Characterization of thiosulfate reductase from Pyrobaculum aerophilum heterologously produced in Pyrococcus furiosus

The genome of the archaeon Pyrobaculum aerophilum (T_opt ~ 100 °C) contains an operon (PAE2859–2861) encoding a putative pyranopterin-containing oxidoreductase of unknown function and metal content. These genes (with one gene modified to encode a His-affinity tag) were inserted into the fermentative anaerobic archaeon, Pyrococcus furiosus (T_opt ~ 100 °C). Dye-linked assays of cytoplasmic extracts from recombinant P. furiosus show that the P. aerophilum enzyme is a thiosulfate reductase (Tsr) and reduces thiosulfate but not polysulfide. The enzyme (Tsr–Mo) from molybdenum-grown cells contains Mo (Mo:W = 9:1) while the enzyme (Tsr–W) from tungsten-grown cells contains mainly W (Mo:W = 1:6). Purified Tsr–Mo has over ten times the activity (V_max = 1580 vs. 141 µmol min⁻¹ mg⁻¹) and twice the affinity for thiosulfate (K_m = ~ 100 vs. ~ 200 μM) than Tsr–W and is reduced at a lower potential (E_peak = − 255 vs − 402 mV). Tsr–Mo and Tsr–W proteins are heterodimers lacking the membrane anchor subunit (PAE2861). Recombinant P. furiosus expressing P. aerophilum Tsr could not use thiosulfate as a terminal electron acceptor. P. furiosus contains five pyranopterin-containing enzymes, all of which utilize W. P. aerophilum Tsr–Mo is the first example of an active Mo-containing enzyme produced in P. furiosus.