Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase

Abstract

Class Ia ribonucleotide reductase (RNR) of Escherichia coli contains an unusually stable tyrosyl radical cofactor in the β₂ subunit (Y₁₂₂^•) necessary for nucleotide reductase activity. Upon binding the cognate α₂ subunit, loaded with nucleoside diphosphate substrate and an allosteric/activity effector, a rate determining conformational change(s) enables rapid radical transfer (RT) within the active α₂β₂ complex from the Y₁₂₂^• site in β₂ to the substrate activating cysteine residue (C₄₃₉) in α₂ via a pathway of redox active amino acids (Y₁₂₂[β] ↔ W₄₈[β]? ↔ Y₃₅₆[β] ↔ Y₇₃₁[α] ↔ Y₇₃₀[α] ↔ C₄₃₉[α]) spanning >35 Å. Ionizable residues at the α₂β₂ interface are essential in mediating RT, and therefore control activity. One of these mutations, E₃₅₀X (where X = A, D, Q) in β₂, obviates all RT, though the mechanism of control by which E₃₅₀ mediates RT remains unclear. Herein, we utilize an E₃₅₀Q-photoβ₂ construct to photochemically rescue RNR activity from an otherwise inactive construct, wherein the initial RT event (Y₁₂₂^• → Y₃₅₆) is replaced by direct photochemical radical generation of Y₃₅₆^•. These data present compelling evidence that E₃₅₀ conveys allosteric information between the α₂ and β₂ subunits facilitating conformational gating of RT that specifically targets Y₁₂₂^• reduction, while the fidelity of the remainder of the RT pathway is retained.

Figure 1.

Radical transfer (RT) pathway of Escherichia coli class Ia RNR. Redox active amino acids known to be involved in PCET, formation of the Fe₂^III-O/Y122• cofactor, or conformational gating are labeled. Residues that are disordered in the X-ray crystallographic structures of β₂ are also labeled. The subject of this paper, E₃₅₀, is shown at the nexus of putative H⁺ transfer (orange arrows) pathways to either Y₃₅₆[β] or Y₇₃₁[α] or a conformational gating network between substrate and effector on α₂ and Y₁₂₂•[β] (green arrow) in β₂. Proton and electron transfer directions are indicated with arrows. Nucleoside diphosphate substrate, NDP, and (deoxy)nucleoside triphosphate allosteric effector, (d)NTP (X = H, OH), are also shown.

Figure 2.

K_D determination for E₃₅₀Q-photoβ₂ with Y₇₃₁F α₂. [Re^I]* emission lifetime measurements as a function of [Y₇₃₁F] concentration (blue circles) and associated fit to experimental data based on eq (2). Error bars indicate one standard deviation among triplicate lifetime measurements. Inset shows activity inhibition K_D assay (orange circles) with associated fit based on eq (1). Error bars indicate one standard deviation from triplicate measurements.

Figure 3.

Photochemical single-turnover assays and WT standard. Photochemical samples (columns 1–4) contained 20 μM β₂ as indicated and 10 μM of either WT (orange) or Y₇₃₁F (blue) α₂ with 0.2 mM [³H]-CDP (32,000 cpm/nmole), 3 mM ATP and 10 mM Ru(NH₃)₆Cl₂ in standard assay buffer and illuminated for 4 min. WT β₂ standard was performed identically except no flash quencher was added.

Figure 4.

Transient absorption spectrum of E₃₅₀Q-photoβ₂ and E₃₅₀Q:Y₃₅₆F-photoβ₂ following laser induced flash quenching. Samples were prepared at 30μM photoβ₂, 50 μM Y₇₃₁F α₂, 1 mM CDP, 3 mM ATP and 10 mM Ru(NH₃)₆Cl₃ in assay buffer pH 7.6. Spectra were generated 1 μs after excitation and represent the average of 3 independent samples of 1000 laser shots.

Figure 5.

Y₃₅₆• decay kinetics within the E₃₅₀Q-photoβ₂-WT and E₃₅₀Q-photoβ₂-Y₇₃₁F interface probed by single wavelength TA (λ_obs = 410 nm). Sample conditions identical to those of Figure 3. Transients represent the average of 1000 shots on a representative sample and associated fit to a single exponential function. Y₇₃₁F data was duplicated and offset (translucent blue) for clearer evaluation of the fit.