Oxygen quenching of protein phosphorescence was determined for the buried Trp residues of apoazurin, liver alcohol dehydrogenase, and alkaline phosphatase as a function of temperature (0-50 degrees C) and applied pressure (up to 3 kbar). Accurate control of the oxygen concentration in solution, by a method that employs an internal protein reference, largely confirms the small bimolecular quenching rate constants (k(q)) reported previously for these proteins. Wide variations in flexibility of the globular fold, as attained from protein to protein or by changing external conditions of temperature and pressure, establish that the magnitude of k(q) is directly correlated to the rigidity of the protein matrix surrounding the chromophore and demonstrates that the quenching rate constant is limited by hindered migration of oxygen through compact regions of the polypeptide. The magnitude of k(q) implies that O-2 diffusion in proteins can be slowed over 1000-fold relative to water and much more than was inferred from the corresponding fluorescence quenching rate. The activation enthalpy for the structural fluctuations underlying O-2 diffusion in proteins ranges between 9 and 12 kcal mol(-1), similar among the three proteins but larger than the 3 kcal mol(-1) for O-2 diffusion in water. The activation volumes, obtained from the pressure dependence of k(q) are largest and positive at 50 degrees C and below 2 kbar, but decrease monotonically at higher pressure and at lower temperature. This behavior, together with a similar magnitude of the activation volumes among proteins with different internal mobility are interpreted as to indicate an essential role of internal water molecules in conferring flexibility to protein structure.