Structural Effects on the Ultrafast Photoisomerization of Photoactive Yellow Protein. Transient Absorption Spectroscopy of Two Point Mutants

International audience; Subpicosecond transient absorption spectroscopy was used to address the role of the local environment on the photoisomerization process of the p-coumaric thioester chromophore in Photoactive Yellow Protein (PYP) by studying two point mutants, T50V and E46Q. These mutations introduce alterations of the hydrogen-bond network involving the amino acids of the active site close to the chromophore and the chromophore phenolate group, respectively. Transient-absorption spectra of T50V and E46Q are found to be qualitatively similar to those of the wild-type PYP (WT) and R52Q, suggesting that the earliest steps of the photoinduced processes in all three mutants remain similar to those of the WT. Target analyses of the transient spectra of T50V, E46Q, R52Q and WT, were successfully performed by using a model based on the one previously published by Larsen et al. (Biophys. J. 2004, 87, 1858), which involves heterogeneous excited-state populations undergoing deactivation along two competitive relaxation pathways. A so-called reactive pathway leads to the sequential formation of the well-characterized cis intermediates, I0 and I1, of the photocycle. The second pathway is non reactive and produces a transient species that restores the initial trans ground state in 3-6 ps. This transient is tentatively attributed to a distorted vibrationally-hot trans ground state. The most prominent effect of mutation is observed for T50V and R52Q which exhibit significantly slower excited-state deactivations, whereas E46Q behaves like the WT protein. This difference is analyzed in terms of a significant decrease, in T50V and R52Q, of the fraction of heterogeneous excited-state population that undergoes isomerization. The quantum yield of isomerization deduced from the target analyses was found to be 0.31 ± 0.08 for WT, 0.22 ± 0.06 for T50V, 0.29 ± 0.08 for E46Q and 0.19 ± 0.05 for R52Q. The decrease of isomerization yield observed in T50V and R52Q is mainly attributed to the loss of rigidity of the protein active site, induced by these mutations, rather than to the deletion of the positive charge of Arg52 in R52Q.