Intramolecular electron transfer in azurin in water and deuterium oxide has been studied over a broad temperature range. The kinetic deuterium isotope effect, k(H)/k(D), is smaller than unity (0.7 at 298 K), primarily caused by the different activation entropies in water (-56.5 J K-1 mol(-1)) and in deuterium oxide (-35.7 J K-1 mol(-1)). This difference suggests a role for distinct protein solvation in the two media, which is supported by the results of voltammetric measurements: the reduction potential (E-0') of Cu2+/+ at 298 K is 10 mV more positive in D2O than in H2O, The temperature dependence of E-0' is also different, yielding entropy changes of -57 J K-1 mol-l in water and -84 J K-1 mol(-1) in deuterium oxide. The driving force difference of 10 mV is in keeping with the kinetic isotope effect, but the contribution to DeltaS(double dagger) from the temperature dependence of E-0' is positive rather than negative. Isotope effects are, however, also inherent in the nuclear reorganization Gibbs free energy and in the tunneling factor for the electron transfer process. A slightly larger thermal protein expansion in H2O than in D2O (0.001 nm K-1) is sufficient both to account for the activation entropy difference and to compensate for the different temperature dependencies of E-0'. Thus, differences in driving force and thermal expansion appear as the most straightforward rationale for the observed isotope effect.
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Publication status||Published - 2001|