<i>Treponema denticola</i> Superoxide Reductase: In Vivo Role, in Vitro Reactivities, and a Novel [Fe(Cys)₄] Site

Abstract

In vitro and in vivo results are presented demonstrating that superoxide reductase (SOR) from the air-sensitive oral spirochete, <i>Treponema denticola</i> (Td), is a principal enzymatic scavenger of superoxide in this organism. This SOR contains the characteristic non-heme [Fe­(His)₄Cys] active sites. No other metal-binding domain has been annotated for Td SOR. However, we found that Td SOR also accommodates a [Fe­(Cys)₄] site whose spectroscopic and redox properties resemble those in so-called 2Fe-SORs. Spectroscopic comparisons of the wild type and engineered Cys → Ser variants indicate that three of the Cys ligands correspond to those in [Fe­(Cys)₄] sites of “canonical” 2Fe-SORs, whereas the fourth Cys ligand residue has no counterpart in canonical 2Fe-SORs or in any other known [Fe­(Cys)₄] protein. Structural modeling is consistent with iron ligation of the “noncanonical” Cys residue across subunit interfaces of the Td SOR homodimer. The Td SOR was isolated with only a small percentage of [Fe­(Cys)₄] sites. However, quantitative formation of stable [Fe­(Cys)₄] sites was readily achieved by exposing the as-isolated protein to an iron salt, a disulfide reducing agent and air. The disulfide/dithiol status and iron occupancy of the Td SOR [Fe­(Cys)₄] sites could, thus, reflect intracellular redox status, particularly during periods of oxidative stress