X-ray structure of a Hg²⁺ complex of mercuric reductase (MerA) and quantum mechanical/molecular mechanical study of Hg²⁺ transfer between the C-terminal and buried catalytic site cysteine pairs

Mercuric reductase, MerA, is a key enzyme in bacterial mercury resistance. This homodimeric enzyme captures and reduces toxic Hg²⁺ to Hg⁰, which is relatively unreactive and can exit the cell passively. Prior to reduction, the Hg²⁺ is transferred from a pair of cysteines (C558′ and C559′ using Tn501 numbering) at the C-terminus of one monomer to another pair of cysteines (C136 and C141) in the catalytic site of the other monomer. Here, we present the X-ray structure of the C-terminal Hg²⁺ complex of the C136A/C141A double mutant of the Tn501 MerA catalytic core and explore the molecular mechanism of this Hg transfer with quantum mechanical/molecular mechanical (QM/MM) calculations. The transfer is found to be nearly thermoneutral and to pass through a stable tricoordinated intermediate that is marginally less stable than the two end states. For the overall process, Hg²⁺ is always paired with at least two thiolates and thus is present at both the C-terminal and catalytic binding sites as a neutral complex. Prior to Hg²⁺ transfer, C141 is negatively charged. As Hg²⁺ is transferred into the catalytic site, a proton is transferred from C136 to C559′ while C558′ becomes negatively charged, resulting in the net transfer of a negative charge over a distance of ∼7.5 Å. Thus, the transport of this soft divalent cation is made energetically feasible by pairing a competition between multiple Cys thiols and/or thiolates for Hg²⁺ with a competition between the Hg²⁺ and protons for the thiolates.