Volume changes associated with CO photodissociation from fully reduced cytochrome aa(3) have been determined using photoacoustic calorimetry (PAC). Analysis of PAC data reveals two intermediate steps in the photodissociation reaction with volume changes of +6.8 and +6.9 mL/mol occurring on time scales of <50 ns and similar to 1.5 mu s, respectively (T = 22 degrees C). The value of Delta V for the fast process can be attributed to conformational dynamics associated with photoinduced transfer of CO from the high-potential heme a(3) to the high-potential Cu-B site, while the slower phase reaction occurs on a time scale consistent with the thermal dissociation of CO from Cu-B. It is speculated that these volume changes may be linked to movement of Glu-242 as part of a proton shuttle mechanism in heme/copper oxidases.