Detection of Catalytically Linked Conformational Changes in Wild-Type Class Ia Ribonucleotide Reductase Using...

The enzyme, ribonucleotide reductase (RNR), is essential for DNA synthesis in all cells. The class Ia Escherichia coli RNR consists of two dimeric subunits, α₂ and β₂, which form an active but unstable heterodimer of dimers, α₂β₂. The structure of the wild-type form of the enzyme has been challenging to study due to the instability of the catalytic complex. A long-range proton-coupled electron-transfer (PCET) pathway facilitates radical migration from the Y122 radical-diiron cofactor in the β subunit to an active site cysteine, C439, in the α subunit to initiate the RNR chemistry. The PCET reactions and active site chemistry are spectroscopically masked by a rate-limiting, conformational gate. Here, we present a reaction-induced Fourier transform infrared (RIFTIR) spectroscopic method to monitor the mechanism of the active, wild-type RNR α₂β₂ complex. This method is employed to obtain new information about conformational changes accompanying RNR catalysis, including the role of carboxylate interactions, deprotonation, and oxidation of active site cysteines, and a detailed description of reversible secondary structural changes. Labeling of tyrosine revealed a conformationally active tyrosine in the β subunit, assigned to Y356β, which is part of the intersubunit PCET pathway. New insights into the roles of the inhibitors, azidoUDP and dATP, and the sensitivity of RIFTIR spectroscopy to detect subtle conformational motions arising from protein allostery are also presented.