1H NMR study of the dynamics of the pH modulation of axial coordination in Aplysia limacina (Val(E7)) and sperm whale double mutant His(E7)-->Val,Thr(E10)-->Arg metmyoglobin.

The molecular and electronic structure, thermodynamics, dynamics, and mechanism of interconversion of the pH-modulated reversible equilibria of Aplysia limacina metmyoglobin, (metMb), have been investigated by 1H NMR spectroscopy. The four identified species which interconvert slowly on the NMR time scale (lifetime > 1 ms) are metMbOH (B) at alkaline pH, five coordinate metMb (N) at acidic to neutral pH, an acidic form, A, near pH approximately 4 and an extremely low pH form, D, attributed to an equilibrium unfolded species. The presence of strong distal hydrogen bonding by Arg (E10) to bound hydroxide is detected via a significant solvent isotope effect on the metMbOH (B) hyperfine shifts. Integration of the peak intensities yields pK values of 7.7 and approximately 4 for the B<-->N and N<-->A equilibria, respectively. Saturation transfer via chemical exchange is observed for B<-->N and N<-->A, where the rates for forming metMbOH (B) and the acidic form A from N are base- and acid-catalyzed, respectively, while the reverse rates are first-order. The much slower interconversion rate for N<-->B in A. limacina metMb than His(E7) containing mammalian metMb is attributed to the fact that a ligand bond is broken rather than just proton transferred and that the equilibrium involves a major rearrangement of the orientation of Arg(E10). This conclusion is supported by 1H NMR data for the sperm whale double mutant His(E7)-->Val/Thr(E10)-->Arg metMb, which exhibits a pK approximately 8.7 for the equilibrium between five-coordinate metMb (N) and metMbOH (B) with an even slower interconversion rate than in A. limacina metMb. This double mutant metMbOH (B) exhibits hydrogen bonding by Arg (E10) with coordinated hydroxide similar to that in A. limacina metMbOH. The slow but acid-catalyzed rates of conversion of A. limacina metMb (N) to the acid species A with significantly weakened bonding of the heme iron to the axial His(F8) residue is consistent with protonation of an inaccessible residue and/or a structural change accompanying the protonation equilibrium. It is concluded that metMb will coordinate water strongly only when there is a distal hydrogen bond acceptor residue, while the hydroxide ion is coordinated strongly only if there is a distal hydrogen bond donor residue.