Fig. 2. Structural and energetic changes of the Asp⁴¹⁰-Arg³⁸¹ salt bridge.

Sausage representation of the computed RMSFs of the backbone atoms in the (FAD)_ox (A) and FAD^−● (B) states. The RMSF values are encoded by both the thickness and color in the illustration. Close-up view of hydrogen bonding interactions of Asp⁴¹⁰ (D410) in (C) the (FAD)_ox state with Arg³⁸¹ (R381) and in (D) the FAD^−● state with Lys²⁶⁴ (K264) and Asn²²² (N222). (E) Time evolution of the distance between Asp⁴¹⁰ and Arg³⁸¹, distance between Asp⁴¹⁰ and Lys²⁶⁴, and gyration of radius (R_g) over all backbone CA atoms in the oxidized (FAD)_ox (magenta), FAD^−● (blue), and FAD/Ade^−● (green) states. The shaded area in yellow indicates concerted motions between the disruption of the Asp⁴¹⁰-Arg³⁸¹ salt bridge and the formation of the Asp⁴¹⁰-Lys²⁶⁴ contact, accompanied by an increase in R_g in the FAD^─● state (blue). (F) Computed potential of mean force (PMF) as a function of the ion-pair separation between Asp⁴¹⁰ and Arg³⁸¹ from umbrella sampling simulations at different cofactor states: oxidized (FAD)_ox (magenta), FAD^−● (blue), and FAD/Ade^−● (green). (G) Computed average nonequilibrium work for separating the Asp⁴¹⁰-Arg³⁸¹ salt bridge through steered MD simulations. The color code is the same as that in (F).