Project Summary/Abstract: Superoxide is a toxic molecule that all organisms exposed to oxygen must cope with. This is particularly true for pathogenic microbes, as the host harnesses the toxic properties of superoxide to combat invaders via the oxidative burst. To detoxify superoxide, nearly all forms of life, including strict anaerobes, produce superoxide dismutase (SOD). Convergent evolution has led to the development of three independent SOD families, all of which are dependent on metals for function. The most widely distributed family of SODs are those which depend on iron (Fe) or manganese (Mn) for function. Members of the Fe/Mn superfamily are present in eukaryotes, archaea, and bacteria. Despite over forty years of study, it is not possible to predict accurately the metal utilized by members of the Fe/Mn superfamily of SODs. Difficulties in predicting metalloprotein metal utilization are not confined to the Fe/Mn SOD superfamily but also occur with other classes of metalloenzymes. This deficiency is driven by relatively low levels of protein sequence identity amongst SODs from different organisms that utilize the same metal cofactor. Additionally, the environmental and molecular factors that dictate the metal used by members of this protein superfamily are also unknown. Members of the Fe/Mn SOD superfamily are canonically thought to use either Fe or Mn, but not both, as a cofactor. This idea arose despite early investigations that identified Fe/Mn SOD family members that are active with both Fe and Mn. The ability of these “cambialistic” SODs (able to use either Fe or Mn as a catalytic cofactor) was dismissed as a quirk of chemistry. At the time, it was thought that intracellular metal concentrations did not change enough to alter the metal bound by a SOD. S. aureus possesses two superoxide dismutases, SodA and SodM, which are ~75% identical. Initially, both SODs were reported to be Mn-dependent. During infection, the host restricts the availability of Mn and inactivates Mn-dependent SODs via the Mn-binding immune protein calprotectin. Recent work discovered that SodM critically contributes to the ability of S. aureus to maintain a defense against oxidative stress when Mn-starved, both in culture and during infection, while SodA is important when Mn is freely available. Biochemical analyses revealed that SodM is not strictly Mn-dependent but is instead cambialistic, and the ability to use Fe enables it to promote resistance to oxidative stress when S. aureus is Mn-limited by the host. These observations support a physiological role for cambialism and the hypothesis that metal availability shapes the repertoire of SODs possessed by an organism. The experiments in this proposal will evaluate this hypothesis and elucidate the molecular features that dictate metal utilization in the Fe/Mn SOD superfamily. Aim I: Elucidate the molecular features that dictate metal utilization of Fe/Mn SOD superfamily members. Aim II: Determine if environmental metal availability promotes retention of a metal-specific and cambialistic SOD by S. aureus. Aim III: Elucidate the broader contribution of cambialistic SODs to maintaining a defense against superoxide.

Project Narrative: Superoxide is a threat to all forms of life and its toxicity is harnessed by the immune system to combat infection. Superoxide dismutases are metal utilizing enzymes that enable us and invading microbes to cope with superoxide, yet we do not know the molecular or environmental factors that dictate the metal utilized by these enzymes. The proposed studies will address this unanswered question, providing insight into how both humans and invading pathogens overcome the universal threat of superoxide.