Kinetics of Hydrogen Atom Abstraction from Substrate by an Active Site Thiyl Radical in Ribonucleotide Reductase

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class Ia RNR is an α[subscript 2]β[subscript 2] complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y[subscript 122] → [β-W[subscript 48]] → β-Y[subscript 356] → α-Y[subscript 731] → α-Y[subscript 730] → α-C[subscript 439]) that spans ∼35 Å. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [ReI] photooxidant site-specifically incorporated at position 355 ([Re]-β[subscript 2]), adjacent to PCET pathway residue Y[subscript 356] in β. [Re]-β[subscript 2] was further modified by replacing Y[subscript 356] with 2,3,5-trifluorotyrosine to enable photochemical generation and spectroscopic observation of chemically competent tyrosyl radical(s). Using transient absorption spectroscopy, we compare the kinetics of Y· decay in the presence of substrate and wt-α[subscript 2], Y[subscript 731]F-α[subscript 2] ,or C[subscript 439]S-α[subscript 2], as well as with 3′-[2H]-substrate and wt-α[subscript 2]. We find that only in the presence of wt-α[subscript 2] and the unlabeled substrate do we observe an enhanced rate of radical decay indicative of forward radical propagation. This observation reveals that cleavage of the 3′-C–H bond of substrate by the transiently formed C[subscript 439·] thiyl radical is rate-limiting in forward PCET through α and has allowed calculation of a lower bound for the rate constant associated with this step of (1.4 ± 0.4) × 10[subscript 4] s[subscript –1]. Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis.