Direct interfacial Y₇₃₁ oxidation in α₂ by a photoβ₂ subunit of E. coli class Ia ribonucleotide reductase

Abstract

Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). In the class Ia RNRs, NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 Å across the interface of the two required subunits (β₂ and α₂) involving multiple PCET steps and the conserved tyrosine triad [Y₃₅₆(β₂)–Y₇₃₁(α₂)–Y₇₃₀(α₂)]. We report the synthesis of an active photochemical RNR (photoRNR) complex in which a Re(I)-tricarbonyl phenanthroline ([Re]) photooxidant is attached site-specifically to the Cys in the Y₃₅₆C-(β₂) subunit and an ionizable, 2,3,5-trifluorotyrosine (2,3,5-F₃Y) is incorporated in place of Y₇₃₁ in α₂. This intersubunit PCET pathway is investigated by ns laser spectroscopy on [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂ in the presence of substrate, CDP, and effector, ATP. This experiment has allowed analysis of the photoinjection of a radical into α₂ from β₂ in the absence of the interfacial Y₃₅₆ residue. The system is competent for light-dependent substrate turnover. Time-resolved emission experiments reveal an intimate dependence of the rate of radical injection on the protonation state at position Y₇₃₁(α₂), which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y₃₅₆ and Y₇₃₁, at the subunit interface.

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Introduction

Enzymatic control of coupled proton and electron transfer^1–4 is critical in managing biological processes ranging from energy storage (photosystem II)^5–9 and conversion (cytochrome c oxidase)¹⁰ to the synthesis of DNA precursors (ribonucleotide reductase).^11–14 To better understand biological PCET, we have undertaken studies of this process in class Ia RNRs, which catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs)—a process required for synthesis and repair of DNA in all organisms.^15,16 Catalysis by the class I RNRs proceeds by a radical mechanism requiring coupling of radical transport over 35 Å involving PCET across the two subunits to substrate turnover. The long distance, reversibility, and rate-limiting conformational gating of radical transport have made study of this process challenging. To overcome this challenge, we have developed two methodologies: photoRNRs^17–21 and site-specific incorporation of unnatural amino acids in place of pathway residues.^22–24

E. coli class Ia RNR has served as the paradigm for this long distance radical transport. It is composed of two homodimeric subunits: α₂ and β₂. A docking model for this complex,²⁵ substantiated by recent biochemical and biophysical studies,^26,27 has provided the working model for the radical transport pathway shown in Fig. 1. The active site for NDP reduction resides in α₂, where the cysteine radical (C₄₃₉˙) must be transiently generated during each turnover by the essential diferric-tyrosyl radical (Y₁₂₂˙) cofactor in β₂. This long range oxidation requires a multi-step radical hopping mechanism that involves a specific pathway including four tyrosines (Y₁₂₂ and Y₃₅₆ in β₂; Y₇₃₁ and Y₇₃₀ in α₂)^11,28 and potentially W₄₈ in β₂.¹¹

Fig. 1 Current model of radical transport pathway in class Ia RNR leading to nucleotide reduction as determined by the docking model¹⁶ and diagonal distance measurements acquired by PELDOR spectroscopy.³⁴ Key redox active amino acids and known distance measurements involved in PCET pathway are shown. Residue W₄₈ is grayed indicating the absence of experimental evidence supporting its participation in the PCET radical mechanism. Residue Y₃₅₆ is shown at the interface for illustrative purposes. Salmon arrows indicate electron transfer (ET) and green arrows represent proton transfer (PT). In α₂, co-linear PCET is denoted by the dual-colored arrow, and the proposed bi-directional PCET in β₂ is indicated by the orthogonality of the ET and PT pathways.

Recent attention has focused on the detection of the proposed transient radical intermediates and identification of the operative PCET mechanism at each site. Mössbauer studies have established that Y₁₂₂˙ reduction in β₂ is triggered by binding of substrate and effector to α₂ ²⁹ and involves proton donation from the water at Fe₁ (Fig. 1). This process involves orthogonal PCET wherein the proton and electron come from different residues. High-field electron paramagnetic resonance (Hf EPR) and deuterium electron nuclear double resonance (ENDOR) have provided atomic level resolution of local hydrogen bond interactions, specifically the co-linearity of the PCET within α₂. Additionally, significant shifts in g_x values together with the assignment of hyperfine coupling features from the ENDOR spectra of various amino-substituted RNR mutants propose an important role for electrostatics at the α₂:β₂ interface.³⁰ However, the disordered C-terminal tail of β₂ where Y₃₅₆ resides has made interrogation of the chemistry at the subunit interface challenging (Fig. 1).

Rate limiting conformational gating in RNR obscures radical transport across the subunit interface, prompting us to develop photoRNRs to trigger radical initiation with light to avoid this gating and to potentially enable the observation of Y˙ at the interface. Radical injection kinetics were initially made possible using a 19mer peptide photoRNR, which corresponded to the identical 19 residues of the C-terminal tail of β₂ along with a modification that appended a photooxidant (rhenium phenanthroline [Re]) adjacent to Y₃₅₆ or fluorinated derivatives. This peptide photoRNR enables nucleotide reduction in the presence of α₂ and light and allows for observation of radical injection into α₂.²¹ Radical injection was only realized in the presence of an intact Y₇₃₁–Y₇₃₀ dyad within α₂, providing important support for co-linear PCET within this subunit.

More recently, photoRNRs have been generated in which the peptide with the photooxidant is replaced by the full-length β₂ containing a site-specifically incorporated [Re] photooxidant at residue 355 using a S₃₅₅C-β₂ mutant.³¹ Transient absorption spectroscopy on the active [Re₃₅₅]-β₂:α₂ complex in comparison to the control [Re₃₅₅]-Y₃₅₆F-β₂:α₂ complex allowed for the assignment of a photogenerated ˙Y₃₅₆.³² In addition, further modification of [Re₃₅₅]-β₂ by installation of an unnatural 2,3,5-F₃Y in place of Y₃₅₆ in β₂ yielded the first direct measurement of ˙Y propagation kinetics through the active RNR complex.³³ Comparison of ˙Y decay transient kinetics in the [Re₃₅₅]-2,3,5-F₃Y₃₅₆-β₂:α₂ complex in the presence of substrate, either [3′-¹H]-CDP or [3′-²H]-CDP, and effector ATP, revealed an observed rate constant of 1.4 × 10⁴ s⁻¹ and unmasked for the first time an isotope effect on cleavage of the 3′ C–H of the substrate. The photoRNRs thus circumvent the masking of radicals by conformational gating and thus have provided insight regarding radical transport and nucleotide reduction chemistry not accessible by any other method.

In this work, the [Re] photooxidant is attached to Cys in the Y₃₅₆C-β₂ mutant and this modified [Re₃₅₆]-β₂ subunit is associated to α₂ in which Y₇₃₁ is site-specifically replaced with 2,3,5-F₃Y. This [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂ complex together with the [Re₃₅₆]-β₂:wt-α₂ and [Re₃₅₆]-β₂:Y₇₃₁F-α₂ control complexes are studied in the presence of CDP and ATP. In contrast to previous photoRNR systems, installation of [Re] at position 356 enables direct interfacial generation of a tyrosyl radical at position 731. Additionally, by leveraging the greater acidity of 2,3,5-F₃Y to enable deprotonation at neutral pH, this residue furnishes an ionizable reporter that varies with experimental pH. In turn, site-selective removal of a single proton at position Y₇₃₁(α) provides the first protein:protein scaffold of RNR that permits the investigation of the effect of a modified proton microenvironment on radical transport on transient time scales (sub μs) at the interface. In the absence of Y₃₅₆, radical injection is only achieved when position Y₇₃₁ is deprotonated. In addition to confirming the complexity of RNR in maintaining a well-organized PCET pathway, this work introduces and highlights the importance of a well-defined proton exit channel out of α₂ involving the key pathway residues, Y₃₅₆ and Y₇₃₁, at the subunit interface.

