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

摘要

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class Ia RNR is an α_2β_2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y_(122) → [β-W_(48)] → β-Y_(356) → α-Y_(731) → α-Y_(730) → α-C_(439)) that spans ～35 A. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [Re~1] photooxidant site-specifically incorporated at position 355 ([Re]-β_2), adjacent to PCET pathway residue Y_(356) in β. [Re]-β_2 was further modified by replacing Y_(356) with 2,3,5-trifluorotyr- osine 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_2, Y_(731)F-α_2 ,or C_(439)S- α_2 as well as with 3'-[~2H]-substrate and wt-α_2. We find that only in the presence of wt-α_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_(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) × 10~4 s~(-1). Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis.