This study characterizes the CO rebinding kinetics after photodissociation of horse heart myoglobin (Mb) and human R-state hemoglobin (Hb) encapsulated in wet silica gels, in the presence of various concentrations of glycerol. The geminate yield for HbCO is scarcely affected by the gel matrix, indicating that the protein can fluctuate as in a homogeneous solution. On the contrary, the geminate yield for gel-embedded MbCO is much higher than that in solution, suggesting that the gel matrix inhibits the movements of the protein. The geminate yield for both proteins increases substantially with the addition of glycerol to the bathing solution. The observed kinetics could be rationalized using a simple three-state model. Rate constants have been modeled using a modified Kramers equation, which indicated that the gel exerts an internal friction on the elementary rate constants. The rate constant for geminate rebinding, kCA, is essentially viscosity independent below 20 cP for both proteins. The internal friction for the ligand escape rate, kCS, is much smaller and is found to be negligible for HbCO and 5 cP for MbCO. The activation barrier for kCS increases with glycerol concentration in response to increased viscosity and reduced ligand solubility. The rate kSC showed a complex behavior that reflects the opposing effects of viscosity and activity arising from molecular confinement and crowding. Accordingly, the corresponding activation barriers show a biphasic behavior, with a minimum at ≈40% glycerol for HbCO and at ≈75% glycerol for MbCO. The results highlight the potential of silica gel encapsulation for in vitro studies aimed to reproduce the crowded and confined environment experienced by proteins in vivo. The diverse response to encapsulation of Mb and Hb could actually reflect physiologically relevant functional properties escaping detection in the diluted solutions normally used for biophysical investigations.
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