Direct Measurement of Proton Transfer as a Probing Reaction for the Microenvironment of the Apomyoglobin Heme‐Binding Site

Aromatic alcohols fluoresce at different wavelengths in their neutral (øOH*) and anionic (øO*) excited states. Consequently, time‐resolved fluorescence measurements, at the respective wavelengths, can be used for measuring the rates of proton dissociation and recombination of the excited molecule. As the lifetime of the excited state is very short (a few nanoseconds), the measured reaction is that which takes place in a volume corresponding to the diffusion distance of the proton during the lifetime of the excited state. 8‐Hydroxypyrene 1,3,6‐trisulfonate (pK°= 7.7, pK*= 0.5) is bound to apomyoglobin with a stoichiometry of 1 : 1. In the bound state its neutral form fluorescence increase 20‐fold. The binding affinity is pH‐dependent. Two protonatable groups, with pK= 6.5, participate in the stabilization of the negatively charged ligand in the binding site. The ligand is bound only to the apoprotein and is displaced from its site by hemin. Thus we suggest that the ligand is bound to the heme binding site of apomyoglobin. Time‐resolved fluorescence of the bound ligand yields the rate constants of proton dissociation and recombination as taking place within the heme binding cavity of apomyoglobin. The rate of proton dissociation is slowed to 7 %, of the rate measured for the free ligand. Such a slow disso‐ ciation indicates a strong interaction of the water in the cavity with the walls [Gutman, M., Huppert, D., and Nachliel, E. (1982) Eur. J. Biochem. 121, 637‐642]. The water activity in the site is equivalent to a (H₂O) = 0.67.