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

초록

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 & Aring;-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 (HBA, alpha beta gamma B) and non-MMOB-bound protomer (HBB, alpha beta gamma). MMOB initiates an allosteric cascade beginning at the N-terminal region of the HBA beta-subunit and extending to the di-iron active site. This structural shift shortens the Fe<middle dot><middle dot><middle dot>Fe distance in HBA to 2.7 & Aring;, consistent with a geometry conducive to O2 activation, while HBB retains a 3.1 & Aring; distance. The gamma-subunit modulates this asymmetry by stabilizing the resting HBB and facilitating the reorganization of HBA. These findings support an asymmetric catalytic cycle that allows continuous hydroxylation and promotes electron transfer, thereby providing a structural basis for future mechanistic studies.

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