Photochemical Generation of a Tryptophan Radical within the Subunit Interface of Ribonucleotide Reductase

Abstract

The E. coli class Ia ribonucleotide reductase (RNR) achieves forward and reverse proton-coupled electron transfer (PCET) over a pathway of redox-active amino acids (β-Y122 ⇌ β-Y356 ⇌ α-Y731 ⇌ α-Y730 ⇌ α-C439) spanning ~35 Å and two subunits every time it turns over. We have developed photoRNRs that allow radical transport to be phototriggered at tyrosine (Y) or fluorotyrosine (FnY) residues along the PCET pathway. We now report a new photoRNR in which photooxidation of a tryptophan (W) residue replacing Y356 within the α/β subunit interface proceeds by a stepwise ETPT (electron transfer then proton transfer) mechanism and provides an orthogonal spectroscopic handle with respect to radical pathway residues Y731/Y730 in α. This construct displays a ~3-fold enhancement in photochemical yield of W• relative to F3Y• and a ~7-fold enhancement relative to Y•. Photogeneration of the W• radical occurs with a rate constant of 4.4 ± 0.2 × 105 s−1, which obeys a Marcus correlation for radical generation at the RNR subunit interface. Despite the fact that the Y → W variant displays no enzymatic activity in the absence of light, photogeneration of W• within the subunit interface results in 20% activity for turnover relative to wt-RNR under the same conditions.

A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes

“…14 Differences in reactivity between wt-α2 and Y 731 F-α2 are also recorded for photo-RNR, which contains a [Re I ] photooxidant appended to the C-terminal tail of β2 (S 355 C). 34,35 We therefore posited that the block at 731 could perturb the reduction potential of Y 356 • compared to the wt enzyme. The equilibration experiments were repeated with Y 730 F-α2, and as seen in Figure 4 (blue dots), variations can be observed between Y 731 F-α2 and Y 730 F-α2, with the former construct generating slightly higher amounts of Y 356 •.…”

“…Three distinct types of experiments are currently the basis for favoring the latter possibility. 29,35,37,59,60 …”

“…In the presence of α2, S, and E and with light initiation, these constructs exert significant control in facilitating PT during oxidation of residue 356, shuttling reactive intermediates between the subunits and in the case of W, rapid PT out of the α/β interface. 35,60 …”

Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase

“…Whereas, photochemical generation Y 356 •(β) by the photoβ 2 has been examined extensively and occurs on the microsecond timescale, 58 the direct generation of Y 731 •(α) to bypass Y 356 has been achieved by the introduction of [Re] at position 356. 59 …”

Thermally Induced Oxidation of [FeII(tacn)2](OTf)2 (tacn = 1,4,7‐triazacyclononane)

“…These reactions have been increasingly observed in reactions catalyzed by metalloenzymes. [1b], Examples of PCET in biological systems include lipoxygenases, oxalate decarboxylase, photosystem II, cytochrome c oxidase, cytochrome P450, methane monooxygenases, and ribonucleotide reductases . Previous studies using [Fe II (H 2 bip)] 2+ and [Fe II (H 2 bim)] 2+ [H 2 bip = 2,2′‐bi(tetrahydropyrimidine); H 2 bim = 2,2′‐bi‐2‐imidazoline] and TEMPO (2,2,6,6‐tetramethyl‐1‐piperdinyloxy) [Equation and Equation ], have demonstrated this type of reaction in iron‐containing model complexes.…”