Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and...

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Enzymatic reactions are crucial biological processes.
• Understanding enzyme mechanisms requires visualizing transient intermediates.
• Time-resolved crystallography offers a method to study these intermediates.

Purpose of the Study:

• To apply time-resolved serial femtosecond crystallography (TR-SFX) with caged compounds to study fungal nitric oxide reductase.
• To determine the structure of an enzyme intermediate during catalysis at room temperature.
• To elucidate the coordination geometry of nitric oxide (NO) bound to the enzyme.

Main Methods:

• Utilized X-ray free electron laser (XFEL) for high-intensity, short X-ray pulses.
• Employed photosensitive caged-NO compounds for controlled NO release.
• Performed serial femtosecond crystallography (SFX) at room temperature.
• Analyzed data to determine enzyme structure at 2.1 Å resolution.

Main Results:

• Captured a NO-bound intermediate of fungal NO reductase 20 milliseconds after NO release.
• Identified a slightly bent Fe-N-O coordination geometry for the bound NO.
• Confirmed the structure is free from X-ray radiation damage.
• Observed a geometry suitable for subsequent enzymatic steps, like H- transfer.

Conclusions:

• The combination of XFEL and caged compounds is a powerful tool for time-resolved enzyme studies.
• This method allows for atomic-level determination of functional enzyme structures during catalysis.
• The observed NO binding geometry provides insights into the NO reductase catalytic mechanism.