NmerA, the Metal Binding Domain of Mercuric Ion Reductase, Removes Hg²⁺ from Proteins, Delivers It to the Catalytic Core, and Protects Cells under Glutathione-Depleted Conditions^,

Abstract

The ligand binding and catalytic properties of heavy metal ions have led to the evolution of metal ion-specific pathways for control of their intracellular trafficking and/or elimination. Small MW proteins/domains containing a GMTCXXC metal binding motif in a betaalphabetabetaalphabeta fold are common among proteins controlling the mobility of soft metal ions such as Cu(1+), Zn(2+), and Hg(2+), and the functions of several have been established. In bacterial mercuric ion reductases (MerA), which catalyze reduction of Hg(2+) to Hg(0) as a means of detoxification, one or two repeats of sequences with this fold are highly conserved as N-terminal domains (NmerA) of uncertain function. To simplify functional analysis of NmerA, we cloned and expressed the domain and catalytic core of Tn501 MerA as separate proteins. In this paper, we show Tn501 NmerA to be a stable, soluble protein that binds 1 Hg(2+)/domain and delivers it to the catalytic core at kinetically competent rates. Comparison of steady-state data for full-length versus catalytic core MerA using Hg(glutathione)(2) or Hg(thioredoxin) as substrate demonstrates that the NmerA domain does participate in acquisition and delivery of Hg(2+) to the catalytic core during the reduction catalyzed by full-length MerA, particularly when Hg(2+) is bound to a protein. Finally, comparison of growth curves for glutathione-depleted Escherichia coli expressing either catalytic core, full-length, or a combination of core plus NmerA shows an increased protection of cells against Hg(2+) in the media when NmerA is present, providing the first evidence of a functional role for this highly conserved domain.

A Novel Mercuric Reductase from the Unique Deep Brine Environment of Atlantis II in the Red Sea

“…As expected, both enzymes have the catalytic redox-active cysteine disulfide pair (Cys-136/Cys-141) in the active site (10,11), and the Hg 2ϩ binding Cys-558/Cys-559 pair located near the C terminus of the polypeptide chain (45,46). In addition, both molecules possess the NmerA domain that contains the pair of cysteine thiols (Cys-11/Cys-14) in the conserved GMTCXXC sequence motif that has been shown to be involved in Hg 2ϩ binding under glutathione-depleted conditions (12,13).…”

“…Furthermore, it has been shown that Hg 2ϩ increases H 2 O 2 formation in mitochondria, leading to increased consumption of reduced glutathione (14). Taken together, these results strongly indicate that the pair of cysteines in the NmerA domain of mercuric reductases does have a functional role in vivo, enhancing mercuric ion detoxification by acting as an accessory pathway for delivery of the substrate to the active site (12).…”

“…In fact, mutant M1 has an inhibitory profile comparable with that of the soil enzyme. Interestingly, this mutation lies right next to the conserved metal-binding domain containing the pair of cysteines (Cys-11/Cys-14) that is involved in the binding of Hg 2ϩ under glutathione-depleted conditions (12).…”

“…S3). The model shows the homodimer structure of the catalytic core of the enzyme, which contains the C-terminal pair of cysteines (Cys-558 and Cys-559) involved in binding of Hg 2ϩ from one subunit and the cysteine redox-active pair of the second subunit (Cys-136 and Cys-141) (12), present in the catalytic pocket of the enzyme (see also supplemental Fig. 3).…”

“…Initially, the NmerA cysteine pair appeared to be dispensable for catalysis in vivo, because a double mutant in which both residues were replaced by alanines was found to be fully functional (11). However, it was later shown that, under physiological conditions in which intracellular thiols are depleted, the NmerA domain binds Hg 2ϩ and transfers it from ligands in cytoplasm to the catalytic core for reduction (12,13). Furthermore, it has been shown that Hg 2ϩ increases H 2 O 2 formation in mitochondria, leading to increased consumption of reduced glutathione (14).…”