UCSF

Mercuric Ion Reductase (MerA) is the primary protein in the mercury resistance mer operon for detoxifying mercury. Specifically, MerA is a disulfide oxidoreductase that uses FAD-mediated NADPH reduction of Hg(II) to elemental Hg(0), an innocuous form of mercury. This is accomplished through a 3-cysteine pair pathway, where the first pair on NmerA, a metallochaperone-like domain, scavenges Hg(II) from the environment, a second pair on the last 10 residues of the protein, denoted the C-terminal tail (CTT), moves the Hg(II) over a 15 Å distance from NmerA to a final cysteine pair in the catalytic core of the protein, where the 2-electron reduction of Hg(II) takes place. Given the ubiquitous nature of C-terminal tails in other oxidoreductases, such as theoredoxin reductase (TrxR), we endeavored to determine the residues that modulate the CTT in and out equilibrium that allows for efficient movement of Hg(II) through the MerA mercury pathway without being the rate limiting step of the enzyme. To elucidate these residues, we implemented the Rosetta FloppyTail protocol to gain insight into the residues that appeared to influence the stability of the CTT through polar and electrostatic interactions. This work resulted in determination of the ionic triad of residues: K99, E446, and K449. Steady-state and pre-equilibrium kinetics were used to determine the rates of Hg(II) acquisition by the CTT in mutants of the ionic triad residues as an indirect probe of in and out conformation of tail. This was completed in conjunction with an attempt at identifying relevant residues to be used a probes in relaxation dispersion NMR techniques for obtaining information regarding the dynamics of the CTT of MerA. The results from these diverse techniques suggested that K99, E446, and K449 all play a role in modulating the tail dynamics, with K99 and K449 acting to constrain the tail to the dimer cleft and E446 acting to liberate the tail. None of our results oppose the notion that these residues also play a role in the chemical catalysis of the enzyme.