Treponema denticola superoxide reductase: in vivo role, in vitro reactivities, and a novel [Fe(Cys)(4)] site.
In vitro and in vivo results are presented demonstrating that superoxide reductase (SOR) from the air-sensitive oral spirochete, Treponema denticola (Td), is a principal enzymatic scavenger of superoxide in this organism. This SOR contains the characteristic non-heme [Fe(His)(4)Cys] 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.
Department of Chemistry, University of Texas at San Antonio, San Antonio, TX 78249, USA.
Jonathan D
JD
Caranto
Linda L
LL
Gebhardt
Charles E
CE
MacGowan
Ronald J
RJ
Limberger
Donald M
DM
Kurtz
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048373:Treponema denticola / Q000201:enzymology*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D007501:Iron; D010088:Oxidoreductases; C121849:superoxide reductase; D003545:Cysteine