Characterization of a Cobalt-Specific P_1B-ATPase

The P_1B-type ATPases are a ubiquitous family of P-type ATPases involved in the transport of transition metal ions. Divided into subclasses based on sequence characteristics and substrate specificity, these integral membrane transporters play key roles in metal homeostasis, metal tolerance, and the biosynthesis of metalloproteins. The P_1B‑4-ATPases have the simplest architecture of the five P_1B-ATPase families and have been suggested to play a role in Co²⁺ transport. A P_1B‑4-ATPase from <i>Sulfitobacter</i> sp. NAS-14.1, designated sCoaT, has been cloned, expressed, and purified. Activity assays indicate that sCoaT is specific for Co²⁺. A single Co²⁺ binding site is present, and optical, electron paramagnetic resonance, and X-ray absorption spectroscopic data are consistent with tetrahedral coordination by oxygen and nitrogen ligands, including a histidine and likely a water. Surprisingly, there is no evidence for coordination by sulfur. Mutation of a conserved cysteine residue, Cys 327, in the signature transmembrane Ser-Pro-Cys metal binding motif does not abolish the ATP hydrolysis activity or affect the spectroscopic analysis, establishing that this residue is not involved in the initial Co²⁺ binding by sCoaT. In contrast, replacements of conserved transmembrane residues Ser 325, His 657, Glu 658, and Thr 661 with alanine abolish ATP hydrolysis activity and Co²⁺ binding, indicating that these residues are necessary for Co²⁺ transport. These data represent the first in vitro characterization of a P_1B‑4-ATPase and its Co²⁺ binding site