The kinetics of thermal unfolding of apo- and holo-Chromobacterium violaceum phenylalanine hydroxylase (cPAH) was investigated using circular dichroism (CD) over the temperature range 44-76 degrees C. In addition to the native cofactor (Fe-II), the unfolding kinetics of holo-cPAH was characterized using Zn-II and Co-II as cofactors. Kinetic profiles for apo- and holo-cPAH showed a single-phase exponential rise in the CD signal at lambda = 222 nm and a first-order dependence on protein concentration, The extrapolated unfolding rate constants (k(u)) at ambient temperature followed the order apo > Fe > Zn >> Co. Transition -state analysis of the activation parameters revealed an isokinetic correlation, which suggests a common mechanism for the enzyme variants. The values of the entropy of activation (Delta S-double dagger) for apo- and Fe-cPAH were negative but small: -34 +/-24 and -32 +/- 18 J mol(-1) K-1, respectively. On the other hand, Delta S-double dagger values for Zn- and Co-cPAH were large and positive: 54 9 and 175 27 J mol-1 K-1, respectively. Therefore, at higher temperatures the unfolding rates of Zn- and Co-cPAH are affected significantly by entropy, while the unfolding rates of apo- and Fe-cPAH are dominated by enthalpy even at higher temperatures. The rate of unfolding of holo-cPAH did not depend on excess metal concentrations and maintained single-phase kinetic profiles, refuting the occurrence of adventitious metal binding and the notion that unfolding occurs via apo-cPAH exclusively. Isothermal titration calorimetry (ITC) was employed to measure cPAH binding affinities for Fe, Zn, and Co as well as the enthalpy of metal coordination. Dissociation constants (K-d) decreased in the order Fe > Zn > Co. The non-native metals, Zn and Co, were bound more tightly than Fe. The activation enthalpy for unfolding (Delta H-double dagger) displayed a linear correlation with the enthalpy of metal binding obtained from ITC measurements (Delta H-ITC). On this basis, a common mechanism (transition state) is suggested for this family of metal cofactors, and the varying enthalpy of activation arises from the differing stabilities of enzyme variants having different metal cofactors.