The particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) is a fascinating enzyme, which can mediate the facile and selective conversion of methane into methanol under ambient conditions. The enzyme is the best methane oxidizer in the nature. In contrast, it is extremely difficult to achieve the efficient conversion of methane into methanol in the laboratory because the C-H bond is very inert (the C-H bond energy is very high, 105 kcal/mol). For that reason, the search for a catalyst or process that can convert methane into methanol under mild conditions has been one of the holy grails of organic chemistry. Moreover, the chemical transformation has a great potential for industry since methanol is much more value-added than methane and the earth has a hug reserve of methane. Understanding the mechanism of methane oxidation in pMMO, therefore, has long been a goal of biochemist in searching for a new catalyst. Despite almost 30 years of extensive research on its biological function and crystal structures, unfortunately, the catalytic site and mechanism of methane oxidation in pMMO enzyme is still under debate.

In Part 1 (Chapter II) of this thesis, two suicide substrates, acetylene (HCCH) and propyne (CH3CCH), have been developed as mechanism-based probes of the catalytic site of methane oxidation within the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath). In addition, trifluoropropyne (CF3CCH) has been shown to be a non-competitive inhibitor of the enzyme. The oxidation of HCCH and CH3CCH by the enzyme is similar to that observed for a biomimetic tricopper complex that has been recently shown to be capable of mediating efficient methane oxidation under ambient conditions. Parallel experiments on the recombinant PmoB subunit anchored in membranes reveal no evidence of reactivity between HCCH (or CH3CCH) and the dicopper center when the latter is activated by O2 in presence of reductant. These results together with in-depth studies on the inhibition of the pMMO by the three alkynes together have culminated in (i) the identification of the hydroxylation site of the enzyme; (ii) the mapping of the substrate/product migration pathways during turnover; and (iii) the elucidation of the catalytic mechanism of methane oxidation. MALDI-TOF MS of the subunits of the chemically modified pMMO complexes together with LC-MS/MS analysis of the peptides derived from in-gel proteolytic digestion of the subunits reveal that the catalytic site of the alkane oxidation resides within the transmembrane domain of the protein. The pattern of chemical modification of the protein suggests the role of the transmembrane subunits (pmoA and pmoC) in controlling the substrate entrance and product exit during enzymatic turnover. These conclusions are corroborated by computer docking of the substrates and products to the protein fold provided by the X-ray crystal structures. Taken together, these results provide strong evidence that the catalytic site is indeed the tricopper cluster sequestered by the pmoA and pmoC subunits.

In Part 2 (Chapter III) of the thesis, I will present the development of nanodiamond particles in improving the mass spectrometric analysis of membrane proteins. This work came about from our original efforts to better characterize pMMO by using proteomic techniques. In fact, we have confirmed the exact molecular weights of pMMO’s subunits by nanodiamond-assisted MALDI-MS. The observed masses (46 kDa, 30 kDa, and 29 kDa) from the MALDI-MS analysis of intact pMMO complex have been assigned confidently to PmoB, PmoC and PmoC respectively. Moreover, we have extended the application of nanodiamond particles to enrich and purify other membrane proteins from highly contaminated buffers. We also demonstrated the outperformance of this approach, comparing to many other current methods, in preparing membrane proteins for both MALDI-MS analysis of membrane protein complexes and membrane protein digestion for “bottom-up” proteomics.

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