Cryo‐EM Structures Reveal the Molecular Basis of Asymmetric Allosteric Activation by MMOB in the Hydroxylase of Soluble Methane Monooxygenase

ABSTRACT Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane at non‐heme di‐iron active sites under ambient conditions. The regulatory component (MMOB) is essential for catalytic activity, inducing conformational changes in the active site and facilitating substrate ingress in hydroxylase (MMOH). Advances in cryogenic electron microscopy (cryo‐EM) have enabled structural studies of sMMO under near‐native conditions. 3D variability analysis reveals that an asymmetric MMOH–MMOB complex predominates in solution, supporting a sequential binding mechanism. Here, we report a 2.85 Å‐resolution cryo‐EM structure of MMOH–1MMOB (H‐1B) complex, in which a single MMOB binds to MMOH and generates two distinct protomers: MMOB‐bound protomer (HB ^A , αβγB) and non‐MMOB‐bound protomer (HB ^B , αβγ). MMOB initiates an allosteric cascade beginning at the N ‐terminal region of the HB ^A β‐subunit and extending to the di‐iron active site. This structural shift shortens the Fe···Fe distance in HB ^A to 2.7 Å, consistent with a geometry conducive to O ₂ activation, while HB ^B retains a 3.1 Å distance. The γ‐subunit modulates this asymmetry by stabilizing the resting HB ^B and facilitating the reorganization of HB ^A . These findings support an asymmetric catalytic cycle that allows continuous hydroxylation and promotes electron transfer, thereby providing a structural basis for future mechanistic studies.