Functional role of an unusual tyrosine residue in the electron transfer chain of a prokaryotic (6–4) photolyase† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc03386a

FAD photoreduction mechanism by different aromatic residues in a phylogenetically ancient photolyase.

: Oligonucleotide sequences for DNA repair studies (first line) and site-directed mutagenesis (2nd to 7th line). The TT pair that yields the (6-4) photoproduct is printed in bold. The triplets of the mutation sites are underlined.

Parameterization of DFTB-FO energies
In order to describe the hole transfer energetics correctly, the DFTB HOMO energies must be corrected compared to ionization potential (IP) calculated at DFT level (ωB97XD 3 / 6-311g** 4,5 ). The relative energies of Trp and Tyr have been determined in a previous work. 6 We use the same protocol for relative energies of Trp and Phe. In each molecule, the DFT IP and the DFTB HOMO energy of the neutral state are considered to determine the correction which will be added to the site energy. We calculate the energy difference between the IP of two sites at DFT level and compare this difference to HOMO energy of DFTB. The obtained value (Table   S1) is added to DFTB HOMO energy to reproduce the DFT difference at DFTB level.
FAD requires a more specific treatment because of the excitation step needing for hole transfer. The interacting orbitals should be the HOMO-1 of FAD * and HOMO of A. We can consider that the HOMO-1 of FAD* correspond to the HOMO of FAD semi-occupied following different approximations: -we neglect the impact of the occupation of the LUMO of FAD on the HOMO energy -we neglect the relaxation of the environment induced by modification of the dipolar moment of FAD after excitation. The hole transfer from FAD* to A is supposed to occur very fast (< 1 ps), the environment has no time to relax before hole transfer.
We thus compare the DFTB HOMO energy with the DFT IP of FAD to determine the shift added to DFTB HOMO energy.

Water molecules between FAD and A backbone
In the PhrB crystallographic structure, a water molecule interacts with FAD O4 or N5 and A backbone. 7 This position is conserved all along the MD simulations of WT and Y391F (Table   S3). In Y391A, the available space between FAD and Trp390 is filled up with 4-5 water molecules ( Figure S2). The water bridge between FAD and A backbone is maintained during MD as five water molecules occupy successively this position during the 100 ns Y391A simulation ( Figure S3).

Interaction between FAD and A in Y391W rotamers
We performed 100 ns simulations for four different systems (WT, Y391F, Y391Wp and Y391Wd). Each simulation shows a stable protein and a small RMSD fluctuation of the mutated A with the exception of a short turn of the W391 in Y391Wp ( Figure S4). This movement happens between the 24 ns and 35 ns and the distance between FAD and Trp391 is increased by around 2 Å. In the proximal position of Trp391, the aromatic planes of A and FAD are parallel and the N5 and indol nitrogen atom are quite close (around 4 Å) but no hydrogen bond between them is observed ( Figure S5). Movement of Trp391 has a small impact on isoalloxazine ring RMSD.

Figure S4: RMSD evolution of FAD (left) and A (right) along Y391Wp (black) and Y391Wd (grey) simulations (compared to final position of Y391Wp). The highest RMSD values for
Y391Wp correspond to the position of A closest to B, similar to Y391Wd position.

Superexchange tunneling through tyrosine and phenylalanine
We calculated the superexchange tunneling with the same method as published in ref 8,9 . We used the HOMO of FAD as donor, A (Tyr or Phe) as bridge and B (Trp390) as acceptor for an electron hole. We also used the pathways plugin in VMD 10,11 to perform a Pathways model analysis 12 taking into account A (Tyr, Phe or Ala) and water molecule as bridge.
The different pathways are given in Figure S6 for WT and Y391F. The pathways involving water molecules represent 80%, 75% and 88% of the strongest pathways in WT, Y391F and Y391A simulations respectively. The pathways through A aromatic cycle and associated electronic coupling damping values have been obtained deleting the water from the bridge. All average electronic coupling damping values are reported in Table S4.  for the different MD simulations are given in Figure S4. Films (also given in SI) show the charge propagation on one simulation of 500 ns of WT or Y391Wp. In this simulation, residues are coloured according to their charge: blue for positive charge, red for neutral and green for intermediate charge.