Reversible, long-range radical transfer in E. coli class Ia ribonucleotide reductase

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversionof nucleotides to 2'-deoxynucleotides and are classified on the basis of the metallo-cofactor used to conduct this chemistry. The class Ia RNRs initiate nucleotide reduction when a stable diferric-tyrosyl radical (Y•, t1/2 of 4 days at 4 °C) cofactor in the β2 subunit transiently oxidizes a cysteine to a thiyl radical (S•) in the active site of the α2 subunit. In the active α2β2 complex of the class Ia RNR from E. coli , researchers have proposed that radical hopping occurs reversibly over 35 Å along a specific pathway comprised of redox-active aromatic amino acids: Y122• ↔ [W48?] ↔ Y356 in β2 to Y731 ↔ Y730 ↔ C439 in α2. Each step necessitates a proton-coupled electron transfer (PCET). Protein conformational changes constitute the rate-limiting step in the overall catalytic scheme and kinetically mask the detailed chemistry of the PCET steps. Technology has evolved to allow the site-selective replacement of the four pathway tyrosines with unnatural tyrosine analogues. Rapid kinetic techniques combined with multifrequency electron paramagnetic resonance, pulsed electron-electron double resonance, and electron nuclear double resonance spectroscopies have facilitated the analysis of stable and transient radical intermediates in these mutants. These studies are beginning to reveal the mechanistic underpinnings of the radical transfer (RT) process. This Account summarizes recent mechanistic studies on mutant E. coli RNRs containing the following tyrosine analogues: 3,4-dihydroxyphenylalanine (DOPA) or 3-aminotyrosine (NH2Y), both thermodynamic radical traps; 3-nitrotyrosine (NO2Y), a thermodynamic barrier and probe of local environmental perturbations to the phenolic pKa; and fluorotyrosines (FnYs, n = 2 or 3), dual reporters on local pKas and reduction potentials. These studies have established the existence of a specific pathway spanning 35 Å within a globular α2β2 complex that involves one stable (position 122) and three transient (positions 356, 730, and 731) Y•s. Our results also support that RT occurs by an orthogonal PCET mechanism within β2, with Y122• reduction accompanied by proton transfer from an Fe1-bound water in the diferric cluster and Y356 oxidation coupled to an off-pathway proton transfer likely involving E350. In α2, RT likely occurs by a co-linear PCET mechanism, based on studies of light-initiated radical propagation from photopeptides that mimic the β2 subunit to the intact α2 subunit and on [(2)H]-ENDOR spectroscopic analysis of the hydrogen-bonding environment surrounding a stabilized NH2Y• formed at position 730. Additionally, studies on the thermodynamics of the RT pathway reveal that the relative reduction potentials decrease according to Y122 < Y356 < Y731 ≈ Y730 ≤ C439, and that the pathway in the forward direction is thermodynamically unfavorable. C439 oxidation is likely driven by rapid, irreversible loss of water during the nucleotide reduction process. Kinetic studies of radical intermediates reveal that RT is gated by conformational changes that occur on the order of >100 s(-1) in addition to the changes that are rate-limiting in the wild-type enzyme (∼10 s(-1)). The rate constant of one of the PCET steps is ∼10(5) s(-1), as measured in photoinitiated experiments.

Figure 1

A docking model of the E. coli α2β2 complex. α2 (pink and red) contains three nucleotide binding sites. β2 (light and dark blue) contains the diferric-Y• cofactor; residues 340 to 375 are not resolved in this structure. A peptide corresponding to the C-terminal 20 amino acids of β is bound to each α, a portion of which (residues 360-375) is resolved in the crystal structure (cyan). The “ATP cone” region of α, which contains the effector site that governs activity, is colored orange. This model separates Y₁₂₂• in β2 from C₄₃₉ in α2 by >35 Å. GDP (green), TTP (yellow) and the Fe₂O core of the diferric cluster (orange) are shown in CPK space-filling models. Residues constituting the RT pathway (green) are shown in sticks.

Figure 2

Mechanism of NDP reduction by RNR. The S• shown on C₄₃₉ of α2 in the first reaction step is reversibly generated by Y₁₂₂• in β2 by the mechanism shown in Figure 3a.

Figure 3

The Nocera/Stubbe elaboration of the Uhlin/Eklund model for RT in E. coli class Ia RNR. (a) The proposed movement of protons (blue arrows) and electrons (red arrows) at each step on the pathway. Distances (Å) are from structures of α2 and β2. E₃₅₀ and Y₃₅₆ are disordered in all β2 structures, and their positions are unknown. There is no direct evidence that W₄₈ and D₂₃₇ participate in RT and thus they are shown in gray. The distance between W₄₈ and Y₇₃₁ is modeled to be 25 Å. (b) The proposed relative reduction potentials of residues on the RT pathway from experiments using the indicated UAAs site-specifically incorporated in place of each Y. NH₂Y has been incorporated at position 356, 730, or 731. Note that the absolute reduction potentials of these residues, the structures of which are shown in Figure 4, are not known, and the relative reduction potentials indicated are our best estimates given current knowledge (see Table 1 and Footnote 1).