Experimental

Modified RNR subunits were constructed, expressed, purified, modified, and characterized as previously reported or with minor modification.^{19,23,24,31,35} Protein concentrations were measured by absorbance at 280 nm using: ε_α2 = 189 000 M⁻¹ cm⁻¹, ε_β2-apo = 121 000 M⁻¹ cm⁻¹, ε_β2-holo = 131 000 M⁻¹ cm⁻¹, and ε_β2-[Re] = 189 000 M⁻¹ cm⁻¹. Purity of protein constructs was assessed by SDS-PAGE (Fig. S1†). All measurements were conducted in assay buffer at pH 7.6 (50 mM HEPES, 15 MgSO₄, 1 mM EDTA; unless otherwise specified). Measurement of the dissociation constant (K_D) between [Re₃₅₆]-β₂ and wt-α₂ was performed by a spectrophotometric competitive inhibition assay as previously reported.^17,21 Measurement of the pK_a of the phenolic proton of 2,3,5-F₃Y₇₃₁ within the assembled 2,3,5-F₃Y₇₃₁-α₂:[Re₃₅₆]-β₂ complex was performed by fluorometric titration as previously reported.²¹ The details of methods that deviate from published procedures are provided in the ESI.† Similarly, photoinitiated nucleotide reduction activity assays were performed according to published methods.^17–19,32 Error bars represent 2σ resulting from photolysis on ≥three independent samples.

Time-resolved spectroscopic measurements were performed using a home-built nanosecond laser system previously described.^21,31–33 Each sample was prepared prior to photolysis and measurements were performed in triplicate. The calculation of the uncertainty in experimental measurements to 95% confidence limits (2σ) is described in the ESI (eqn S1–S5†).

Results and discussion

PhotoRNRs: [Re₃₅₆]-β₂:α₂ and [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂

To probe radical initiation across the α₂:β₂ interface, specific variants of each subunit were required. To directly target the intersubunit radical transport step of Y₇₃₁ oxidation and subsequent radical injection into α₂, we chose to circumvent Y₃₅₆ oxidation entirely. In contrast to previous systems where photooxidants were placed adjacent to Y₃₅₆ at position 355, [Re] replaces Y₃₅₆ in this study. The new construct maintains the mutations C₂₆₈S and C₃₀₅S, and preserves catalytic activity. Additionally, the mutation, Y₃₅₆C, thus enables alkylation with [Re]-Br to yield [Re₃₅₆]-β₂. To examine the effect of a proton at position Y₇₃₁, this residue was replaced with 2,3,5-F₃Y, solution pK_a = 6.4, compared with Y (pK_a = ∼10). The photoRNR construct is illustrated in Fig. 2. These two subunit modifications allow for direct oxidization of Y₇₃₁ by a photoβ₂.

Fig. 2 PhotoRNR design schematic depicting the intersubunit photochemical oxidation of 2,3,5-F₃Y₇₃₁ by [Re₃₅₆]-β₂, incorporation of 2,3,5-F₃Y at position Y₇₃₁ allowed radical generation to be examined in the absence of a proton at this residue.

Construction, expression, isolation, and labelling to generate [Re₃₅₆]-β₂ were performed as previously reported with minor modifications.³¹ Unlabelled and reconstituted Y₃₅₆C-β₂ (holo) is inactive (0.16(5) U) towards nucleotide reduction (wt-β₂ activity = 6000–8000 U), as expected given the absence of Y₃₅₆. We note that labelling does not preclude binding (K_D, = 0.43(11) μM), as measured in the competitive inhibition assay shown in Fig. S2.† This value is in agreement with previously reported values for active photoRNR β₂ mutants,³² and not significantly altered from wt-RNR.^12,36

[Re₃₅₆]-β₂ displays similar spectroscopic and photophysical properties, as reported previously for [Re₃₅₅]-β₂.³¹ Fig. S3† shows typical absorption and emission spectra for [Re₃₅₆]-β₂; the absorption spectrum is dominated by the characteristic features of rhenium(I) tricarbonyl bisimine compounds and the emission originates from a triplet metal-to-ligand charge transfer state. The absorption spectrum of [Re₃₅₆]-β₂ is accurately re-constructed as the sum of Y₃₅₆C-β₂ and twice the [Re]-Br absorption spectrum, as expected for a construct with two [Re] molecules per β₂ dimer (Fig. S3†).

