Conversion of hydrophobic substrates such as polyaromatic hydrocarbons (PAHs) was studied in aqueous-organic media using transformation of pyrene by cytochrome c. The experiments were conducted in pure solvents and aqueous-acetonitrile mixtures. The reaction rates dropped gradually as the solvent concentration was increased and were negligible in pure solvents. Thermodynamic calculations show that substrate desolvation was the governing factor in this reaction. The decrease in free energy of activation upon increasing the solvent concentration was found to be proportional to the substrate transfer free energy. This suggests that in order to make such reactions feasible in organic media, it will be necessary to improve the binding between the proteins and the substrates.