Addressing Ligand-Based Redox in Molybdenum-Dependent Methionine Sulfoxide Reductase.

Area of Science:

• Biochemistry
• Biophysical Chemistry
• Enzymology

Background:

• Methionine sulfoxide reductases (MsrP) are crucial enzymes involved in cellular redox homeostasis.
• Understanding the active site structure of MsrP is key to elucidating its oxygen atom transfer mechanism.

Purpose of the Study:

• To investigate the geometric and electronic structure of the E. coli periplasmic molybdenum-dependent methionine sulfoxide reductase (MsrP).
• To characterize the active site coordination and redox properties of MsrP in its as-isolated state.

Main Methods:

• Pulsed Electron Paramagnetic Resonance (EPR) spectroscopy (including 17O and 1H labeling).
• Continuous Wave (CW) EPR spectroscopy.
• X-ray Absorption Spectroscopy (XAS), specifically Extended X-ray Absorption Fine Structure (EXAFS).

Main Results:

• The as-isolated Mo(V) MsrP does not contain an exchangeable H2O/OH- ligand at the molybdenum center, unlike related sulfite oxidizing enzymes.
• Re-evaluation of CW EPR spectra, supported by data from a MsrP-N45R variant and small-molecule analogues, indicates a novel "thiol-blocked" active site structure: [(PDT)MoVO(SCys)(thiolate)]-.
• EXAFS data corroborate the proposed thiol-blocked coordination environment.

Conclusions:

• The findings reveal a unique active site architecture in MsrP, characterized by a "thiol-blocked" molybdenum center.
• This structural feature has implications for understanding the enzyme's catalytic mechanism, particularly its oxygen atom transfer capabilities.
• The study contributes to the broader understanding of molybdenum-dependent enzymes and their redox chemistry.