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. 2024 Jun 6;128(22):5320-5326.
doi: 10.1021/acs.jpcb.4c00710. Epub 2024 May 28.

Understanding the Red Shift in the Absorption Spectrum of the FAD Cofactor in ClCry4 Protein

Katarina Kretschmer  1 , Anders Frederiksen  1 , Peter Reinholdt  2 , Jacob Kongsted  2 , Ilia A Solov'yov  1   3   4
Affiliations

Understanding the Red Shift in the Absorption Spectrum of the FAD Cofactor in ClCry4 Protein

Katarina Kretschmer et al. J Phys Chem B. .

Abstract

It is still a puzzle that has not been entirely solved how migratory birds utilize the Earth's magnetic field for biannual migration. The most consistent explanation thus far is rooted in the modulation of the biological function of the cryptochrome 4 (Cry4) protein by an external magnetic field. This phenomenon is closely linked with the flavin adenine dinucleotide (FAD) cofactor that is noncovalently bound in the protein. Cry4 is activated by blue light, which is absorbed by the FAD cofactor. Subsequent electron and proton transfers trigger radical pair formation in the protein, which is sensitive to the external magnetic field. An important long-lasting redox state of the FAD cofactor is the signaling (FADH•) state, which is present after the transient electron transfer steps have been completed. Recent experimental efforts succeeded in crystallizing the Cry4 protein from Columbia livia (ClCry4) with all of the important residues needed for protein photoreduction. This specific crystallization of Cry4 protein so far is the only avian cryptochrome crystal structure available, which, however, has great similarity to the Cry4 proteins of night migratory birds. The previous experimental studies of the ClCry4 protein included the absorption properties of the protein in its different redox states. The absorption spectrum of the FADH• state demonstrated a peculiar red shift compared to the photoabsorption properties of the FAD cofactor in its FADH• state in other Cry proteins from other species. The aim of this study is to understand this red shift by employing the tools of computational microscopy and, in particular, a QM/MM approach that relies on the polarizable embedding approximation.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
FADH• cofactor and the residues W395, W372, W318, and W369 involved in the ET process inside of the ClCry4 protein. The residue N391, specific for the ClCry4 protein, is indicated. The atoms inside the marked QM-region were treated quantum mechanically.
Figure 2
Figure 2
Time evolution of the rmsd of the ClCry4 protein backbone during the equilibration (NPT) (A) and the production (NVT) (B) simulations computed relative to the starting protein configuration in the respective simulations. For the NPT simulation, the rmsd of the protein and the FADH• cofactor was plotted with (black line) and without (red line) the phosphate binding loop residues 220–245. The rmsd time evolution for the NVT simulation is shown for the complete protein backbone.
Figure 3
Figure 3
Photoabsorption spectra computed for the FADH• cofactor inside AtCry1 (A) and ClCry4 (B). The experimental absorption spectrum for the FADH• cofactor inside the AtCry1 (orange) and ClCry4 protein (green) was measured in previous studies., The computational photoabsorption spectra for AtCry1 (blue) were reused from a previous study. The thin lines represent the computed absorption spectra obtained for the different snapshots, while the solid lines represent the averaged absorption spectra over all snapshots for the AtCry1 and ClCry4 proteins, respectively. The arrows represent the pronounced peaks in the calculated and experimental absorption spectra of both proteins. The black vertical lines mark the peaks in both experimentally obtained absorption spectra, to which the computationally obtained absorption spectra for AtCry1 and ClCry4 were red-shifted. The green and orange vertical lines in (B) emphasize the experimentally observed red shift inside the ClCry4 protein compared to the absorption spectrum of AtCry1. The orange dashed line features a fragment of the AtCry1 absorption spectrum from (A).
Figure 4
Figure 4
Normalized probability density distribution ρ(r) of the sampled distances between the flavin part of the FADH• cofactor (N5 atom) and the oxygen atoms OD1 and OD2 of the D396 residue in the AtCry1 protein (A) and the flavin part of the FADH• cofactor (N5 atom) and the OD1 atom of the N391 residue in the ClCry4 protein (B).
Figure 5
Figure 5
Normalized probability density distribution ρ(ϑ) of the sampled angles between the flavin part of the FADH• cofactor (N5–H5 bond) and the OD1/OD2-C bond of the D396 residue in the AtCry1 protein (A) and the flavin part of the FADH• cofactor (N5–H bond) and the OD1-C bond of the N391 residue in the ClCry4 protein (B).
Figure 6
Figure 6
Averaged absorption spectra from the photoabsorption spectra computed for the FADH• cofactor inside the ClCry4 protein: wild type (blue), excluded N391 residue (red), and N391D mutation (black). The peaks of the individual absorption spectra are highlighted here to illustrate the shift of the spectra in relation to each other. The spectra were recorded for a wavelength between 500 and 700 nm. All spectra from the individual snapshots are shown in Figure S1 in the Supporting Information.
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