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., < approximately 50 ns), a second phase occurring with a lifetime of approximately 100 ns and a third phase occurring with a lifetime of approximately 2 micros. The corresponding volume and enthalpy changes for these processes are: DeltaH1, DeltaV1 = +79 +/- 10 kcal mol(-1), +9 +/- 1 mL mol(-1); DeltaH2, DeltaV2 = -79 +/- 5 kcal mol(-1), -9 +/- 2 mL mol(-1); DeltaH3, DeltaV3 = +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 a3 to heme a followed by proton transfer from Glu242 to Arg38 on an approximately 100 ns timescale. The slow phase is likely a combination of CO thermal dissociation from CuB and additional ET between heme a3 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.