Construction of 2,3,5-F₃Y₇₃₁-α₂ was achieved using in vivo nonsense suppression methodology previously developed to install fluorotyrosine reporters in class Ia RNR.²⁴ The specific activity of 2,3,5-F₃Y₇₃₁-α₂ under catalytic conditions (375(5) U) is diminished relative to wt-α₂ (1800–2500 U), while under single-turnover activity (2.75(4) equiv. dCDP/α₂) is comparable to wt-α₂ (∼3 dCDP equiv./α₂). This decrease in catalytic activity, though comparable to that of wild-type (wt), is consistent with previous reports of this mutant.³⁷

The deprotonation of 2,3,5-F₃Y₇₃₁ is expected to perturb the rate of radical generation at position Y₇₃₁. The kinetic penalty associated with proton transfer is alleviated by removal of the proton when experimental pH > pK_a, thus requiring measurement of the precise pK_a of 2,3,5-F₃Y₇₃₁ in the assembled construct. As previously reported,^{19–21,32,33,38} the increase in the rate of photooxidation for tyrosinate relative to tyrosine,³⁹ makes [Re] emission a reporter of the protonation state of nearby tyrosine residues. Fluorometric titration of the [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂ complex reveals the pK_a of 2,3,5-F₃Y₇₃₁ to be 6.7(1), Fig. 3. Accordingly, ∼90% of 2,3,5-F₃Y₇₃₁ residues are deprotonated under experimental conditions at the optimal operating pH for RNR (pH 7.6). Given the thermodynamically unfavourable acidity of the tyrosyl radical cation (pK_a = −2) a PCET process managing both the electron and proton transfers is mandated.^40,41

Fig. 3 Measurement of the pK_a of the phenolic proton of 2,3,5-F₃Y₇₃₁ in the [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂ complex by steady-state emission. Fluorometric titrations of 2.5 μM 2,3,5-F₃Y₇₃₁-α₂, 3.75 μM [Re₃₅₆]-β₂, 1 mM CDP, and 3 mM ATP were carried over a pH range of 5 to 9. Samples were illuminated with λ_exc = 315 nm, scanned from 420–700 nm at 0.5 nm intervals, integrated for 1 s at each data point, and averaged from three scans. The collected emission plots were integrated for fluorescence intensity and plotted against pH. Data were fit to an internal sigmoidal logistic function in OriginPro 8.5. The inflection point of the monoprotic pH-titration curve (x₀) is the pK_a:.

Photoinitiated substrate turnover

To establish that the photoRNR construct is competent to generate dCDP, the [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂ complex in the presence of [³H]-CDP and ATP was photolyzed for 10 min (λ > 313 nm) and dCDP was measured by scintillation counting. The results of this single turnover experiment are shown in Fig. 4. Perturbation of the enzyme by the introduction of [Re] results in a reduced level of turnover that is 5–10% relative to wt-RNR under the same pH conditions. Notwithstanding, the presence of photogenerated products establish the relevance of [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂ complex to the natural enzyme. Attenuated enzymatic activity is also detected at pH > 7.6 which is consistent with the observed pH rate profiles for the wt enzyme and fluorotyrosine derivatized RNR constructs.⁴²

Fig. 4 Photoinitiated nucleotide reduction assay monitored (left) as a function of pH in the [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂ complex and (right) at pH = 7.6 for the control, [Re₃₅₆]-β₂:Y₇₃₁F-α₂. A solution containing 25 μM [Re₃₅₆]-β₂, 200 μM [³H]-CDP (21 148 cpm nmol⁻¹), 3 mM ATP in assay buffer, and a separate solution of 10 μM α₂ were incubated at 25 °C. Following two min incubation the two solutions were mixed, transferred to the photolysis cuvette, and illuminated (λ > 313 nm) for 10 min.

Radical injection kinetics

The photogeneration of product for the [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂ complex prompted us to undertake radical injection kinetics studies. Using the [Re]* emission lifetime as a reporter for radical injection, ns TA laser spectroscopy on the [Re₃₅₆]-β₂:α₂ complexes in the presence of CDP and ATP was conducted. The emission decay lifetimes for each construct were measured at pH = 7.6, where maximum turnover was observed, and are summarized in Table 1; representative traces are included in Fig. S4.† As previously observed for [Re₃₅₅]-β₂,³² the [Re]* lifetime in [Re₃₅₆]-β₂ (τ = 507(3) ns) increases upon binding to Y₇₃₁F-α₂ (τ_o = 652(4) ns), consistent with the more hydrophobic environment engendered by the protein environment. The lifetime τ_o of the Y₇₃₁F-α₂ variant provides a reference for excited-state decay of [Re]* when it is located at the interface, but in the absence of quenching by the tyrosine located at position 731. Upon introduction of Y₇₃₁, the [Re]* emission (τ = 629(4) ns) is quenched relative to Y₇₃₁F-α₂ according to the following equation:

