Exploring the Impact of Membrane Environments on Particulate Methane Monooxygenase - Methane monooxygenase enzymes (MMOs) perform the chemically challenging reaction of the oxidation of methane to methanol. MMOs are metalloenzymes that can either be soluble (sMMO) or membrane-bound (particulate MMO, pMMO). The pMMO is a copper-dependent enzyme that is found in methanotroph intracytoplasmic membranes, organized into hexagonal arrays. pMMO is only active in a lipid environment, indicating the essential role of the membrane in the methane oxidation activity. Previous studies of pMMO in DDM micelles, native lipid nanodiscs, synthetic lipid nanodiscs, and native membranes enabled comparisons of activity in different lipid environments. The goal of this research is to determine why the native membrane is so important for optimal methane oxidation activity using a proteoliposome system. Liposomes provide several advantages over nanodiscs, including increased enzymatic activity as well as the retention of the native array patterning. In Aim 1, pMMO will be reconstituted into liposomes of different sizes and with different lipid compositions, and the effects on activity will be assessed. In Aim 2, the structures of pMMO arrays and their surrounding lipids in liposomes of varying compositions and sizes will be determined via cryogenic electron microscopy. Finally, in Aim 3, the liposomes will be utilized to test the hypothesis that pMMO translocates protons. These combined studies will elucidate how the membrane environment and its components influence the structure and function of pMMO.
