Cryoprotectants Severely Exacerbate X-ray-Induced Photoreduction

Area of Science:

• Biochemistry
• Structural Biology
• Spectroscopy

Background:

• Metalloenzymes utilize essential transition metal ions for catalysis.
• X-ray methods like macromolecular crystallography and X-ray absorption spectroscopy are crucial for determining active site structures.
• X-ray irradiation can alter metal oxidation states and structures, complicating analysis.

Purpose of the Study:

• To investigate the effect of common cryoprotectants on X-ray-induced photoreduction of transition metal ions.
• To determine if cryoprotectants like PEG or glycerol impact the integrity of metal active sites during X-ray analysis.

Main Methods:

• X-ray absorption spectroscopy (XAS) was employed to study copper ions.
• Samples containing copper were analyzed with and without cryoprotectants (polyethylene glycol, glycerol).
• The rate of photoreduction of Cu(II) to Cu(I) under X-ray irradiation was measured.

Main Results:

• Cryoprotectants, specifically alcohol and ether types, significantly increased the rate of photoreduction.
• A 10-fold exacerbation in the rate of Cu(II) to Cu(I) photoreduction was observed in the presence of cryoprotectants.
• This indicates cryoprotectants enhance X-ray-induced damage to metal centers.

Conclusions:

• The routine use of cryoprotectants in X-ray studies of metalloenzymes may lead to artifacts.
• This exacerbation of photoreduction can result in erroneous determination of enzyme structures and catalytic mechanisms.
• Careful consideration of cryoprotectant use is necessary for accurate structural biology studies.