Active site heterogeneity in dimethyl sulfoxide reductase from Rhodobacter capsulatus revealed by Raman spectroscopy

Area of Science:

• Biochemistry
• Spectroscopy

Background:

• DMSO reductase from Rhodobacter capsulatus contains a molybdenum cofactor crucial for its enzymatic activity.
• Understanding the structural and functional states of this cofactor is essential for elucidating enzyme mechanisms.

Purpose of the Study:

• To investigate the structural heterogeneity of the molybdenum cofactor in different redox states of DMSO reductase from Rhodobacter capsulatus using Raman spectroscopy.
• To characterize the specific forms present in enzyme preparations and their relationship to redox cycling.

Main Methods:

• Raman spectroscopy with 752 nm laser excitation was employed.
• Studies included three oxidized forms ('redox cycled', 'as prepared', DMSOR(mod)D) and two reduced forms (DMS, dithionite).
• Isotopic labeling (DMS(18)O) and deuteration experiments were performed.

Main Results:

• The 'redox cycled' form exhibits a Mo=O stretch at 865 cm(-1), with the oxo ligand exchangeable and showing hydroxo character due to hydrogen bonding.
• The 'as prepared' enzyme shows an additional band at 818 cm(-1), assigned to the S=O stretch of coordinated DMSO.
• The DMSOR(mod)D form, also present in 'as prepared' enzyme, is absent after redox cycling, indicating active site heterogeneity.

Conclusions:

• The 'as prepared' DMSO reductase exhibits significant active site heterogeneity.
• Multiple molybdenum cofactor forms, including coordinated DMSO and modified dithiolene sulfur, coexist in the enzyme.
• Raman spectroscopy is a powerful tool for distinguishing these forms and understanding cofactor structure.