Abstract 1198 Modifications to the [4Fe4S] microenvironment in human DNA primase probe the influence of size, polarity, and charge on the substrate-dependent redox behavior of the regulatory [4Fe4S] cluster

The production of short ribonucleotide primers required for processive DNA replication is carried out by the DNA-dependent, RNA polymerase, DNA primase. Electrochemical studies conducted with the [4Fe4S]-coordinating domain of human DNA primase (p58C) as well as with the full-length enzyme have demonstrated that substrate binding activates the otherwise inert [4Fe4S] cluster towards reversible redox activity in the presence of ribonucleotide triphosphates (rNTPs). Given the 25 Å distance between the [4Fe4S] cluster and rNTP binding site, this observation implies that the binding of rNTPs allosterically modulates the redox properties of the [4Fe4S] cluster; however, the structural basis for this effect has not been well-defined. Herein, we have sought to understand how protein residues in the immediate environment of the [4Fe4S] cluster influence its substrate-dependent redox behavior. Previously, we have demonstrated that mutation of proline residues 286 and 385 to isoleucine does not affect the cluster-loading or secondary structure of p58C. Mutation of these residues does, however, affect the electronic properties and substrate-dependent redox behavior of the [4Fe4S] cluster. To further investigate the roles of prolines 286 and 385 in allostery, we have generated additional site-directed variants to probe how size, polarity, and charge influence the response of [4Fe4S] cluster redox properties to substrate binding using UV-Visible, Circular Dichroism, and Electron Paramagnetic Resonance spectroscopies. Our results hint at the involvement of several additional residues at the rNTP binding site in an allosteric mechanism responsible for the communication of redox state information between the [4Fe4S] and DNA binding sites. This work was funded by the University of Alabama start-up and NSF grant number 1950855 awarded to MKT.