Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger

Abstract

Photochemical radical initiation is a powerful tool for studying radical initiation and transport in biology. Ribonucleotide reductases (RNRs), which catalyze the conversion of nucleotides to deoxynucleotides in all organisms, are an exemplar of radical mediated transformations in biology. Class Ia RNRs are composed of two subunits: α2 and β2. As a method to initiate radical formation photochemically within β2, a single surface-exposed cysteine of the β2 subunit of Escherichia coli Class Ia RNR has been labeled (98%) with a photooxidant ([Re ] = tricarbonyl(1,10-phenanthroline)(methylpyridyl)rhenium(I)). The labeling was achieved by incubation of S355C-β2 with the 4-(bromomethyl)pyridyl derivative of [Re] to yield the labeled species, [Re]-S355C-β2. Steady-state and time-resolved emission experiments reveal that the metal-to-ligand charge transfer (MLCT) excited-state (3)[Re ](∗) is not significantly perturbed after bioconjugation and is available as a phototrigger of tyrosine radical at position 356 in the β2 subunit; transient absorption spectroscopy reveals that the radical lives for microseconds. The work described herein provides a platform for photochemical radical initiation and study of proton-coupled electron transfer (PCET) in the β2 subunit of RNR, from which radical initiation and transport for this enzyme originates.

Fig. 1.

Key amino acids along a putative radical transport pathway in class Ia RNR. The pathway is ascertained from docking models of the α2 (gold) and β2 (blue) subunits. Substrate turnover requires charge transfer across α2 and β2 subunits over a distance of 35 Å. Radical hopping via the indicated redox-active amino acids is proposed to account for the observed rate of turnover. Distances are known for proposed hopping steps within individual subunits; distances involving Y356 have not been determined. Graphics were generated from Protein Data Bank entries 1RLR (α2) (16) and 1RIB (β2) (18).

Fig. 2.

X-ray crystal structure of [Re]-Br. Thermal ellipsoids are reported at a 50% probability level. Hydrogen atoms, solvent molecules, and an additional molecule of [Re]-Br are omitted for clarity. Selected metric parameters are reported in Table S2.

Fig. 3.

Spectroscopic comparison of [Re]-Br and [Re]-S355C-β2. The UV-vis absorption (brown) and emission spectra (λ_ex = 355 nm) (brown) (50 μM) of [Re]-Br (MeCN solution) and the UV-vis absorption (blue) and emission (blue) (10 μM) spectra of [Re]-S355C-β2 (50 mM HEPES, 1 mM EDTA, pH 8.0) are shown. The absorption spectrum of S355C-β2 (gray) (50 mM HEPES, 1 mM EDTA, pH 8.0) is also included for reference. The simulated absorption spectrum (red) of [Re]-S355C-β2 by summing the absorption spectrum of twice the absorption spectrum of [Re]-Br and S355C-β2. The similarity of the actual (blue) and simulated (red) [Re]-S355C-β2 UV-vis absorption traces suggests that binding has no significant impact on the spectroscopic properties of [Re].

Fig. 4.

Strategy for the construction of a photoRNR-β2. The attachment of the PO, [Re ] = [Re(phen)(CO)₃(PyCH₂Br)] via the methylene group of the pyridyl ligand of the rhenium center is shown schematically. Radical initiation at Y356: (1) a 355 nm laser pulse generates the [Re]* excited state, which (2) is quenched to produce (3) the [Re^II] species, which oxidizes Y356, to regenerate the [Re^I] ground state and the radical. (4) The photogenerated Y356 radical is observed and monitored by transient absorption spectroscopy. Graphics were generated from Protein Data Bank entry 1MXR (β2) (15).

Fig. 5.

Photochemically generated •Y356. Time-resolved spectroscopic data are recorded after excitation (λ_ex = 355 nm) of met-[Re]-S355C-β2. Top: Transient absorption spectrum of [Re]-S355C-β2 collected 1 μs after excitation (65 μM in 50 mM HEPES, 32.5 mM Ru^III(NH₃)₆Cl₃, 1 mM EDTA, pH 8.0). The spectrum shown is obtained from 2,500 four-spectrum sequences taken on two samples (1,250 four-spectrum sequences each), averaged, and smoothed using a low-pass filter on the basis of a fast Fourier transform (FFT). Bottom: Transient absorption kinetics for transient •Y356 (λ_obs = 408 nm) and a biexponential fit (blue) (50 μM in 69% 50 mM HEPES, 25 mM Ru^III(NH₃)₆Cl₃, 1 mM EDTA, pH 8.0) (τ₁ = 8.1 ± 1.1 μs, τ₂ = 2.0 ± 0.8 μs 31%). The trace shown is obtained from 5,000 sweeps (averages) taken on a single sample.