Semiquinone-induced maturation of Bacillus anthracis ribonucleotide reductase by a superoxide intermediate

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides, and represent the only de novo pathway to provide DNA building blocks. Three different classes of RNR are known, denoted I-III. Class I RNRs are heteromeric proteins built up by α and β subunits and are further divided into different subclasses, partly based on the metal content of the β-subunit. In subclass Ib RNR the β-subunit is denoted NrdF, and harbors a manganese-tyrosyl radical cofactor. The generation of this cofactor is dependent on a flavodoxin-like maturase denoted NrdI, responsible for the formation of an active oxygen species suggested to be either a superoxide or a hydroperoxide. Herein we report on the magnetic properties of the manganese-tyrosyl radical cofactor of Bacillus anthracis NrdF and the redox properties of B. anthracis NrdI. The tyrosyl radical in NrdF is stabilized through its interaction with a ferromagnetically coupled manganese dimer. Moreover, we show through a combination of redox titration and protein electrochemistry that in contrast to hitherto characterized NrdIs, the B. anthracis NrdI is stable in its semiquinone form (NrdIsq) with a difference in electrochemical potential of ∼110 mV between the hydroquinone and semiquinone state. The under anaerobic conditions stable NrdIsq is fully capable of generating the oxidized, tyrosyl radical-containing form of Mn-NrdF when exposed to oxygen. This latter observation strongly supports that a superoxide radical is involved in the maturation mechanism, and contradicts the participation of a peroxide species. Additionally, EPR spectra on whole cells revealed that a significant fraction of NrdI resides in its semiquinone form in vivo, underscoring that NrdIsq is catalytically relevant.

Keywords: Bacillus; Cyclic Voltammetry; Free Radicals; Manganese; Maturase; NrdF; NrdI; Ribonucleotide Reductase; Semiquinone; Superoxide Ion.

FIGURE 1.

B. anthracis NrdI reduced through titration with dithionite. Top: formation of the semiquinone state (NrdI_sq, thick black line) from NrdI_q (dashed line); Bottom: continued titration resulting in the formation of the fully reduced hydroquinone state (NrdI_hq, dotted black line). The concentration of NrdI was 20 μm in 50 mm Tris-HCl buffer, pH 7.0.

FIGURE 2.

A, cyclic voltammetry traces of B. anthracis NrdI and of free FMN. Recorded under anaerobic conditions using a graphite electrode in 50 mm Tris-HCl, pH 7.6. [NrdI] = 180 μm, scan rate 60 mV s⁻¹ (a); [NrdI] = 180 μm, scan rate 100 mV s⁻¹ (b); [FMN] = 150 μm, scan rate 10 mV s⁻¹ (c). B, cyclic square wave voltammetry traces of B. anthracis NrdI (solid black line) and of free FMN (solid gray line). Recorded under anaerobic conditions using a gold electrode in 50 mm Tris-HCl, pH 7.6. a and a′ anodic and cathodic scans, respectively, of NrdI (180 μm); b and b′ anodic and cathodic scans respectively of free FMN (150 μm). Square wave voltammograms were obtained using a square wave frequency of 10 Hz, pulse height = 25 mV, pulse width = 50 ms, and step height = 15 mV.

FIGURE 3.

X-band EPR spectra of B. anthracis NrdI_sqin vivo and in vitro. In vivo EPR recorded on a frozen cell pellet obtained prior to IPTG addition (a); and following 4 h overexpression of NrdI in E. coli (b); NrdI_sq generated in vitro through dithionite reduction (c). The spectra were recorded at 98K under non-saturating conditions. In a and b recording conditions were identical, (c) has been scaled to the same signal amplitude as for NrdI_sq in b for comparison.

FIGURE 4.

X-band EPR spectra of B. anthracis Mn^III₂-Y·-NrdF at 77K (A) and in the temperature interval T = 6.7 to 40 K (T = 6.7, 10, 14, 20, 25, 35, and 40 K) (B). Arrows in Fig. 4B indicate the temperature dependence at the five lines of the “split tyrosyl radical” with increasing temperature. Recording conditions: modulation amplitude, 0.5 mT; 4 scans; microwave power was 10 milliwatt in A and 20 milliwatt in B.

FIGURE 5.

Power of half saturation (P_1/2) for the Mn and Fe reconstituted forms of B. anthracis NrdF and for E. coli NrdB. B. anthracis Mn^III₂-Y·-NrdF (squares); purified Mn^III₂-Y·-NrdF (half-filled square); Fe^III₂-Y·-NrdF (triangles); E. coli Fe^III₂-Y·-NrdB (filled circles). Dashed lines are extrapolation from the fitted curves for the different relaxation regimes for Fe^III₂-Y·-NrdF.

FIGURE 6.

UV-visible absorption spectra of Mn reconstituted NrdF and NrdI_q. Starting spectrum containing NrdI_q (100 μm), NrdF (200 μm), and Mn^II (300 μm) in Tris-HCl, pH 7.0 (dashed line, a); Formation of Mn^III₂-Y·-NrdF (≈6 μm) from NrdI_sq (black solid line, b); Formation of Mn^III₂-Y·-NrdF (≈10 μm) from NrdI_hq (gray solid line, c). Inset: absorption difference spectra (obtained by subtraction of the starting spectrum from the respective end spectra) highlighting changes attributable to the formation of Mn^III₂-Y·. Oxidation product formed from NrdI_sq (b-a) (black line); oxidation product formed from NrdI_hq (c-a) (gray line).

FIGURE 7.

UV-visible spectra following the reoxidation of NrdI_hq in the presence of Mn^II₂-NrdF. Starting spectrum containing NrdI_q (38 μm), NrdF (65 μm), and Mn^II (100 μm) in Tris-HCl, pH 7.0 (dotted black line); NrdI reduced to NrdI_hq (dashed black line); Spectra recorded during the re-oxidation of NrdI_hq, showing the appearance and disappearance of NrdI_sq (solid lines). Direction of change indicated by arrows, initial changes observed 10 s after exposure to oxygen (dashed arrows); subsequent changes during 15 min (solid arrows). Spectra recorded after 10, 80, 150, 240, 600, and 900 s. Inset: absorbance at 572 nm followed over time as a measure of NrdI_sq concentration (triangles, left Y-axis); Yield of Mn^III₂-Y·-NrdF with regards to NrdF concentration (circles, right Y-axis), time points as above.

SCHEME 1.

Suggested reaction pathway for NrdI-catalyzed maturation of Mn-NrdF, where Mn-NrdF is activated by a superoxide radical formed from NrdI_sq. Alternatively NrdI_hq can generate either two equivalents of the active O₂^⨪ species (right), or regenerate NrdI_q through formation of one equivalent of the unproductive H₂O₂ (left, dashed line). Note that the scheme is not balanced with regard to protons.