Abstract
Anaerobic respiration using elemental sulfur as the terminal electron acceptor is a crucial metabolic process in sulfur-reducing bacteria and some archaea, playing an indispensable role in the biogeochemical sulfur cycle. This study aimed to elucidate the molecular mechanism of sulfur respiration in Geobacter sulfurreducens PCA. The protein encoded by the esuD gene was identified as a novel polysulfide reductase, MccSep, containing five c-type hemes and one Sec residue. The X-ray crystal structure of MccSep solved at 1.90Å revealed that Cys239 and Sec325, positioned near the His/Cys-coordinated heme, are crucial for the catalytic activity. The reduction of elemental sulfur and the growth of the ΔesuD strain on elemental sulfur media were significantly impaired compared to the parent strain, highlighting the crucial role of MccSep in sulfur respiration. The esuD gene was part of the esu operon, which includes esuABCDEFGHIJ on the G. sulfurreducens PCA genome. The lack of halo formation on the elemental sulfur agar medium by the ΔesuA, ΔesuB, ΔesuC, and ΔesuEFGHIJ strains showed the involvement of the operon in sulfur respiration. Expression of the operon genes was upregulated in response to elemental sulfur in the medium. Enzymatic analysis identified EsuA as a sulfur transferase with high catalytic efficiency for polysulfides using Cys330 as the catalytic residue. The ΔesuB strain expressing the plasmid-derived C147A mutant of EsuB did not complement sulfur respiration ability, demonstrating the critical role of Cys147 in EsuB for sulfur respiration. Similarly, introducing a plasmid expressing the ΔC104 mutant of EsuC into the ΔesuC strain did not restore sulfur respiration, indicating that Cys104 of EsuC is also essential for this process. Subcellular localization analysis showed that EsuA predominantly localized to the outer and inner membrane fractions, EsuB to the outer membrane, and ExtJ to the periplasm. These findings offer valuable insights into the molecular mechanisms underlying bacterial sulfur respiration.
