Distal pocket mutations at the E7 position (His64) of sperm whale deoxymyoglobin (deoxyMb) are used as a probe of distal pocket polarity and hydration. Changes of two key spectroscopic markers, the Fe-His(F8) stretch in the resonance Raman spectrum and the position of band III in the absorption spectrum, are monitored as the His64Tyr, His64Phe, His64Leu, and His64Gly mutations alter the distal heme pocket environment. The Fe-His vibration for the Phe, Leu, and Gly mutants is shifted to a lower frequency by 1-2 cm-1 relative to the Tyr mutant, wild type (WT), and native deoxyMb. Band III shifts to the red by ~4 nm (~70 cm-1) relative to WT and native deoxyMb for all the His64 mutants examined in this work. We correlate the small shift in the Fe-His frequency to the local electrostatic environment directly above the heme iron, affected by the presence of a localized water molecule in the heine pocket that is hydrogen-bonded to the E7 residue. The position of hand III is roughly correlated to the displacement of the iron from the heme plane; however, the relatively large scatter in this correlation, as well as its dependence on distal pocket mutations, suggests that the heine pocket environment, particularly the E7 residue, also affects the energy of this transition.
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