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

Abstract

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., < ∼50 ns), a second phase occurring with a lifetime of ∼100 ns and a third phase occurring with a lifetime of ∼2 µs. The corresponding volume and enthalpy changes for these processes are: ΔH₁, ΔV₁ = +79 ± 10 kcal mol⁻¹, +9 ± 1 mL mol⁻¹; ΔH₂, ΔV₂ = −79 ± 5 kcal mol⁻¹, −9 ± 2 mL mol⁻¹; ΔH₃, ΔV₃ = +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–Cu_B⁺ 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 ∼100 ns timescale. The slow phase is likely a combination of CO themal dissociation from Cu_B 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.