Table 1 Radical injection rates from [Re₃₅₆]-β₂ to α₂ variants modified at position 731 at pH = 7.6

[Re356]-β2: X731-α2	τ 600 nm ns^−1	k q/10^4 s^−1^b
a Lifetime of emission decay measured on 10 μM [Re356]-β2, 25 μM α2 (as indicated), 1 mM CDP, 3 mM ATP in assay buffer (pH 7.6), λexc = 355 nm, λobs = 600 nm. Errors shown in parentheses represent 2σ resulting from measurement on ≥3 independent samples. b Emission quenching rate constant, kq, determined from eqn (1). Error in quenching rate constants calculated as shown in ESI.
F	652(4)	—
Y	629(4)	5.6 (1.4)
2,3,5-F 3 Y	593(6)	15.4 (2.2)

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Accordingly, this quenching rate constant, k_q, is equivalent to the radical generation rate, and from eqn (1) it is calculated to be 5.6(1.4) × 10⁴ s⁻¹. In comparison to previously observed photooxidation of Y₃₅₆ in [Re₃₅₅]-β₂ (k_q = 4.1(1) × 10⁵ s⁻¹), the direct oxidation of Y₇₃₁ by [Re₃₅₆]*-β₂ is slower possibly owing to an increased charge transfer distance, which is occurring across the subunit interface vs. at adjacent positions in previous investigations. Further analysis of the emission decay kinetics of [Re]* reveal a dependency on the rate of radical injection and the protonation state of tyrosine at position Y₇₃₁(α). Replacement of Y₇₃₁ with 2,3,5-F₃Y₇₃₁ enhances the quenching rate constant by a factor of three (τ = 593(7) ns (k_q = 15.4(2.2) × 10⁴ s⁻¹). This acceleration of radical injection into α₂ is in accordance with the differing protonation states of tyrosine in the two constructs. Under the experimental conditions of pH = 7.6, 2,3,5-F₃Y₇₃₁ is deprotonated and hence quenching occurs by ET rather than PCET, which results in faster tyrosine oxidation, despite being ∼50–100 mV more difficult to oxidize than native tyrosine at this pH (as determined from solution peak potentials (E_p) of N-acetyl and C-amide protected fluorotyrosines measured by differential pulse voltammetry).²² For clarity, obtaining precise single reside midpoint potentials in protein constructs is extremely challenging, and thus thermodynamic considerations must be guided from these measured E_p values,¹¹ despite recent findings that suggest significant deviations of the formal reduction potential and solution E_p values.⁴³

To investigate how the proton at position Y₇₃₁ affects interfacial PCET and subsequent interfacial radical injection kinetics, emission quenching of the [Re]* within the [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂ complex was monitored over the activity accessible pH region of RNR. A plot of the [Re]* decay lifetimes for [Re₃₅₆]-β₂ alone and in the three [Re₃₅₆]-β₂:α₂ variant complexes monitored as a function of pH are shown in Fig. S5;† representative emission decay traces are provided in Fig. S4.† The quenching rate constants, k_q, as a function of pH may be determined from eqn (1); these rate constants are plotted in Fig. 5. Quenching by Y₇₃₁(wt)-α₂ (red circles, ) and 2,3,5-F₃Y₇₃₁-α₂ (light blue squares, ) are referenced to the control, Y₇₃₁F-α₂. Within our error limits, little to no dependence of k_q is observed for the [Re₃₅₆]-β₂:Y₇₃₁(wt)-α₂ complex (), as native tyrosine is protonated throughout the pH window. Conversely, a large pH dependence is observed for k_q when [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂ is compared to Y₇₃₁F (). Guided by our measurement of the pK_a of 2,3,5-F₃Y₇₃₁-α₂, shown in Fig. 3, we ascribe the observed differences in quenching to the relative ratio of deprotonated:protonated forms of 2,3,5-F₃Y₇₃₁ as pH is varied. The large k_qs at high pH is expected owing to deprotonation of F₃Y₇₃₁ whereas quenching at low pH approaches that of Y₇₃₁ where both of the tyrosines are protonated.

