A Stable Mercury-Containing Complex of the Organomercurial Lyase MerB: Catalysis, Product Release, and Direct Transfer to MerA ² Gregory C. Benison, ‡ Paola Di Lello, § Jacob E. Shokes, ‡ Nathaniel J. Cosper, ‡,| Robert A. Scott, ‡,§,| Pascale Legault,* ,§ and James G. Omichinski* ,‡,§ Department of Chemistry, Department of Biochemistry and Molecular Biology, and Center for Metalloenzyme Studies, UniVersity of Georgia, Athens, Georgia 30602 ReceiVed February 13, 2004; ReVised Manuscript ReceiVed May 4, 2004 ABSTRACT: Bacteria isolated from organic mercury-contaminated sites have developed a system of two enzymes that allows them to efficiently convert both ionic and organic mercury compounds to the less toxic elemental mercury. Both enzymes are encoded on the mer operon and require sulfhydryl-bound substrates. The first enzyme is an organomercurial lyase (MerB), and the second enzyme is a mercuric ion reductase (MerA). MerB catalyzes the protonolysis of the carbon-mercury bond, resulting in the formation of a reduced carbon compound and inorganic ionic mercury. Of several mercury-containing MerB complexes that we attempted to prepare, the most stable was a complex consisting of the organomercurial lyase (MerB), a mercuric ion, and a molecule of the MerB inhibitor dithiothreitol (DTT). Nuclear magnetic resonance (NMR) spectroscopy and extended X-ray absorption fine structure spectroscopy of the MerB/Hg/DTT complex have shown that the ligands to the mercuric ion in the complex consist of both sulfurs from the DTT molecule and one cysteine ligand, C96, from the protein. The stability of the MerB/Hg/DTT complex, even in the presence of a large excess of competing cysteine, has been demonstrated by NMR and dialysis. We used an enzyme buffering test to determine that the MerB/Hg/ DTT complex acts as a substrate for the mercuric reductase MerA. The observed MerA activity is higher than the expected activity assuming free diffusion of the mercuric ion from MerB to MerA. This suggests that the mercuric ion can be transferred between the two enzymes by a direct transfer mechanism. Mercury compounds, especially organomercurial com- pounds such as methylmercury (MeHg), have been respon- sible for a significant amount of human poisoning and environmental degradation (1). Mercury and its compounds are introduced into the environment by both natural and anthropogenic processes (2, 3). Although the highly toxic compound MeHg has been introduced into the environment directly by industrial processes, this compound can also be produced from less toxic forms of mercury by bacteria in aquatic sediments (4, 5). MeHg can appear in humans, fish, and other animals in high concentrations because of its tendency to biomagnify (2, 6). These concerns have led to regulation of the uses of mercury as well as efforts to clean up mercury pollution (7). A variety of methods for the remediation of mercury pollution are proposed or in use (8). Some methods involve physical removal or decontamination of soil or water. An alternative is to use the naturally occurring mercury- detoxifying capability of certain bacterial strains in bio- remediation schemes. Bioremediation schemes have been developed using the microorganisms themselves (9) or by incorporating the bacterial mercury resistance genes into plants (10, 11). The method of using plants to remediate pollution, known as phytoremediation, has several desirable properties: plants have a large biomass, can grow in many of the polluted habitats, and are inexpensive (12, 13). Mercury resistant bacterial strains possess a set of genes known as the mer operon, which is usually found on plasmids (14). These genes allow resistant strains to thrive in the presence of ionic or organic mercury compounds that are highly toxic to nonresistant bacteria or other forms of life. Ionic mercury is detoxified by a series of transport steps culminating in reduction by MerA to elemental mercury [Hg(0)]. Transport begins when ionic mercury [Hg(II)] is bound to MerP in the periplasm. Hg(II) is then transferred to the membrane protein MerT and from there to the enzyme mercuric reductase (MerA) in the cytosol. MerA reduces the mercuric ion to elemental mercury. It has been proposed that the transfer of Hg(II) both from MerP to MerT and from ² G.C.B. was funded by a National Science Foundation Graduate Research Fellowship. This work was supported by American Cancer Society Grant RPG LBC-100183 (J.G.O.). XAS work in the laboratory of R.A.S. is supported by the National Institutes of Health (GM 42025). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program. * To whom correspondence should be addressed. Current address: Universite ´ de Montre ´al, De ´partement de Biochimie, C. P. 6128, Succ. Centreville, Montre ´al, Quebec H3C 3J7, Canada. Tel: 514-343-7341. Fax: 514-343-2210. E-mail (J.G.O.): jg.omichinski@umontreal.ca. E-mail (P.L.): pascale.legault@umontreal.ca. ‡ Department of Chemistry. § Department of Biochemistry and Molecular Biology. | Center for Metalloenzyme Studies. 8333 Biochemistry 2004, 43, 8333-8345 10.1021/bi049662h CCC: $27.50 © 2004 American Chemical Society Published on Web 06/12/2004