Time-resolved thermodynamic profiles for CO photolsysis from the mixed valence form of bovine heart cytochrome c oxidase

Photoacoustic calorimetry has been utilized to probe the thermodynamics accompanying photodissociation of the CO mixed valence form of bovine heart cytochrome c oxidase (COMV CcO). At pH's below 9 photolysis of the COMV CcO results in three kinetic phases with the first phase occurring faster than the time resolution of the instrument (i.e.,< similar to 50 ns), a second phase occurring with a lifetime of similar to 100 ns and a third phase occurring with a lifetime of similar to 2 mu s. The corresponding volume and enthalpy changes for these processes are: Delta H-1, Delta V-1 = +79 +/- 10 kcal mol(-1) =+ 9 +/- 1 mL mol(-1); Delta H-2, Delta V-2 =-79 +/- 5 kcal mol(-1), -9 +/- 2 mL mol(-1); Delta H-3, Delta V-3 =+ 54 +/- 7 kcal mol(-1), + 8 +/- 1 mL mol(-1). At pH's above 9 only one phase is observed, a prompt phase occurring in < 50 ns. The overall volume change is negligible above pH 9 and the enthalpy change is + 29 +/- 5 kcal mol(-1). The data are consistent with the prompt phase being associated with CO-Fe-a3 bond cleavage, CO-CuB + bond formation, Fe-a3 low-spin to high-spin transition and fast electron transfer (ET) from heme a(3) to heme a followed by proton transfer from Glu242 to Arg38 on an similar to 100 ns timescale. The slow phase is likely a combination of CO themal dissociation from Cu-B and additional ET between heme a(3) to heme a. Interestingly, this phase is not evident above pH 9 suggesting linkage between CO dissociation/ET and the protonation state of a group or groups near the binuclear center.