Orchestration of diverse components in soluble methane monooxygenase for methane hydroxylation

doi:10.1016/S1872-2067(24)60192-0

Abstract

Abstract:

Methane (CH₄) has a higher heat capacity (104.9 kcal/mol) than carbon dioxide (CO₂), and this has inspired research aimed at reducing methane levels to retard global warming. Hydroxylation under ambient conditions through methanotrophs can provide crucial information for understanding the harsh C-H activation of methane. Soluble methane monooxygenase (sMMO) belongs to the bacterial multi-component monooxygenase superfamily and requires hydroxylase (MMOH), regulatory (MMOB), and reductase (MMOR) components. Recent structural and biophysical studies have demonstrated that these components accelerate and retard methane hydroxylation in MMOH through protein-protein interactions. Complex structures of sMMO, including MMOH-MMOB and MMOH-MMOD, illustrate how these regulatory and inhibitory components orchestrate the di-iron active sites located within the four-helix bundles of MMOH, specifically at the docking surface known as the canyon region. In addition, recent biophysical studies have demonstrated the role of MmoR, a σ⁵⁴-dependent transcriptional regulator, in regulating sMMO expression. This perspective article introduces remarkable discoveries in recent reports on sMMO components that are crucial for understanding sMMO expression and activities. Our findings provide insight into how sMMO components interact with MMOH to control methane hydroxylation, shedding light on the mechanisms governing sMMO expression and the interactions between activating enzymes and promoters.

Key words: Soluble methane monooxygenase, Non-heme di-iron active site, Methane oxidation, C-H activation, O₂ activation

Yunha Hwang, Dong-Heon Lee, Seung Jae Lee. Orchestration of diverse components in soluble methane monooxygenase for methane hydroxylation[J]. Chinese Journal of Catalysis, 2025, 68: 204-212.

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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(24)60192-0

https://www.cjcatal.com/EN/Y2025/V68/I1/204

Figures/Tables 5

Fig. 1. Gene clusters and structures of sMMO. (a) Gene clusters of sMMO operon from type II and Type X methanotrophs. X-ray crystallography structure of MMOH (PDB: 1MTY) (b), MMOH-MMOB (PDB: 4GAM) (c), and MMOH-MMOD (PDB: 6D7K) (d). MMOH has a homodimer structure (α2β2γ2) with two canyon regions, which allow the binding of other components, MMOB or MMOD. MMOH: methane monooxygenase hydroxylase; MMOB: methane monooxygenase regulatory protein; MMOD: methane monooxygenase inhibitory protein; sMMO: soluble methane monooxygenase.

Fig. 2. Positional shifts in the four-helix bundle through the complex generation of MMOH and other components. (a) Schematic model depicting the allosteric effect on MMOH induced by MMOB and MMOD. Root-mean square-deviations (RMSD) between MMOH (PDB: 1MTY) and complexes, including MMOH-MMOB (PDB: 4GAM) (b) and MMOH-MMOD (PDB: 6D7K) (c). MMOH: methane monooxygenase hydroxylase; MMOB: methane monooxygenase regulatory protein; MMOD: methane monooxygenase inhibitory protein; sMMO: soluble methane monooxygenase.

Fig. 3. Di-iron active sites in sMMO structure. (a) X-ray, oxidized MMOH (PDB: 1MTY). (b) X-ray, reduced MMOH (PDB: 1FYZ). (c) X-ray, MMOH-MMOB (PDB: 4GAM). (d) XFEL, oxidized MMOH-MMOB (PDB: 6YD0). (e) reduced MMOH-MMOB (PDB: 6YDI). (f) MMOH-MMOD (PDB: 6D7K). Rotameric shifts by the sidechains of T213 (g) and Glu243 (h) induced by MMOB. Rotameric shifts by the sidechains of T213 (i) and Glu243 (j) induced by MMOD. MMOH: methane monooxygenase hydroxylase; MMOB: methane monooxygenase regulatory protein; MMOD: methane monooxygenase inhibitory protein; sMMO: soluble methane monooxygenase; XFEL: X-ray free electron laser.

Fig. 4. Substrate ingress and egress pathways based on the complex formation. Cavities 1, 2, and 3 are shown as surfaces within the interior of MMOH (PDB: 1MTY) (a) and MMOH-MMOB (PDB: 4GAM) (b). (c) The binding of MMOB initiates a cascade reaction that leads to conformational changes in residues associated with cavities. The cavities visualized using PyMOL 2.5.2 are represented as surface models. MMOH: methane monooxygenase hydroxylase; MMOB: methane monooxygenase regulatory protein; sMMO: soluble methane monooxygenase.

Fig. 5. Transcriptional regulator MmoR for sMMO expression. (a) Schematic of the transcriptional mechanism of sMMO mediated by MmoR and Sigma-54. (b) Domain organization of M. sporium 5 MmoR. (c) Upstream activator sequence (UAS) and promoter regions located between mmoG and mmoX in M. sporium 5. (d) Conserved enhancer and promoter sequences from the upstream of mmoX in Methylosinus trichosporium OB3b, Methylosinus sporium 5, and Methylococcus capsulatus Bath. MmoR: methane monooxygenase transcriptional regulator; RNAP: RNA polymerase; IHF: integration host factor; sMMO: soluble methane monooxygenase.

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