f Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative "cap helix" region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin. M ethylmercury (MeHg), a neurotoxin present in the environment, is a significant risk to human health in many regions of the world (1). Sources of the mercury (Hg) substrate from which MeHg is derived are numerous and include both natural and anthropogenic sources (2, 3). Currently, only anaerobic microbes are known to produce MeHg (4), predominantly in sediments of aquatic environments. Once MeHg is produced, it accumulates in the aquatic food chain, becoming concentrated in top predators (5). Human exposure to MeHg results from eating those predators, for example, in marine food webs, shark, swordfish, albacore tuna, and eel. Once in the body, MeHg passes through the intestinal epithelium, usually complexed with thiol groups in proteins (6). Eventually, MeHg buildup in humans can lead to neuropathies in adults and developmental disorders in children exposed in utero (2).Anaerobic Deltaproteobacteria are thought to be the major contributors of MeHg found in the environment (7, 8), although potentially significant contributions by methanogens and fermenting bacteria have recently been discovered (9-11). In 2013, we showed that the proteins encoded by two genes in two bacteria of the Deltaproteobacteria phylum, Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA, are essential for the conversion of Hg(II) to MeHg (12). That study was stimulated by the work of Choi and colleagues (13-15) implicating a 40-kDa corrinoid protein i...