV lower in the variant.
- The three-dimensional structure reaffirmed the five-coordinate heme iron center, showing that the Tyr ligand could be incorporated via a shift in the F helix of the protein. This shift also caused minor changes in the positions of Leu29, a heme propionate, and a heme vinyl group, and altered hydrogen bonding interactions with the heme prosthetic group.
Cite This Article
APA
Hildebrand DP, Burk DL, Maurus R, Ferrer JC, Brayer GD, Mauk AG.
(1995).
The proximal ligand variant His93Tyr of horse heart myoglobin.
Biochemistry, 34(6), 1997-2005.
https://doi.org/10.1021/bi00006a021 Publication
Researcher Affiliations
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
MeSH Terms
- Animals
- Binding Sites
- Crystallography, X-Ray
- Electrochemistry
- Electron Spin Resonance Spectroscopy
- Histidine / chemistry
- Horses
- Hot Temperature
- Hydrogen Bonding
- Hydrogen-Ion Concentration
- Magnetic Resonance Spectroscopy
- Models, Molecular
- Molecular Structure
- Mutagenesis, Site-Directed
- Myocardium / chemistry
- Myoglobin / chemistry
- Myoglobin / genetics
- Protein Denaturation
- Spectrophotometry
- Tyrosine / chemistry
Citations
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- San Francisco B, Bretsnyder EC, Rodgers KR, Kranz RG. Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF. Biochemistry 2011 Dec 20;50(50):10974-85.
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- Perera R, Sono M, Voegtle HL, Dawson JH. Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant. Arch Biochem Biophys 2011 Mar 1;507(1):119-25.
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