Kinetics and Dynamics Controlling Proton-Coupled Electron Transfer in Ribonucleotide Reductase

Abstract

Proton-coupled electron transfer (PCET) reactions comprise a fundamental mechanism for energy transduction in nature. In catalyzing the conversion of ribonucleotides to deoxyribonucleotides, ribonucleotide reductase (RNR) performs reversible, long-range PCET over a pathway of redox active amino acids (β-Y₁₂₂ [special characters omitted] β-Y₃₅₆[special characters omitted] α-Y₇₃₁ [special characters omitted]α-Y₇₃₀[special characters omitted] α-C₄₃₉) that spans ~35 Å and two subunits. As such, RNR serves as a paradigm for the study of PCET in biology.

Subunit interaction dynamics, examined by fluorescence spectroscopy, exposed mechanisms underlying allosteric control over PCET and contributed to an expanded kinetic model for turnover. Trapped meta-stable states of the active α₂β₂ complex are dictated by the translocation of a single charge and attenuate dissociation 10⁴-fold. These trapped states were leveraged to resolve the stoichiometric distribution of the Y₁₂₂^· cofactor from its ensemble average of 1.2 Y^·/β₂, revealing that 02 contain either 2 or 0 Y^·.

Circumventing rate-limiting conformational changes that gate turnover, photoinitiated RNRs were prepared to allow photochemically driven Y₃₅₆ oxidation, and spectroscopic resolution of the ensuing reactivity. A series of photoRNRs containing unnatural F_nYs (n = 2-3) and W in place of β-Y₃₅₆ were prepared. All of these photoβ₂s give rise to transient absorption (TA) spectra consistent with their oxidized forms and undergo photochemically driven turnover.

Time-resolved emission spectroscopy allowed examination of ET kinetics as a function of driving force within the α/β subunit interface. Marcus-inverted kinetics were observed, providing reorganization and electronic coupling energies. Comparing ET and PCET kinetics as a function of pH, buffer concentration, oligomeric state, and buffer isotopic composition revealed new insights into biological control over PCET reactions and implicate a role of α₂ in facilitating proton transfer from β-Y ₃₅₆.

Single wavelength TA kinetics provided direct measure of the rate constant for PCET through α, assignment of the rate-determining step as 3'-C–H bond cleavage by C₄₃₉^·, and a lower bound of 7 for the associated 1° ME. The pK_a of proton acceptor(s) at the subunit interface, and the relative energies of individual radical intermediates were determined, revealing matched tuning to the surrounding environment and highlighting the subtlety of precision control underlying RNR catalysis. (Copies available exclusively from MIT Libraries, libraries.mit.edu/docs - [email protected])