Enzyme catalysis captured using multiple structures from one crystal at varying temperatures.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• High-resolution enzyme crystal structures reveal catalytic mechanisms.
• X-rays can drive reactions in crystals at cryogenic temperatures, creating 'structural movies'.
• Low temperatures suppress protein conformational motion, limiting previous studies.

Purpose of the Study:

• To apply the Multiple Serial Structures from One Crystal (MSOX) approach to copper nitrite reductase at room temperature and 190 K.
• To capture high-resolution structural states of enzyme catalysis across a range of temperatures.
• To investigate the impact of temperature on enzyme reaction dynamics and structural movie generation.

Main Methods:

• Application of the MSOX technique to copper nitrite reductase.
• X-ray crystallography at room temperature (293 K) and 190 K.
• Density functional theory (DFT) calculations to interpret structural and electronic states.

Main Results:

• Nitrite reductase reaction intermediates were captured at 190 K and room temperature.
• Nitrite binding geometry transitioned from 'top-hat' to 'side-on' during catalysis.
• 'Top-hat' geometry correlates with the oxidized Type 2 copper site; 'side-on' with the reduced state.
• Substrate-to-product conversion occurred at lower radiation doses at 190 K compared to 100 K.

Conclusions:

• The MSOX approach enables capturing enzyme catalytic cycles at multiple temperatures (MSOX-VT).
• Higher temperatures (190 K) allow for more complete catalytic cycle capture with reduced radiation damage.
• This technique provides unprecedented structural insights into redox enzyme catalysis.