Structural, spectroscopic and functional investigation into Fe-substituted MnSOD from human pathogen Clostridium difficile

Abstract

Clostridium difficile, which inhabits the human digestive tract, is an etiological agent that causes pseudomembranous colitis and antibiotic-associated diarrhea. The oxidative stress tightly relates to its virulence, which highlights the function of its superoxide dismutase (SOD). The SOD from Clostridium difficile (SOD_cd) is a Mn/Fe cambialistic SOD with MnSOD_cd exhibiting an optimal activity while Fe-sub-MnSOD_cd showing 10-fold less activity. To explain why the Fe-loaded protein exhibits a much lower activity than the Mn-loaded form, Fe-sub-MnSOD_cd and MnSOD_cd were expressed in E. coli using M9 minimal medium, and characterized by X-ray crystallography, metal analysis, optical and EPR pH titration, azide binding affinity, etc. The pK_a values of the active site residues and substrate affinities determined by spectroscopic titrations indicated that MnSOD_cd has a higher affinity for the substrate compared to Fe-sub-MnSOD_cd, while Fe-sub-MnSOD_cd has more affinity for OH⁻. The different tendency of the anion ligation may be ascribed to the electronic configurations of Fe³⁺ in d⁵vs. Mn³⁺ in d⁴, and it could be tuned by the hydrogen-bonding network around the active site of SOD_cd. Furthermore, the free energy for the O₂˙⁻ oxidation–reduction transition state from DFT calculation demonstrated that MnSOD_cd could disproportionate O₂˙⁻ more easily than Fe-sub-MnSOD_cd. These results revealed that SOD_cd could exquisitely differentiate between the Mn- and Fe-based activity. This metal specificity for SOD_cd may benefit the pathogenicity of C. difficile and pave a fundamental way for retarding C. difficile associated diseases.