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 α2β2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 → [β-W48] → β-Y356 → α-Y731 → α-Y730 → α-C439) 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]-β2), adjacent to PCET pathway residue Y356 in β. [Re]-β2 was further modified by replacing Y356 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-α2, Y731F-α2 ,or C439S-α2, as well as with 3′-[2H]-substrate and wt-α2. We find that only in the presence of wt-α2 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 C439· 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) × 104 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.

Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase

“…41 In practice, the maximal turnover observed is <4 dCDP/α 2 ; ~1.8 dCDP/α 2 is typically observed for non-photochemical experiments (wt β 2 with Ru(NH 3 ) 6 Cl 3 flash quencher) and 0.1–0.6 dCDP/α 2 when the single turnover experiment is driven by light under the photochemical experimental conditions where Ru(NH 3 ) 6 Cl 3 is employed as a “flash quench” reagent. 38,42 Figure 3 shows that the photochemical turnover performance for four photoβ 2 variants: photoβ 2 , Y 356 F-photoβ 2 , E 350 Q-photoβ 2 , and E 350 Q:Y 356 F-photoβ 2 in complex with both WT and Y 731 F α 2 is similar to previous photoβ 2 experiments. 38,42 These mutants were chosen to examine the role of Y 356 (vs. the Y 356 F variant), Y 731 (vs. the Y 731 F) and E 350 (vs. E 350 Q).…”

“…38,42 Figure 3 shows that the photochemical turnover performance for four photoβ 2 variants: photoβ 2 , Y 356 F-photoβ 2 , E 350 Q-photoβ 2 , and E 350 Q:Y 356 F-photoβ 2 in complex with both WT and Y 731 F α 2 is similar to previous photoβ 2 experiments. 38,42 These mutants were chosen to examine the role of Y 356 (vs. the Y 356 F variant), Y 731 (vs. the Y 731 F) and E 350 (vs. E 350 Q). The photoβ 2 in complex with WT α 2 produces ~0.2 dCDP/α 2 , corresponding to ~10% of WT β 2 turnover in the presence of flash quencher, whereas no turnover is observed in the presence of Y 731 F α 2 .…”

“…The fractional dCDP formation of photoβ 2 relative to the WT β 2 is ascribed to off pathway photochemical degradation targeting non-RT pathway associated aromatic sidechains both in α and β (vida infra) . 38,42,43 Photochemical turnover is not observed with either WT or Y 731 F α 2 in complex with Y 356 F-photoβ 2 . Remarkably, by comparison, E 350 Q-photoβ 2 displayed similar dCDP formation activity relative to the standard photoβ 2 .…”

Charge-Transfer Dynamics at the α/β Subunit Interface of a Photochemical Ribonucleotide Reductase

“…Upon oxidation, α-C 439 • initiates active site chemistry by hydrogen atom abstraction from substrate. 22–24 Multi-step substrate-based radical chemistry follows, 25,26 after which reverse PCET carries the radical “hole” back to its stable resting state at Y 122 in β via the same PCET pathway. 27 …”

“…28 This methodology has enabled detailed studies of photoinitiated substrate turnover, 29 spectroscopic observation of photogenerated radicals, 30 and measurement of both radical injection rates into α 2 , 31 and radical propagation rates through α 2 to the active site. 24 A photochemically competent β 2 subunit 32 (photoβ 2 ) is prepared by installing three mutations (C 268 S, C 305 S, and S 355 C) to render a single cysteine residue surface-exposed, and facilitate site-specific conjugation of a bromomethylpyridyl rhenium(I) tricarbonyl phenanthroline complex ([Re I ]) to position β 355 via an S N 2 reaction. 33 Recently, we extended this methodology by incorporating 2,3,5-F 3 Y at position β 356 via nonsense codon suppression methodology (Figure 1, inset).…”