Conformational gating governs ligand recognition in Bacillus subtilis cytochrome P450 CypX

Bacterial cytochrome P450s are cornerstone monooxygenases that enable selective oxidation across the fields of metabolism and biocatalysis. Yet, for the orphan Bacillus subtilis enzyme CYP134A1 (CypX), how ligand identity governs conformational readiness remains unclear despite its solved structure. Here we combine atomistic simulations of apo CypX and four ligand-bound complexes with principal-component analysis, two-dimensional free energy surfaces, and an operational three-state classification based on two gating coordinates: d_mouth, the distance between the centers of geometry of the BC-loop and FG-loop Cα sets that reports opening at the channel entrance, and d_cover, the distance from the FG-loop Cα centroid to the heme Fe that reports the cover position over the distal pocket. These descriptors are complemented by reactive-geometry and hydrogen-bond analyses and by MM-PBSA component analysis as a supportive energetic readout. The simulations reveal a conserved mouth-cover landscape with three recurrent basins, open, intermediate, and closed. Ligands mainly redistribute populations across these pre-existing states and reshape gate geometry rather than generating new conformational states. Together, these findings support a conformational-selection mechanism linking ligand recognition, gating, and catalytic readiness in this orphan bacterial P450.