Spectroscopic, electrochemical, and ligand binding properties of the horse heart metmyoglobin His64-Tyr variant.
The distal histidine (E7) of horse heart myoglobin (Mb) has been replaced by tyrosine using site-specific mutagenesis.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 degrees C). The midpoint reduction potential of the variant is 20 mV (vs. SHE at pH 7, 25 degrees 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.
Tang
H L
HL
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Chance
B
B
Mauk
A G
AG
Powers
L S
LS
Reddy
K S
KS
Smith
M
M
D000818:Animals; D004563:Electrochemistry; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine; D006736:Horses; D008024:Ligands; D008786:Metmyoglobin / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D013057:Spectrum Analysis; D014443:Tyrosine
D008024:Ligands; D008786:Metmyoglobin; D014443:Tyrosine; D006639:Histidine