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

Area of Science:

• Biochemistry
• Microbiology
• Structural Biology

Background:

• Clostridium difficile causes severe gastrointestinal infections.
• Oxidative stress and superoxide dismutase (SOD) are crucial for C. difficile virulence.
• SODcd exhibits Mn/Fe cambialistic activity, with distinct differences in Mn- and Fe-loaded forms.

Purpose of the Study:

• To elucidate the structural and functional basis for the lower activity of Fe-substituted MnSODcd compared to MnSODcd.
• To understand the metal specificity of SODcd and its implications for C. difficile pathogenicity.

Main Methods:

• Expression and purification of MnSODcd and Fe-sub-MnSODcd in E. coli.
• Characterization using X-ray crystallography, metal analysis, optical and EPR spectroscopy, and pH titrations.
• Computational analysis using Density Functional Theory (DFT) for transition state energy calculations.

Main Results:

• Fe-sub-MnSODcd displayed significantly lower substrate affinity and higher affinity for hydroxide ions compared to MnSODcd.
• Differences in anion ligation tendencies were attributed to Fe(3+) vs. Mn(3+) electronic configurations and active site hydrogen bonding.
• DFT calculations confirmed that MnSODcd more readily disproportionates superoxide radicals than Fe-sub-MnSODcd.

Conclusions:

• SODcd exhibits exquisite metal specificity, favoring manganese over iron for optimal activity.
• This metal preference may contribute to the pathogenicity of C. difficile.
• Understanding this specificity offers potential therapeutic strategies against C. difficile-associated diseases.