The particulate methane monooxygenase from Methylococcus capsulatus (Bath)
The isolation procedure for the pMMO complex has been optimised to obtain a high specific activity enzyme from Methylococcus capsulatus (Bath). The enzyme is comprised of the pMMO hydroxylase (pMMOH) consisting of polypeptides 47,26 and 23kDa molecular mass. In addition to this, a putative pMMO reductase (pMMOR) was also found to be necessary to maintain propylene oxidising activity. This component was found to consist of two polypeptides of approximately 63 and 8kDa. Preliminary Nterminal sequence data of the large subunit ofpMMOR indicates that the sequence bears 70% similarity to the methanol dehydrogenase (MDH) from Methylococcus capsulatus (Bath). Therefore, we tentatively propose that the" MDH can act as a reductase component to the pMMOH. The significance of this result prompted investigations into the previous published proposals that electrons derived from the methanol oxidation reaction can be channelled back into the methane oxidation reaction by the methanol dehydrogenase, independent of NADH. Any effect of methanol to act as a reductant to pMMO in membrane preparations was lost upon isolation of the pMMO complex, indicating the necessity to maintain a fully functional methanol dehydrogenase (MDH) upon isolation. In addition to this, the in vitro electron donors of pMMO, NADH and duroquinol were found to act via distinct pathways of electron transfer (electron transport inhibitor studies). Electron paramagnetic resonance (EPR) spectroscopy data provided evidence that the copper in the active site of pMMO existed as a mononuclear copper (II) centre not a trinuclear copper centre suggested by Chan and coworkers (Chan et al., 1993; Nguyen et al., 1994, 1996a, 1996b, 1998). In addition to this preliminary data also indicates the presence of an iron centre which is only EPR visible after reduction of the complex suggesting the majority of iron in the complex is EPR invisible. The exact nature of this iron centre is still unclear. A structural study of the pMMO complex has also been undertaken using electron microscopy studies in conjunction with single particle analysis. This allowed low resolution projection maps of different views of the pMMO complex to be generated. The complex appears to exist in a polymeric state of at least a dimer, possibly a tetramer if the molecular weight analysis calculated by sedimentation equilibrium analysis is taken into account. This study has provided some insight into basic characteristics and the structure of a duroquinol-driven pMMO complex and its interaction with other electron transfer proteins.