We have studied the equilibrium fluctuation dynamics of Zn-substituted myoglobin and its His64→Leu (H64L) mutant in the pH range from 5 to 9 by using time-resolved transient-hole-burning (TRTHB) spectroscopy. In the H64L mutant, we have observed a largely reduced width of the absorption spectrum and only a slight temporal shift of the hole-burning spectrum. These observations both reflect the suppressed conformational fluctuation in the mutant. On the other hand, the pH-dependent change in the absorption spectrum could not be solely explained by the change in the protonation state of His64 induced by the pH change. These results suggest that although the fluctuation dynamics observed by the TRTHB experiment of the native sample mainly reflects the conformational motion around His64, the interconversion process of His64 between its protonated and unprotonated states has a minor contribution. Instead, we have proposed a tentative interpretation that the motion of the water molecule around His64 is the main source of the observed dynamics in the TRTHB technique.
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