Membrane properties modulate methane oxidation by particulate methane monooxygenase

Area of Science:

• Biochemistry and Structural Biology
• Microbiology
• Membrane Biophysics

Background:

• Copper-dependent membrane monooxygenases, particulate methane monooxygenase (pMMO) and ammonia monooxygenase (AMO), are crucial for methane and ammonia oxidation, respectively.
• These enzymes are vital biotechnological targets and form hexagonal arrays within intracytoplasmic membranes (ICMs).
• The influence of specific lipids and membrane morphology on pMMO and AMO activity remains poorly understood.

Purpose of the Study:

• To investigate how membrane ultrastructure, lipid composition, and curvature affect pMMO and AMO activity.
• To determine the structural basis of pMMO-pMMO interactions within ICM arrays.
• To establish a platform for studying these enzymes in controlled lipid environments.

Main Methods:

• Cryoelectron tomography (cryoET) to analyze ICM ultrastructure in different bacterial species.
• Reconstitution of purified pMMO into liposomes of varying sizes and lipid compositions.
• High-resolution cryo-electron microscopy (cryoEM) to determine the structure of pMMO arrays.
• Methane solubility measurements to assess lipid-specific effects.

Main Results:

• Distinct ICM ultrastructures were observed across three species of methane- and ammonia-oxidizing bacteria.
• pMMO activity in reconstituted proteoliposomes was inversely correlated with liposome diameter, suggesting enhanced function in curved membranes.
• Phosphoethanolamine (PE), particularly unsaturated forms, and cardiolipin significantly increased pMMO activity.
• A 6 Å cryoEM structure revealed the arrangement of neighboring pMMO trimers and mediating lipids/residues.

Conclusions:

• Membrane curvature and specific lipids, notably PE and cardiolipin, play critical roles in modulating pMMO activity.
• The findings provide insights into the functional impact of the native membrane environment on monooxygenase activity.
• This work establishes a versatile platform for future studies on pMMOs and AMOs in tunable lipid environments.