The time-resolved thermodynamics of the flavin mononucleotide (FMN)-binding LOV1 domain of Chlamydomonas reinhardtii phot (phototropin homolog) was studied by means of laser-induced optoacoustic spectroscopy. In the wild-type protein the early red-shifted intermediate LOV715 exhibits a small volume contraction, V715 =-1.50 ml/mol, with respect to the parent state. LOV715 decays within few micros into the covalent FMN-Cys-57 adduct LOV390, that shows a larger contraction, V390 =-8.8 ml/mol, suggesting a loss of entropy and conformational flexibility. The high energy content of LOV390, E390 =180 kJ/ mol, ensures the driving force for the completion of the photocycle and points to a strained photoreceptor conformation. In the LOV-C57S mutated protein the photoadduct is not formed and V390 is undetected. Large effects on the measured Vs are observed in the photochemically competent R58K and R58K/D31Q mutated proteins, with V390 =-2.0 and -1.9 ml/mol, respectively, and V715 0. The D31Q and D31N substitutions exhibit smaller but well-detectable effects. These results show that the photo-induced volume changes involve the protein region comprising Arg58, which tightly interacts with the FMN phosphate group.
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