The Phylogeny and Active Site Design of Eukaryotic Copper-only Superoxide Dismutases

Abstract

In eukaryotes the bimetallic Cu/Zn superoxide dismutase (SOD) enzymes play important roles in the biology of reactive oxygen species by disproportionating superoxide anion. Recently, we reported that the fungal pathogen Candida albicans expresses a novel copper-only SOD, known as SOD5, that lacks the zinc cofactor and electrostatic loop (ESL) domain of Cu/Zn-SODs for substrate guidance. Despite these abnormalities, C. albicans SOD5 can disproportionate superoxide at rates limited only by diffusion. Here we demonstrate that this curious copper-only SOD occurs throughout the fungal kingdom as well as in phylogenetically distant oomycetes or "pseudofungi" species. It is the only form of extracellular SOD in fungi and oomycetes, in stark contrast to the extracellular Cu/Zn-SODs of plants and animals. Through structural biology and biochemical approaches we demonstrate that these copper-only SODs have evolved with a specialized active site consisting of two highly conserved residues equivalent to SOD5 Glu-110 and Asp-113. The equivalent positions are zinc binding ligands in Cu/Zn-SODs and have evolved in copper-only SODs to control catalysis and copper binding in lieu of zinc and the ESL. Similar to the zinc ion in Cu/Zn-SODs, SOD5 Glu-110 helps orient a key copper-coordinating histidine and extends the pH range of enzyme catalysis. SOD5 Asp-113 connects to the active site in a manner similar to that of the ESL in Cu/Zn-SODs and assists in copper cofactor binding. Copper-only SODs are virulence factors for certain fungal pathogens; thus this unique active site may be a target for future anti-fungal strategies.

Eukaryotic copper-only superoxide dismutases (SODs): A new class of SOD enzymes and SOD-like protein domains

“…Mycobacterium SodC retains loop VII/ESL sequences and shows similar loop VII-Cu site interactions as seen with Cu/Zn-SOD1 (15). There is no equivalent to SOD5 Glu-110 in Mycobacterial SodC; instead the invariant aspartate (equivalent to SOD5 Asp-113) interacts with the dynamic histidine (15,70). Mt SodC appears to be a hybrid of Cu/Zn and fungal Cu-only SODs.…”

“…1 right). Evidence indicates that Asp-113 in the SOD5 active helps circumvent the need for the ESL in stabilizing Cu binding (70). Interestingly, Asp-113 is invariant among all Cu-containing SODs reported to date: in Cu/Zn-SODs, this aspartate is a Zn ligand and in Cu-only SODs the aspartate functions through interactions with the Cu site.…”

“…1). Disruption of this interaction through Glu-110 mutations alters the orientation of the dynamic His-93 and dramatically decreases the pH range of activity (70). Thus, SOD5 Glu-110 appears to act analogous to Zn, interacting with and correctly orienting the dynamic His-93 to promote rapid catalysis up to pH 8 (70).…”

“…Cu-only SODs are widely distributed throughout the fungal kingdom and in all cases examined thus far, are predicted to be extracellular and attached to the cell wall through GPI-anchors (70). Like C. albicans SOD5, all fungal extracellular SODs lack Zn binding and ESL sequences and retain the equivalents to Glu/Gln-110 and Asp-113 at the active site.…”

“…Attempts to load SOD5 with Zn were unsuccessful, demonstrating that SOD5 functions with a single atom of Cu and no other metals (16). Despite such striking deviations from canonical Cu/Zn-SODs, Cu-only SOD5 is an extremely efficient SOD enzyme capable of disproportionating superoxide at rates approaching diffusion limits, k cat =1.8 x 10 9 M -1 s-1 at pH 6.0 (16,70). How does Cu-only SOD5 function in the absence of Zn and the ESL?…”