Why the Flavin Adenine Dinucleotide (FAD) Cofactor Needs To Be Covalently Linked to Complex II of the Electron-Transport Chain for the Conversion of FADH2 into FAD.

A covalently bound flavin cofactor is predominant in the succinate:ubiquinone oxidoreductase (SQR, Complex II), an essential component of the aerobic electron transport, and in the menaquinol:fumarate oxidoreductase (QFR), the anaerobic counterpart, albeit being only present in ~10% of the known flavoenzymes. Here, we investigated the role of this 8α-N(3)-histidyl linkage between the flavin dinucleotide (FAD) cofactor and the respiratory Complex II. After parameterization with DFT we performed classical molecular dynamics simulations and quantum mechanics calculations of Complex II:FAD and Complex II:FADH2, covalently bound and unbound to His-A57. We observed that the covalent bond is essential for the active center arrangement of the FADH2/FAD cofactor. Removal of this bond causes a displacement of the isoalloxazine group which influences the interactions with the protein, flavin solvation and the possible proton transfer pathways. Specifically, for the non-covalently bound FADH2 cofactor the N1 atom moves away from the His-A365 and His-A254 and the N5 atom moves away from glutamine 62A. Both histidines and the glutamine interact with a chain of water molecules that cross the enzyme which is most likely involved in proton transfer. Breaking this chain of water molecules could thereby compromise proton transfer across the two active sites of Complex II.