Here, we report the volume and enthalpy changes accompanying CO photodissociation from the mixed valence form of cytochrome bo 3 oxidase from Escherichia coli. The results of photoacoustic calorimetry indicate two kinetic phases with distinct volume and enthalpy changes accompanying CO photodissociation from heme o 3 and its transfer to Cu B . The first phase occurring on a timescale of <50ns is characterized by a volume decrease of −1.3±0.3mLmol −1 and enthalpy change of 32±1.6kcalmol −1 . Subsequently, a volume increase of 2.9mLmol −1 with an enthalpy change of −5.3±2.5kcalmol −1 is observed with the lifetime of ∼250ns (this phase has not been detected in previous optical studies). These volume and enthalpy changes differ from the volume and enthalpy changes observed for CO dissociation from fully reduced cytochrome bo 3 oxidase indicating that the heme o 3 /Cu B active site dynamics are affected by the redox state of heme b.