Detection of Catalytically Linked Conformational Changes in Wild-Type Class Ia Ribonucleotide Reductase Using Reaction-Induced FTIR Spectroscopy

Abstract

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.

Radical Transport Facilitated by a Proton Transfer Network at the Subunit Interface of Ribonucleotide Reductase

“…The E 326 [α] and E 326 [α′] residues from both α subunits reside at the terminus of the polar amino acid network. In line with this, the results of infrared spectroscopy suggest that E 326 plays a regulatory role in a proton exit channel of the α/β interface, though the complexity arising from the presence of numerous glutamates precludes unequivocal assignment to a specific residue. The empty free space at the exit of the asymmetric channel (Figure ) that E 326 blocks is sizable, suggesting an extensive water network.…”

Conformational Control over Proton-Coupled Electron Transfer in Metalloenzymes

“…Again, the state that forms upon radical translocation is distinct from that of met-β2, 113 and though comparisons between resting state β2 and met-β2 reveal significant changes in the position of Y122, 115,119,120 as well as changes to the H-bond network within β, 121 these changes may not be relevant to those taking place upon radical translocation. 122 By incorporating FnY residues with varied redox potentials and pKas, the thermodynamic reaction landscape of the PCET pathway has been mapped (Figure 4.2c). 123 In the forward direction, PCET between Y122 and Y356 is approximately 100 meV thermodynamically uphill.…”

Conformational Control over Proton-Coupled Electron Transfer in Metalloenzymes

“…Again, the state that forms upon radical translocation is distinct from that of met-β2, 110 and though comparisons between resting state β2 and met-β2 reveal significant changes in the position of Y122, 112,116,117 as well as changes to the H-bond network within β, 118 these changes may not be relevant to those taking place upon radical translocation. 119 By incorporating FnY residues with varied redox potentials and pKas, the thermodynamic reaction landscape of the PCET pathway has been mapped (Figure 4.2c). 120 In the forward direction, PCET between Y122 and Y356 is approximately 100 meV thermodynamically uphill.…”