Figure 4

UAAs incorporated into E. coli class Ia RNR. (a) Compounds 1-2 and 4-8 have been incorporated in position 356 of β2 by EPL, while compounds 1, 3-7 have been incorporated to sites in both α2 and β2 by in vivo nonsense suppression. (b) Purified protein yields achieved by in vivo nonsense suppression are given in parenthesis for each UAA at each position (in mg protein/g cells).

Figure 5

Experimental design, kinetic techniques, and detection methods for studying RT.

Figure 6

Spectroscopic characterization of the diferric-Y₁₂₂• cofactor from E. coli class Ia RNR. (a) The UV-vis spectrum has contributions from the diferric cluster at 325 and 365 nm and Y₁₂₂• at 411 nm. (b) The 9 GHz EPR spectrum of Y₁₂₂• with the origin of its hyperfine couplings indicated. (c) The 140 GHz EPR spectrum of Y₁₂₂• resolves three distinct g tensors. Adapted from reference 22.

Figure 7

Spectroscopic characterization of NH₂Y•s. (a) A point-by-point reconstruction of the absorbance spectra of NH₂Y₇₃₀• (blue) and NH₂Y₇₃₁• (red) formed 1.5 s after reacting Y₇₃₁NH₂Y-α2 (or Y₇₃₀NH₂Y-α2) with wt-β2, CDP, and ATP. (b) Averaged single-wavelength SF UV-vis traces for the Y₇₃₁NH₂Y-α2 reaction described in (A). Loss of Y₁₂₂• (red, 410 nm] correlates with the formation of NH₂Y₇₃₁• (blue, 320 nm]. Biexponential fits to the data are shown as black lines. (c) The 9 GHz EPR spectrum (black) of an identical reaction frozen after 10 s is a ~1:1 composite of residual Y₁₂₂• (blue) and NH₂Y₇₃₁• (red). Figure adapted from reference 17.

Figure 8

Validating the docking model by PELDOR spectroscopy. By exploiting the half-sites reactivity of RNR, diagonal distances between Y₁₂₂• on one α/β pair and DOPA₃₅₆•, NH₂Y₇₃₁•, NH₂Y₇₃₀•, and an active-site N• on the second α/β pair have been measured.^, The pathway residues and S were built in from the docking model, in which Y₃₅₆ is invisible.

Figure 9

Photo-initiated radical propagation in PO-Y-βC19 photopeptides. A photooxidant (PO, black circle) is appended to the N-terminus of a peptide corresponding to residues 356-375 of β2 (βC19, cyan), with either Y or F_nY at position 356. Light excitation (step 1) induces oxidation of the residue at position 356 (2), a fraction of which is reduced by Y₇₃₁ of α2 (3). The photopeptide/wt-α2 complex is capable of generating dNDP (4), providing direct evidence for radical injection from the peptide. Figure constructed from PDB ID 1RLR.

Figure 10

The 94 GHz [2H]-MIMS ENDOR spectrum of NH₂Y₇₃₀• in the active α2β2 complex reveals the details of its intra- and intermolecular H-bonds. Experimental data are shown in black and a simulation in red. The two prominent peaks centered around 0.6 MHz fall in a region expected for coupled H bonds. The detailed analysis of the experimental spectrum and its simulation is described in reference 21.

Figure 11

Solution peak potentials (E_ps) of Y, W, and UAAs blocked with N-acetyl and C-amide functionalities.^,

Figure 12

Assembly of the diferric-Y₁₂₂• (or NO₂Y₁₂₂• or F_nY₁₂₂•) cofactor from diferrous-β2, O₂ and reductant.

Figure 13

(a) X-band EPR spectra (77 K) for E. coli Y₁₂₂• and F_nY₁₂₂•s normalized for radical concentration. Dashed lines highlight the increased spectral width of the F_nY₁₂₂•s.^, (b) EPR spectrum of the reaction of Y₁₂₂(2,3,5)F₃Y-β2, wt-α2, CDP, and ATP quenched at 25 s. The reaction spectrum (black) is a composite of two species: 2,3,5-F₃Y₁₂₂• (pink) and a new radical (blue, magnified in inset), assigned as Y₃₅₆•.

Scheme 1

Kinetic model for radical initiation in the reaction of Y₁₂₂NO₂Y-β2 with wt-α2, S, and E.