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

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 (SODcd) is a Mn/Fe cambialistic SOD with MnSODcd exhibiting an optimal activity while Fe-sub-MnSODcd showing 10-fold less activity. To explain why the Fe-loaded protein exhibits a much lower activity than the Mn-loaded form, Fe-sub-MnSODcd and MnSODcd 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 MnSODcd has a higher affinity for the substrate compared to Fe-sub-MnSODcd, while Fe-sub-MnSODcd has more affinity for OH-. The different tendency of the anion ligation may be ascribed to the electronic configurations of Fe3+ in d(5) vs. Mn3+ in d(4), and it could be tuned by the hydrogen-bonding network around the active site of SODcd. Furthermore, the free energy for the O-2(center dot-) oxidation-reduction transition state from DFT calculation demonstrated that MnSODcd could disproportionate O-2(center dot-) more easily than Fe-sub-MnSODcd. These results revealed that SODcd could exquisitely differentiate between the Mn- and Fe-based activity. This metal specificity for SODcd may benefit the pathogenicity of C. difficile and pave a fundamental way for retarding C. difficile associated diseases.