Time-resolved thermodynamic analysis of the oat phytochrome A phototransformation. A photothermal beam deflection study
2001
Michler, I. | Braslavsky, S.E.
The time-resolved enthalpy and the structural volume changes after excitation of native oat phytochrome A were studied in the micro- to milliseconds range by photothermal beam deflection (PBD), a technique that follows the time-resolved refractive index changes upon decay of the excited species. The first set of intermediates, I700(1) and I700(2), stores ca 83% of the energy of the first excited state, in agreement with previous optoacoustic data, whereas the second set stores only ca 18%. The temperature dependence of the amplitudes ratio for the optical absorbances of the (I700(1) + I700(2)) intermediates set is explained on the basis of the thermochromic equilibrium between P(r,657) and P(r,627), which also is in line with the present 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 intermediates, I700(1) and I700(2), as well as the second set of intermediates, Ibl(1) and Ibl(2). Thus, the final steps toward P(fr) should be largely driven by positive entropic changes brought about by protein movements, in line with previous resonance Raman data. For the production of the first set of intermediates (I700(1) and I700(2)) an expansion of 18 +/- 13 mL mol(-1) was determined, and a further expansion greater than or equal to mL mol(-1) was estimated for the decay from I700(i) to the set of Ibl, intermediates, indicating that the far red-absorbing form of phytochrome (Pfr) has a larger volume than the red-absorbing form of phytochrome. This is in agreement with previous chromatographic and circular dichroism data according to which Pfr shows a larger volume and the chromophore shows a higher accessibility, respectively, in the P(fr) state.
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