Functional role of MrpA in the MrpABCDEFG Na⁺/H⁺ antiporter complex from the archaeon Methanosarcina acetivorans

The multisubunit cation/proton antiporter 3 family, also called Mrp, is widely distributed in all three phylogenetic domains (Eukarya, Bacteria, and Archaea). Investigations have focused on Mrp complexes from the domain Bacteria to the exclusion of Archaea, with a consensus emerging that all seven subunits are required for Na⁺/H⁺ antiport activity. The MrpA subunit from the MrpABCDEFG Na⁺/H⁺ antiporter complex of the archaeon Methanosarcina acetivorans was produced in antiporter-deficient Escherichia coli strains EP432 and KNabc and biochemically characterized to determine the role of MrpA in the complex. Both strains containing MrpA grew in the presence of up to 500 mM NaCl and pH values up to 11.0 with no added NaCl. Everted vesicles from the strains containing MrpA were able to generate a NADH-dependent pH gradient (ΔpH), which was abated by the addition of monovalent cations. The apparent K_m values for Na⁺ and Li⁺ were similar and ranged from 31 to 63 mM, whereas activity was too low to determine the apparent K_m for K⁺. Optimum activity was obtained between pH 7.0 and 8.0. Homology molecular modeling identified two half-closed symmetry-related ion translocation channels that are linked, forming a continuous path from the cytoplasm to the periplasm, analogous to the NuoL subunit of complex I. Bioinformatics analyses revealed genes encoding homologs of MrpABCDEFG in metabolically diverse methaneproducing species. Overall, the results advance the biochemical, evolutionary, and physiological understanding of Mrp complexes that extends to the domain Archaea.