Enzymatic Mercury Detoxification: The Regulatory Protein MerR

Mercury ions and organomercurial reagents are extremely toxic due to their affinity for thiol groups. Many bacteria contain an elaborate detoxification system for a metabolic conversion of toxic Hg$^{2+}$ or organomercurials to less toxic elemental Hg$^0$. The main components of the enzymatic mercury detoxification (see Fig. 1) are the regulatory protein MerR (mercury responsive genetic switch), the organomercurial lyase MerB (cleavage of carbon mercury bonds), and the mercuric ion reductase MerA (reduction of mercuric ions). In these proteins Hg$^{2+}$ is usually coordinated by the thiol groups of cysteines. We utilize the nuclear quadrupole interaction (NQI) of ${\rm^{199m}}$Hg detected by time differential perturbed angular correlation (TDPAC) to identify the Hg metal site geometries in these proteins in order to elucidate the molecular origin of the ultrasensitivity, selectivity and reaction mechanism of this detoxification system. The short lived TDPAC probe ${\rm^{199m}}$Hg ($\tau_{1/2} =$ 43 min) is supplied by the isotope separator ISOLDE. The MerR protein controls the expression of the other detoxification proteins, like MerA or MerB. In the absence of Hg$^{2+}$, MerR binds to the DNA and inhibits DNA transcription; in the presence of trace amounts of Hg$^{2+}$ the DNA bound Hg-MerA complex activates description. In this almost inaccessible concentration range we have identified the Hg coordination in the MerR protein as 3-fold unambiguously. At present, we investigate the binding of the Hg$^{2+}$-MerR complex to the DNA. The MerA enzyme uses NADPH (nicotinamide adenine dinucleotide phosphate) as a two electron reducing agent. It is supposed that the electrons are transferred to one of the four cysteines which constitute together with two tyrosines the active center of the protein. Three different coordination geometries were revealed: two different 2-fold coordinations, and a tetrahedra 4-fold coordination. On the addition of NADPH to the enzyme also Hg$^{0}$ was found. The further investigation of the reaction path with the help of MerA mutants in which the active reaction center was modified by genetic engineering is currently under way.