Radical-translocation intermediates and hurdling of pathway defects in "super-oxidized" (Mn(IV)/Fe(IV)) Chlamydia trachomatis ribonucleotide reductase

Abstract

A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y(•)) or a Mn(IV)/Fe(III) cluster in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit, generating a thiyl radical that abstracts hydrogen (H(•)) from the substrate. With either oxidant, the inter-subunit "hole-transfer" or "radical-translocation" (RT) process is thought to occur by a "hopping" mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wild-type (wt) RNR. The Mn(IV)/Fe(III) cofactor of Chlamydia trachomatis RNR assembles via a Mn(IV)/Fe(IV) intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when α is present, accumulating radicals with EPR spectra characteristic of Y(•)'s. The dependence of Y(•) accumulation on the presence of substrate suggests that RT within this "super-oxidized" enzyme form is gated by the protein, and the failure of a β variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y(•)(s) in the wt enzyme reside(s) within the RT pathway. Remarkably, two variant β proteins having pathway substitutions rendering them inactive in their Mn(IV)/Fe(III) states can generate the pathway Y(•)'s in their Mn(IV)/Fe(IV) states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the "hurdling" of engineered defects in the RT pathway.

Figure 1

X-band EPR spectroscopic evidence for Y• accumulation in the super-oxidized Ct RNR holoenzyme complex. At ambient temperature (22 ± 2 °C), an O₂-free solution of β (∼ 2 mM) containing 0.75 equiv each of Mn^II and Fe^II was mixed with a solution containing the other RNR reaction components to give final concentrations of: 0.2 mM β; 0 (i) or 0.4 mM (ii-vi) α; 0 (i-iii) or 1 mM (iv-vi) CDP; and 0 (i, ii, and iv) or 0.5 mM (iii, v, and vi) ATP. Reaction solutions were transferred to an EPR tube and frozen in cold iso-pentane (T∼ 125 K) 5 seconds after being thoroughly mixed. (A) Delineation of the requirements for radical formation, with spectra acquired over a wide range of the magnetic field. The spectrometer conditions were: T = 14 ± 0.2 K, microwave frequency = 9.47 GHz, microwave power = 20 μW, modulation frequency = 100 KHz, modulation amplitude = 10 G, time constant = 167 ms, scan time = 167 s. (B) Spectra acquired over a narrow range of the magnetic field with a smaller modulation amplitude (2 G) and 5 scans summed per spectrum to provide better resolution of the g ∼ 2 region. In B, the contribution of the spectrum of the Mn^IV/Fe^IV complex has been subtracted to resolve the signal of the organic radical component.

Figure 2

Tests of two possible explanations for the persistence of a fraction of the Mn^IV/Fe^IV complex even after maximal accumulation of the putative Y•(s). (A) Kinetics of decay of the putative Y•(s) (circles) and the Mn^IV/Fe^IV complex (squares). Traces were fit by the equation describing a single-exponential decay process, giving rate constants of 0.017 s^-1 and 0.1 s^-1, respectively. The difference in decay rate constants rules out the possibility that the two species are in rapid equilibrium. (B) X-band EPR of samples prepared with ratios of α:β ranging from 1:1 to 6:1. Samples were prepared as described for Figure 1, except they contained 0.14 mM β and 0.14 – 0.84 mM α. The failures of the radical signal to increase and the Mn^IV/Fe^IV signal to be diminished rules out the possibility that weak binding between the two subunits results in a fraction of the Mn^IV/Fe^IV-β remaining unbound and unable to generate the radical. Spectrometer conditions are the same as for Figure 1.

Figure 3

X-band EPR spectra illustrating the effects of substitutions of residues in the two orthogonal electron-relay pathways in Ct β on the nature of accumulating radical species in reactions of the Mn^II/Fe^II-β complexes with O₂ in the presence of α, CDP, ATP, DTT, and MgSO₄. A schematic of the electron-relay pathways is provided at the left, with the non-native cluster redox state and substituted amino acid residues highlighted in red. Sample preparation was as described in the legend of Figure 1. For samples containing the Mn^IV/Fe^III states, each β protein was pre-treated by adding air-saturated 100 mM HEPES, 10% (v/v) glycerol buffer to the Mn^II/Fe^II-β complex, and incubating for 60 minutes at 5 °C. This solution was then mixed with the remaining components (α, CDP, ATP, MgSO₄, and DTT) and frozen after ∼ 5 s. A displays the “raw” experimental spectra, and, in B, the contribution of the Mn^IV/Fe^IV intermediate has been removed to resolve the spectra of the radical components. The red and blue dashed spectra in B are simulations with parameters provided in Supporting Information. Spectrometer conditions are provided in the legend of Figure 1.

Figure 4

X-band EPR spectra of samples employing the radical-trapping substrate analogue, 2′-azido-2′-deoxyuridine-5′-diphosphate (N₃-UDP), to provide evidence for the functional competence of the RT-pathway radicals in the super-oxidized enzyme for 3′-H• abstraction. (A) Raw experimental spectra acquired over a wide range of the magnetic field and (B) spectra acquired over a narrow range of the magnetic field and processed by subtraction of the contributions from either the Mn^III/Fe^III complex (i) or the Mn^IV/Fe^IV complex (ii, iv, and vi) to resolve the spectra of the organic-radical components. The method of sample preparation and the spectrometer conditions are the same as for Figures 1 and 2.

Figure 5

Product (dCDP) yields demonstrating the functional competence of RT-pathway radicals in the super-oxidized states of Ct β variants with pathway substitutions rendering them inactive for CDP reduction in their stable Mn^IV/Fe^III states. Reactions were initiated by mixing the indicated Mn^II/Fe^II-β complex or the pre-formed Mn^IV/Fe^III-β complex with an air-saturated solution containing the other reactions components, as in preparation of the EPR samples, but with [³H]-CDP (the site of labeling and specific activity are provided in the Experimental Procedures section) included as radioactive tracer for product quantification. After a one-minute reaction, samples were processed (as described in Procedures) based on a published method to separate substrate and product. The dCDP product was then quantified by scintillation counting.

Scheme 1

Orthogonal activation- and catalysis-specific electron-relay pathways in Ct RNR. At bottom is a simple schematic that is used throughout this work to assist in interpretation of the figures.