Geometric and electronic structure of the Mn(IV)Fe(III) cofactor in class Ic ribonucleotide reductase: correlation to the class Ia binuclear non-heme iron enzyme

Abstract

The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) utilizes a Mn/Fe heterobinuclear cofactor, rather than the Fe/Fe cofactor found in the β (R2) subunit of the class Ia enzymes, to react with O2. This reaction produces a stable Mn(IV)Fe(III) cofactor that initiates a radical, which transfers to the adjacent α (R1) subunit and reacts with the substrate. We have studied the Mn(IV)Fe(III) cofactor using nuclear resonance vibrational spectroscopy (NRVS) and absorption (Abs)/circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD spectroscopies to obtain detailed insight into its geometric/electronic structure and to correlate structure with reactivity; NRVS focuses on the Fe(III), whereas MCD reflects the spin-allowed transitions mostly on the Mn(IV). We have evaluated 18 systematically varied structures. Comparison of the simulated NRVS spectra to the experimental data shows that the cofactor has one carboxylate bridge, with Mn(IV) at the site proximal to Phe127. Abs/CD/MCD/VTVH MCD data exhibit 12 transitions that are assigned as d-d and oxo and OH(-) to metal charge-transfer (CT) transitions. Assignments are based on MCD/Abs intensity ratios, transition energies, polarizations, and derivative-shaped pseudo-A term CT transitions. Correlating these results with TD-DFT calculations defines the Mn(IV)Fe(III) cofactor as having a μ-oxo, μ-hydroxo core and a terminal hydroxo ligand on the Mn(IV). From DFT calculations, the Mn(IV) at site 1 is necessary to tune the redox potential to a value similar to that of the tyrosine radical in class Ia RNR, and the OH(-) terminal ligand on this Mn(IV) provides a high proton affinity that could gate radical translocation to the α (R1) subunit.

Figure 1

(a) NRVS spectra of the Mn^IVFe^III cofactor; and representative simulations for structures having (b) two carboxylate bridges (bridging E₂₂₇); (c) one carboxylate bridge (terminal E₂₂₇) with Fe^III at site 1; and (d) one carboxylate bridge (terminal E₂₂₇) with Mn^IV at site 1.

Figure 2

(Left) Spectra obtained from NRVS (experimental, top; simulated, bottom) and (Right) related core motions contributing to the features in the simulated spectrum.

Figure 3

Abs, CD and MCD spectra of the Mn^IVFe^III cofactor in Ct RNR-β (R2). The arrows indicate the energies where the VTVH data were collected, and the * indicates a minor heme contaminant in the sample.

Figure 4

VTVH data for different bands, as indicated in Figure 3. Fits producing |D| = 2.3 cm⁻¹E/D = 0.33 are shown as black lines.

Figure 5

(Left) Comparison of the energies of the calculated d-d transitions (from TD-DFT) for four different structural models of the Mn^IVFe^III cofactor (a–d) and the experimental d-d transitions from MCD spectra, and (Right) relative Mn^IV d orbital energies for each structural model.

Figure 6

LMCT Abs spectra of Mn^IVFe^III RNR-β (the three lowest CT bands are labeled bands 5–7) correlated to TD-DFT calculated µ-oxo → Mn^IV (blue), terminal OH⁻ → Mn^IV (green) and µ-oxo → Fe^III (red) CT transitions for two possible structures, with either OH⁻ or H₂O as the terminal ligand on Mn at site 1. The orbitals for (i), (ii), (iii) transitions are shown in Figure 7 (vide infra).

Figure 7

LMCT processes associated with pseudo-A term features. For pseudo-A term MCD behavior, there must be 2 perpendicular CT transitions and SOC along a 3rd mutually-orthogonal direction. SOC is a single center 1e⁻ operator. Therefore, 2 CTs must be “from” the same orbital or “to” the same orbital. Among the CT transitions in the TD-DFT calculations, there are 3 transitions (i, ii, iii) involving orbitals which could SOC via a 3rd perpendicular direction. For Mn-centered transitions, the terminal OH⁻ → Mn CT transition is x-polarized, and the µ-oxo → Mn CT transition is z-polarized. The ligand donor orbitals are mixed with d_xy and d_yz, which SOC via L_y (bottom left). For µ-oxo ligand-centered transitions, the CT to Mn is z-polarized and the transition to Fe is y-polarized. These metal d orbitals are mixed with the O_pz and O_py ligand orbitals, which SOC via L_x (bottom right)

Figure 8

Calculated ZFS tensor orientation and intensity for the Mn^IVFe^III cofactor. Initial ZFS orientation is shown with blue dotted arrows, where x and y orientations rotate to red arrows due to the antisymmetric ZFS arising from hetero-bimetallic center.

Figure 9

DFT-optimized structural model for the Mn^IVFe^III cofactor from this study.

Scheme 1

(a) Function of the Mn^IVFe^III cofactor in catalysis by a class Ic ribonucleotide reductase: substrate reduction in α (R1) following radical initiation by Mn^IVFe^III cofactor in β (R2) through a radical-translocation process; (b) structure of the Mn^IVFe^III cofactor site in class Ic ribonucleotide reductase (x = 1 or 2, M1 = metal site 1, and M2 = metal site 2); and (c) structure of the Fe^IIIFe^IV intermediate X in class Ia ribonucleotide reductase (x = 1 or 2).