Structure and Substrate Specificity of the Pyrococcal Coenzyme A Disulfide Reductases/Polysulfide Reductases (CoADR/Psr): Implications for S⁰‑Based Respiration and a Sulfur-Dependent Antioxidant System in <i>Pyrococcus</i>

FAD and NAD­(P)­H-dependent coenzyme A disulfide reductases/polysulfide reductases (CoADR/Psr) have been proposed to be important for the reduction of sulfur and disulfides in the sulfur-reducing anaerobic hyperthermophiles <i>Pyrococcus horikoshii</i> and <i>Pyrococcus furiosus</i>; however, the form(s) of sulfur that the enzyme actually reduces are not clear. Here we determined the structure for the FAD- and coenzyme A-containing holoenzyme from <i>P. horikoshii</i> to 2.7 Å resolution and characterized its substrate specificity. The enzyme is relatively promiscuous and reduces a range of disulfide, persulfide, and polysulfide compounds. These results indicate that the likely <i>in vivo</i> substrates are NAD­(P)H and di-, poly-, and persulfide derivatives of coenzyme A, although polysulfide itself is also efficiently reduced. The role of the enzyme in the reduction of elemental sulfur (S₈) <i>in situ</i> is not, however, ruled out by these results, and the possible roles of this substrate are discussed. During aerobic persulfide reduction, rapid recycling of the persulfide substrate was observed, which is proposed to occur via sulfide oxidation by O₂ and/or H₂O₂. As expected, this reaction disappears under anaerobic conditions and may explain observations by others that CoADR is not essential for S⁰ respiration in <i>Pyrococcus</i> or <i>Thermococcus</i> but appears to participate in oxidative defense in the presence of S⁰. When compared to the homologous Npsr enzyme from <i>Shewanella loihica</i> PV-4 and homologous enzymes known to reduce CoA disulfide, the <i>ph</i>CoADR structure shows a relatively restricted substrate channel leading into the sulfur-reducing side of the FAD isoalloxazine ring, suggesting how this enzyme class may select for specific disulfide substrates