Time-resolved thermodynamic analysis of the oat phytochrome A phototransformation. A photothermal beam deflection study

The time-resolved enthalpy and the structural volume changes after excitati on of native oat phytochrome A were studied in the micro- to milliseconds r ange by photothermal beam deflection (PBD), a technique that follows the ti me-resolved refractive index changes. upon decay of the excited species. Th e first set of intermediates, I-700(1) and I-700(2), stores ca 83% of the e nergy of the first excited state, in agreement with previous optoacoustic d ata, whereas the second set stores only ca 18%. The temperature dependence of the amplitudes ratio for the optical absorbances of the (I-700(1) + I-70 0(2)) intermediates set is explained on the basis of the thermochromic equi librium between P-r,P-657 and P-r,P-672, which also is, in line with the pr esent PBD data. These data were best fitted with a parallel mechanism (with equal yield in each branch) for the production of the first set of interme diates, I-700(1), and I-700(2), as well as the second set of intermediates, I-bI(1), and F-bl-(2). Thus, the final steps toward, P-fr should be largel y driven by positive entropic changes brought about by protein movements, i n line with previous resonance Raman data. For the production of the first set of intermediates (I-700(1) and I-700(2)) an expansion of 18 +/- 13 mL m ol(-1) was determined, and a further expansion greater than or equal to7 mL mol(-1) was estimated for the decay from I-700(i) to the set of I-bI inter mediates, indicating that the far red-absorbing form of phytochrome (P-fr) has a larger volume than the red-absorbing form of phytochrome. This is in agreement with previous chromatographic and circular dichroism. data accord ing to which P-fr shows a larger volume and the chromophore shows a, higher accessibility, respectively, in the P-fr state.