SPECTROSCOPIC, ELECTROCHEMICAL, AND LIGAND-BINDING PROPERTIES OF THE HORSE HEART METMYOGLOBIN HIS(64)-TYR VARIANT

The distal histidine (E7) of horse heart myoglobin (Mb) has been repla ced by tyrosine using site-specific mutagenesis. The resulting green M b variant (His(64)-Tyr) was expressed in Escherichia coli JM101, isola ted and purified to homogeneity. Spectrophotometric pH titrations of t he variant exhibit a change in spectrum that occurs with a pK(a) of 4. 7 (25 degrees C). The midpoint reduction potential of the variant is 2 0 mV (vs. SHE at pH 7, 25 degrees C). Cyanide and azide binding measur ements indicate that the oxidized variant binds these anionic ligands with much greater affinity at pH 4.0 than at neutral pH. Extended X-ra y absorption fine structure (EXAFS) spectroscopy establishes that the variant is six coordinate at pH 7.0 and pH 4.2. Higher shell contribut ions 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 li gand 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 sp ectra, electronic absorption spectra, and ligand binding properties of the His(64)-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 replace ment of Tyr-64 by a water molecule at low pH with a pK(a) of 4.7.