Diffusion of small molecules through the structure of myoglobin. Environmental effects

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Abstract
The effect of the ambient solvent viscosity on the mobility of small molecules within myoglobin was studied by substituting Zn-protoporophyrin (ZnPP) for the native Fe-protoporphyrin and using it as an optical probe in the protein (ZnPPMb). The quenching of the ZnPPMb triplet state by oxygen, by anthraquinonesulfonate, and by methyl viologen was followed by exciting it with a laser flash and measuring its decay rate as a function of quencher concentration. The quenching rate constants were taken to measure the diffusion rate of the quencher within the protein. At room temperature, these constants were determined in aqueous and in 37% and 55% (by weight) glycerol-water solutions by measuring the ZnPPMb-delayed fluorescence at 606 nm. It was found that although the quenching rate constants varied the activation energies in the protein were very similar for the different quenchers. In aqueous solution, Ea = 6.0-7.4 kcal/mol; in 37% glycerol, Ea = 6.8-7.5 kcal/mol; and in 55% glycerol, Ea = 8.5-9.2 kcal/mol. The quenching rate of ZnPPMb by oxygen was also measured between 190K and 293K in 80% glycerol, and its triplet decay in the absence of oxygen was determined down to 120K in 88% glycerol. In all experiments, the quenching rates in the protein were compared to those of Zn-hematoporphyrin in the same solvent. The results are discussed in terms of Northrup and McCammon''s gated reaction theory. It is concluded that (a) the mobility within the protein persists at least down to the temperature where the solvent solidifies and (b) the activation energies obtained measure the energetics of the conformational change in the protein which enable large and small quencher molecules to penetrate the protein with equal ease.