Research in the Seefeldt Laboratory is focused on elucidating the mechanism and structure of complex metalloenzymes involved in global energy cycling.

Research in our laboratory is focused on elucidating the mechanism of the metalloenzyme nitrogenase. Nitrogenase is the microbial enzyme responsible for biological reduction of N₂ to ammonia, and thus is a key reaction in the global nitrogen cycle. Mo-based nitrogenase is the most widely distributed enzyme and is the focus of research in our laboratory. This enzyme is composed of two component proteins called the MoFe protein and the Fe protein. The MoFe protein contains the site of N₂ binding (called FeMo-cofactor) and an electron transfer 8Fe-7S cluster (called the P-cluster). The Fe protein component contains a single 4Fe-4S cluster and two nucleotide binding sites. We are using a multidisciplinary approach, including genetics, kinetics, spectroscopy, and X-ray crystallography to address outstanding open questions about the mechanism of this important enzyme. These include: 1) How N₂ is bound at the active site and what roles the protein plays in defining reactivity, 2) the mechanism for controlling electron transfer between the two component proteins, and 3) the mechanism of coupling ATP binding and hydrolysis to the reaction mechanism.

Active Site

One of the outstanding questions about nitrogenase is where and how N₂ interacts with FeMo-cofactor. In collaboration with Dennis Dean's group at Virginia Tech, we are using site-directed mutagenesis, rapid quench methods, and substrate analogs to trap substrates and inhibitors at the active site. Using various spectroscopic methods (i.e., EPR, ENDOR) and X-ray crystallography, we are characterizing the nature of several bound intermediates. Such studies are providing insights into the way that substrates interact with the active site. From such studies, we have recently realized that one FeS face of FeMo-cofactor is likely to be the site of substrate binding. We also have determined that an Arg amino acid residue (a-96^Arg) located near the FeMo-cofactor is likely acting as a gatekeeper, opening and closing during catalytic turnover to expose the active site to substrates. We are currently in a position to fully characterize bound substrates and inhibitors at the active site, and thus to gain detailed insights into the mechanism of this important enzyme.

Roles of ATP

ATP hydrolysis to ADP and Pi is linked to the transfer of an electron from the Fe protein to the MoFe protein. These two coupled reactions are the only known way to reduce the MoFe protein to support substrate reduction, so it is evident that ATP hydrolysis plays a central role in the nitrogenase mechanism. Our current working hypothesis is that ATP binding and hydrolysis on the Fe protein plays several different functions in the total reaction mechanism. Since the Fe protein binds two ATP molecules, one on each subunit, and these binding sites are located some distance (~15 A) away from the 4Fe-4S cluster, it is concluded that ATP must act through protein conformational changes. This general theme of ATP signal transduction is similar to the situation in several other nucleotide binding proteins, such as the GTP binding P-21 protein or the ATP hydrolyzing myosin. We are addressing how ATP binding and hydrolysis on the Fe protein is controlling the binding of the Fe protein with the MoFe protein, the transfer of an electron between the Fe protein and the MoFe protein, and the reactivity within the MoFe protein. It appears that the Fe protein is working as a nanomachine, with a series of ATP controlled protein conformational changes being sent over long distances to control many aspects of the nitrogenase mechanism.

Publications

Funding