Figure 1. A) Examples of several chemical reactions and the respective metalloenzymes involved in performing them that our group is interested in. B) Crystal structure of (PDB id:4WZA) Mo Nitrogenase (Mo N₂ase) from Azotobacter vinelandii, one of our proteins of interest. C) The two metal clusters and some of the important residues involved in the catalysis in Mo N₂ase.

Almost every metalloenzyme, in addition to the catalytic active site, involves additional metal clusters/sites which act as structural sites or are involved in electron/substrate transfers to the active site. Some examples of the additional sites are the 4Fe-4S clusters in Hydrogenases or the unique 8Fe-7S cluster, known as the P-cluster, in N₂ases (Figure 1B, C) in addition to the catalytic site (FeMo-co, Figure 1B, C). Moreover, metalloenzymes are often assisted by other metalloproteins for electron transfers and substrate (and co-substrate) diffusions. Our group recognizes the importance of these additional sites/metalloproteins and aims to study them for better understanding of biological catalysis.

A major limitation in studying many metalloenzymes is many times the lack of efficient/suitable heterologous expression systems for their purification. Additionally, their purification from native organisms is not only complicated and time taking but is also of low yield. This especially affects spectroscopic investigations such as X-ray based techniques because it can be very sample intensive.  In collaboration with Dr. Decamps group, our group is developing and optimizing recombinant expression systems of metalloenzymes, particularly sMMO, in order to have better yields. sMMO or soluble Methane Monooxygenase (Figure 2) is a metalloenzyme which activates O₂ and inserts one atom into an unactivated C–H bond of methane to yield methanol (CH₃OH). Our biochemical approaches will be key for enabling advanced spectroscopic studies within our group and more broadly within the Department of Inorganic Spectroscopy, which should ultimately shed light on the key catalytic intermediates in these important energy converting enzymes.

Figure 2. Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b (PDB id: 1MHZ); the enlarged view of the diiron active site (right).

The “Metalloenzyme Mechanistic Explorations” (MeME) team is always looking for highly motivated and dedicated Bachelor, Master or PhD students and Postdoctoral researchers who are interested and would love to work with proteins and inorganic spectroscopy. If you wish to join the team, please contact Dr. Kushal Sengupta with your CV and cover letter.