Fig. 5 Quenching rate constant of [Re]* by tyrosine, k_q, monitored as function of pH: Y₇₃₁(wt) relative to Y₇₃₁F () and 2,3,5-F₃Y₇₃₁ relative to Y₇₃₁F (). τ_o corresponds to the lifetime of [Re₃₅₆]-β₂:Y₇₃₁F-α₂. Error bars in k_q represent 2σ resulting from error propagation on lifetime measurements as described in ESI.†

Scheme 1 summarizes the decay pathways for [Re]* in the different variants. The presence of tyrosine introduces an excited-state decay pathway via radical generation. For the case of Y₇₃₁(wt)-α₂, oxidation occurs by PCET whereas for 2,3,5-F₃Y₇₃₁-α₂ occurs by ET. These differing mechanisms of tyrosine oxidation for the two variants in the absence of Y₃₅₆ introduce the possibility that Y₃₅₆ may be involved with facilitating proton removal at the interface, though future experiments are needed to establish its specific role. While a co-linear Y₃₅₆–Y₇₃₁ π-stacked mode where Y₃₅₆ acts directly as the proton acceptor for Y₇₃₁ is unlikely in light of recent Hf EPR/ENDOR data on amino-substituted tyrosine derivatives at various pathway positions, the strongly perturbed g_x values of NH₂Y₃₅₆˙ indicate that Y₃₅₆ may communicate with Y₇₃₁ through a network of water molecules.³⁰ Additional evidence supporting the involvement of Y₃₅₆ in modulating Y₇₃₁ oxidation was also observed in [Re₃₅₅]-β₂ construct.³² The photooxidation kinetics of Y₇₃₁, which are summarized in Table S1,† indicate that Y₇₃₁ radical generation is enhanced by the presence of Y₃₅₆ as [Re₃₅₅]-β₂ oxidation of Y₇₃₁ is 2.1(1.2) times faster for Y₃₅₆ than for F₃₅₆. The presence of Y₃₅₆ may facilitate proton removal from α₂via the interface, thus assisting in PCET.

Scheme 1 Excited-state deactivation pathways for [Re₃₅₆]-β₂ in the presence of indicated α₂ subunit.

In this directed study, whereby Y₃₅₆ is absent by virtue of its replacement with [Re₃₅₆], efficient injection of a radical into α₂ is realized only when a proton is removed from the pathway by the introduction of 2,3,5-F₃Y₇₃₁. While this result does not implicate Y₃₅₆ directly as the proton acceptor for Y₇₃₁, it supports the contention that Y₃₅₆ is in communication with Y₇₃₁ at the α₂:β₂ subunit interface and that Y₃₅₆ enables the PCET required for efficient radical transport. Further investigations of this contention are underway along with studies to assess the role of possible contributions from other residues, or perhaps metal ions that may also be involved in managing protons at the interface.

Conclusions

Replacement of Y₃₅₆ by a [Re] photooxidant and installation of 2,3,5-F₃Y at position 731 in α₂ furnishes a photoRNR that specifically targets intersubunit radical transport. This construct supports photoinitiated substrate turnover, confirming its fidelity to the natural system. Time-resolved emission studies reveal that 2,3,5-F₃Y₇₃₁ is oxidized at a rate 3 times faster than native Y₇₃₁ even though the non-natural amino acid is thermodynamically more difficult to oxidize at pH 7.6 (ΔE_p ∼ 50–100 mV). These results emphasize the enzymatic imperative for coupling the proton and electron to allow for efficient radical transport. In conjunction with the parallel studies of [Re₃₅₅]-β₂, these results suggest the importance of a well-coordinated proton exit channel involving Y₃₅₆ and Y₇₃₁ as key interfacial residues for radical transport across the α₂:β₂ interface.

Acknowledgements

DYS acknowledges the National Science Foundation's Division of Graduate Education Grant DGE-1144152. PGH thanks the National Institute of Health for a Post-Doctoral Fellowship (GM087034). This research was supported by the U.S. National Institute of Health Grants GM047274 (DGN) and GM029595 (JS).

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedures, calculation of uncertainty in data analysis, purity gels, determination of K_D, spectroscopic characterization, time-resolved emission decay traces, and table from reference S8 are provided. See DOI: 10.1039/c5sc01125f

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