Revealing the Functional States in the Active Site of BLUF Photoreceptors from Electrochromic Shift Calculations

Photoexcitation with blue light of the flavin chromophore in BLUF photoreceptors induces a switch into a metastable signaling state that is characterized by a red-shifted absorption maximum. The red shift is due to a rearrangement in the hydrogen bond pattern around Gln63 located in the immediate proximity of the isoalloxazine ring system of the chromophore. There is a long-lasting controversy between two structural models, named Q63A and Q63J in the literature, on the local conformation of the residues Gln63 and Tyr21 in the dark state of the photoreceptor. As regards the mechanistic details of the light-activation mechanism, rotation of Gln63 is opposed by tautomerism in the Q63A and Q63J models, respectively. We provide a structure-based simulation of electrochromic shifts of the flavin chromophore in the wild type and in various site-directed mutants. The excellent overall agreement between experimental and computed data allows us to evaluate the two structural models. Compelling evidence is obtained that the Q63A model is incorrect, whereas the Q63J is fully consistent with the present computations. Finally, we confirm independently that a keto–enol tautomerization of the glutamine at position 63, which was proposed as molecular mechanism for the transition between the dark and the light-adapted state, explains the measured 10 to 15 nm red shift in flavin absorption between these two states of the protein. We believe that the accurateness of our results provides evidence that the BLUF photoreceptors absorption is fine-tuned through electrostatic interactions between the chromophore and the protein matrix, and finally that the simplicity of our theoretical model is advantageous as regards easy reproducibility and further extensions.


Text S1: Calculation of the Electrochromic Shifts for the Different Mutants S41A, N44A and N45A
Ser41 is present in all 25 coordinates sets. But more than one side-chain conformation is reported. All 10 coordinates sets of 1X0P show a hydrogen bond between Ser41 and the backbone oxygen of Ile37, which orients the side-chain hydroxyl dipole such that the negatively charged oxygen lies in the negative region of highest intensity of the difference potential of the chromophore. On the other side, it places the polar hydrogen closer to the neutral region of the difference potential ( Figure 4). The S41A mutation, based on the described Ser41 wild type conformation, should, therefore, lead to a strongly redshifted absorption maximum. We obtain quantitative agreement with the measured value (Table S2) based on the structural information of 1X0P. We conclude that the measured value of 12 nm to the red is consistent with the side-chain conformation proposed in 1X0P.
The 10 coordinates sets of 2HFN show three distinct side-chain conformations of Ser41.
The conformations in the coordinates sets B, D and J are as in 1X0P. Again, computed absorption shifts meet the experimental value quantitatively (Table S2). The same side-chain conformation is reported in the two coordinates sets of 2IYG and in coordinates sets A and B of 1YRX. All four computed values agree with the measured 12 nm redshift S1 (Table S2).
The computation of the S41A absorption shift shows that only one out of four proposed side-chain conformations of Ser41 explains the measured wild type-minus-mutant absorption shift. In the remaining three conformations the influence of the polar side-chain hydrogen atom of Ser41 is more pronounced as it moves from the neutral to the more negative region of the difference potential. The distinguished conformation, besides explaining the spectral shift, has the advantage that it is stabilized by a hydrogen bond, which is missing in the remaining three conformations.
Asn44 is not completely conserved among the BLUF proteins (Table S2). In our data set it is present in all coordinates sets of 1X0P and 2HFN, with a reported side-chain conformation S2 that places the carbonyl oxygen atom away from the isoalloxazine ring system. A hydrogen bond is formed between the C2 − − O group of the isoalloxazine and the side-chain amide group of Asn44 (Figure 1b). With the exception of this hydrogen atom, the electrostatic influences of the remaining polar side-chain of Asn44 on the absorption maximum of the photoreceptor seem to be balanced. The N44A mutation can therefore be conceived as the elimination of a positive charge from the positive region of intermediate strength of the difference potential, which should yield a redshift, as for S41A, but weaker in magnitude. The quantitative agreement between the computed and measured values for all 20 coordinates sets confirms this hypothesis (Table S2).
Asn45 is a critical residue for the photoreaction and hence it is completely conserved within the BLUF proteins ( Figure 3). Its side-chain conformation is similar in the two structural models, Trp in and Trp out . In all 25 structural templates the side-chain conformation is such that two hydrogen bonds are present between the residue and the flavin chromophore. One hydrogen bond is formed between the the side-chain carbonyl and the N2H group of the chromophore; the second hydrogen bond is between a hydrogen atom of the amide group and the C4 − − O group of the isoalloxazine. Since the rest of the side-chain is located in regions of weaker difference potential compared to the hydrogen, we assume that the latter is the dominating atom ( Figure 4). The N45A mutation should, therefore, result in a blueshifted absorption maximum. Computed absorption shifts meet the experimental value in all cases but we note that absorption shifts based on structures proposing the Trp in configuration agree somewhat better with the experimental value (Table S2). But we also observe quantitative agreement for 2IYG coordinates set A, which is in the Trp out configuration. Overall differences are certainly to small to be of any significance for the distinction of Trp in and Trp out structural models.    Table S7)    Figure S3: Difference dipole moment of the methyl-isoalloxazine ring system in dependence on the XC functional used in the (TD)DFT calculations S13 (blue) compared to the experimentally determined value S14 (red).   The side-chain orientation of Gln63 has been manipulated in order to simulate the hydrogen bond distance between the side-chain amide and the C4− −O of the isoalloxazine as related for the light-adapted state according to spectroscopic data (see text).