Problems and Future Research
Structural Biology, Biochemistry, and Molecular Biology
There are currently only two known crystal structures for the pMMO protein from two different organisms. More crystal structures at higher resolution will allow for better models. This, however, is problematic because integral membrane proteins are very difficult to work with. Integral membrane proteins are particularly fragile and hard to crystallize. Another common method for obtaining structures is NMR spectroscopy which is not possible on pMMO because it is such a large protein. Obtaining more crystal structures from more methanotrophs will also determine how conserved the protein is between different species.
While the genes which produce pMMO are known, almost nothing is known about how these genes are regulated. Especially in organisms that produce both pMMO and sMMO, there is a lot of work to be done in determining a preference for expressing one over the other. Understanding the mechanism by which pMMO is regulated with also illucidate more information about the enzyme and it's biochemistry.
While the genes which produce pMMO are known, almost nothing is known about how these genes are regulated. Especially in organisms that produce both pMMO and sMMO, there is a lot of work to be done in determining a preference for expressing one over the other. Understanding the mechanism by which pMMO is regulated with also illucidate more information about the enzyme and it's biochemistry.
Mechanism and Synthetic Analogues
A number of problems exist when considering how to best determine the mechanism of action for this reaction. The first is the lack of a clear and precise crystal structure. It is unknown for the most part what side chain residues are actually responsible as ligands for the copper centers. It is also unknown as to wether the conversion to methanol takes place at the dicopper site or the monocopper site. Although the calculated values certainly seem to suggest that dicopper site would be energetically the favored one. Another large problem facing the determination of mechanism is the lack of understanding what transitions metals are actually responsible for the chemistry in question. Copper is a popular candidate, but Fe and Zn also curry some favor in this area.
It is also difficult to produce working synthetic models, based off the aforementioned problems.
Determining the mechanism is of great importance in this protein. The conversion of methane to methanol could prove to be an effective method for storing energy in small molecule sense. It is unlikely that we will grow the proteins ourselves and harvest the methanol, nor would we want it to be that site specific. (Certainly this has synthetic applications as well.) We must learn how the reaction proceeds if we are to effectively use it.
It is also difficult to produce working synthetic models, based off the aforementioned problems.
Determining the mechanism is of great importance in this protein. The conversion of methane to methanol could prove to be an effective method for storing energy in small molecule sense. It is unlikely that we will grow the proteins ourselves and harvest the methanol, nor would we want it to be that site specific. (Certainly this has synthetic applications as well.) We must learn how the reaction proceeds if we are to effectively use it.
Spectroscopic Data
The problem with an enzyme like pMMO, one which is poorly understood, is knowing how to characterize it. Further spectroscopic data is needed to reveal the active site of this enzyme. From current spectroscopic data it seems either the dinuclear copper site in the pmoB subunit or an uncharacterized hydrophilic site in the membrane region are the best guesses for the active site. Further spectroscopic studies are needed to reveal detailed coordination environments of the different metal centers. It is also important to distinguish which of the metal centers activates O2. These are the questions we are left with and further analysis must be done if we are to understand the methane hydroxylation mechanism. Progress is being made and now that researchers have an educated guess of which if the metal centers constitutes the active site it should not be long before new spectroscopic data is revealed.
