Redetermination of the quantum yield of photoisomerization and energy content in the K-intermediate of bacteriorhodopsin photocycle and its mutants by the photoacoustic technique

Data obtained previously in our lab for the quantum yield retinal photoisomerization and the energy content of the K-intermediate formed in the bacteriorhodopsin and its mutants (bR) photocycle are reexamined using time-resolved transient spectroscopy and laser-induced photoacoustic spectroscopy. In the present experiment both nanosecond and subpicosecond laser pulses are used for excitation, with different reference compounds. From these new results it can be concluded that using CoCl2 as a reference compound in the photoacoustic experiment with subpicosecond laser pulses gives a large amount of prompt heat release resulting from multiphoton absorption processes. This results in an overestimated energy content of the K-intermediate of bR and its mutants. Using different reference compounds, the corrected values are 40 +/- 10 kJ/mol, which is in agreement with previously reported values. The apparent quantum yield and energy content values (for each of the isomeric compositions) of the mutants D212N, D85N, R82Q, A53G, W182F, V49A, deionized blue bR, and acid purple bR are also recalculated using bR as a reference compound and are similar to those of bR. (Phi is in the range 0.55-0.65, and E-K is in the range 40-50 kJ/mol.) The relative insensitivity of the apparent average quantum yield value is discussed.