By using site-directed mutagenesis of recombinant sperm whale (SW) myoglobin, the native distal histidine residue, at position 64 (the helical position E7), has been replaced with a tyrosine. The mutation of His64Tyr SW myoglobin has an analogous heme iron electronic structure as that of native hemoglobins M Boston and M Saskatoon. Optical spectroscopy showed that the distal tyrosine bound to the heme iron had a pK value of 5.6. In the pH range of 4.7–11.0, electron spin resonance spectroscopy suggested the presence of two heme iron ligation schemes: the heme iron bound to a distal water molecule or to a distal tyrosine residue. The heme iron coordination in the wild-type myoglobin and in the His64Tyr SW Mb mutant was studied by X-ray absorption near-edge structure (XANES) spectroscopy. Indeed, the heme iron K-edge reflects the electronic organization of the metal inside the six-coordinated complex. Comparative analysis of X-ray absorption heme iron K-edge shapes showed that the heme iron of His64Tyr SW myoglobin is bound to the oxygen atom from the phenol group of the distal tyrosine residue (Fe-OHϕ). When the pH value decreased from pH 7 to 5.6, the Fe-OHϕ bond strength decreased, resulting in an increase of the heme iron high-spin population of His64Tyr SW myoglobin. At low pH values, the Fe-OHϕ bond can be disrupted with the possibility of heme iron binding of another ligand having a higher affinity. When the heme Fe-OHϕ interaction was strong in His64Tyr SW myoglobin, the heme iron was bonded to the oxygen atom of the phenol group of tyrosine without displacement of the hydrogen atom of the phenol group.
|Original language||English (US)|
|Number of pages||6|
|State||Published - Sep 1 1994|
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