Figure 4. Model of electron transfer in the eNOS holoenzyme.

A. Conformation of eNOS with bound CaM as visualized by cryo-EM. For clarity only one oxygenase domain (red) and the reductase domain of the other monomer (blue) with bound CaM (green) are shown. The orientation is identical to that shown in the right panel of Figure 2B. The FMN domain is shown in yellow. In order to transfer electrons to the heme in the oxygenase domain, the FMN domain needs to swing from its position towards the oxygenase (curved yellow arrow).

B. A model of the conformation in which electrons are transferred from the FMN domain to the heme in the presence of NADPH. This conformational change can be achieved by a rigid body swing of the FMN domain, pivoted at the CaM binding helix (transition between yellow and blue) and tethered to the rest of the reductase domain at the C-terminus of the FMN domain. To target this movement, CaM is necessary to lock the eNOS CaM-binding domain into place to provide a stable pivot.

C. Close-up of the contact area between the modeled FMN domain and the oxygenase domains. In this geometry the edge-to-edge distance between the aromatic rings of the flavin and the heme is 13.5 Å.

D. View of the reductase dimer interface from the ‘bottom’ in relation to Figures A–C, along the two-fold symmetry axis. The cofactors are shown in green (NADPH), yellow (FAD) and orange (FMN). The two reductase domains are shown in red and blue. The CaMs are just visible in pale green blending into the background (labeled ‘CaM’). Note that the FMN domain is located in between the bulk of the reductase domain and the CaM. As a consequence, the FMN and the FMN domain are largely obstructed by the rest of the reductase domain. Several of the potentially interacting residues are shown and labeled, including the most C-terminal residue resolved in the reductase crystal structure (labeled ‘F1178 (C)’) and many oppositely charged residues.