Binding and Translocation of Substrate Allosterically Promotes Functional Interactions Within the AlkB–AlkG Electron Transfer Complex

The alkane monooxygenase AlkB and rubredoxin AlkG form an electron transfer complex that hydroxylates terminal alkanes to produce alcohols. The recent cryoEM study of Fontimonas thermophila AlkB–AlkG complex revealed its architecture, including a dodecane (D12) substrate at the active site. However, FtAlkBG molecular mechanism of action remains unknown. Here, we examined its dynamics and interactions by multiscale computations, including molecular dynamics simulations, elastic network models, and QM/MM of the oxygen activation mechanism at the AlkB catalytic site. D12 maintained stable interactions within the catalytic site during two MD runs, coordinated by hydrophobic residues L263–L264, I267, I133. A third extended run revealed that D12 could translocate to a membrane-exposed site near S49/F46 along a hydrophobic channel gated by I54. During this translocation, D12 was temporarily stabilized at intermediate sites IS1 (lined by I27/L30–G31/G50/L53–I54/P59/S124/A127–V128) and IS2 (I33–G34/L37/L45–F46/S49) before nearly exiting the protein, and diffused back to the active site, assisted by L30. Substrate binding and translocation across those intermediate sites affects the coupling between the iron centers in AlkBG, and interfacial interactions between AlkB–AlkG. The channel was further connected to the cytosol, near two surface-exposed arginines, potentially allowing for O₂ passage. The allosteric effects between D12 putative entry site, catalytic site and AlkB–AlkG interface were analyzed by ENM-based methods which confirmed the cooperative perturbation responses and strongly correlated movements of residues belonging to those distal regions. Our study provides new mechanistic insights into key sites and their interactions that could be targeted for developing AlkB-variants with desirable alkane conversion functions.