Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase.

Area of Science:

• Biochemistry
• Enzymology
• Protein Dynamics

Background:

• Ribonucleotide reductases (RNR) are essential enzymes for DNA synthesis.
• Class 1a RNRs utilize a radical transfer (RT) mechanism involving a cysteine thiyl radical.
• Radical transfer occurs across the α2:β2 subunit interface via proton-coupled electron transfer (PCET).

Purpose of the Study:

• Investigate the role of conformational dynamics in RNR radical transfer.
• Characterize the mechanism of radical transfer in the R411A mutant of E. coli class 1a RNR.
• Determine the kinetics of radical transfer across the subunit interface.

Main Methods:

• Site-directed mutagenesis (R411A).
• Amber codon suppression to install 3-amino tyrosine (NH2Y) as a radical trap.
• HYSCORE spectroscopy to study the trapped radical state.
• Photochemical radical generation for kinetic studies.

Main Results:

• The R411A mutation, which disrupts Y731 H-bonding, retains enzymatic activity, indicating conformational flexibility.
• Y731 in the R411A mutant dynamically reforms H-bonds, allowing radical transfer propagation.
• Radical transfer across the interface is conformationally dependent, with rates increasing in the R411A mutant.
• Y731 conformational changes occur on the ns-μs timescale, faster than the catalytic rate.

Conclusions:

• Conformational flexibility of tyrosine residues is critical for efficient radical transfer in RNR.
• Dynamic H-bond reformation facilitates radical propagation along the PCET pathway.
• Enzyme kinetics are modulated by the interplay between protein dynamics and radical transport.