Use of Noncanonical Tyrosine Analogues to Probe Control of Radical Intermediates during Endoperoxide Installation by Verruculogen Synthase (FtmOx1)

Abstract

Important bioactive natural products, including prostaglandin H₂ and artemisinin, contain reactive endoperoxides. Known enzymatic pathways for endoperoxide installation require multiple hydrogen-atom transfers (HATs). For example, iron(II)- and 2-oxoglutarate-dependent verruculogen synthase (FtmOx1; EC 1.14.11.38) mediates HAT from aliphatic C21 of fumitremorgin B, capture of O₂ by the C21 radical (C21•), addition of the peroxyl radical (C21-O-O•) to olefinic C27, and HAT to the resultant C26•. Recent studies proposed conflicting roles for FtmOx1 tyrosine residues, Tyr224 and Tyr68, in the HATs from C21 and to C26•. Here, analysis of variant proteins bearing a ring-halogenated tyrosine or (amino)phenylalanine in place of either residue establishes that Tyr68 is the hydrogen donor to C26•, while Tyr224 has no essential role. The radicals that accumulate rapidly in FtmOx1 variants bearing a HAT-competent tyrosine analog at position 68 exhibit hypsochromically shifted absorption and, in cases of fluorine substitution, ¹⁹F-coupled electron-paramagnetic-resonance (EPR) spectra. By contrast, functional Tyr224-substituted variants generate radicals with unaltered light-absorption and EPR signatures as they produce verruculogen. The alternative major product of the Tyr68Phe variant, which forms competitively with verruculogen also in wild-type FtmOx1 in ²H₂O and in the variant with the less readily oxidized 2,3-F₂Tyr at position 68, is identified by mass spectrometry and isotopic labeling as the 26-hydroxy-21,27-endoperoxide compound formed after capture of another equivalent of O₂ by the longer lived C26•. The results highlight the considerable chemical challenges the enzyme must navigate in averting both oxygen rebound and a second O₂ coupling to obtain verruculogen selectively over other possible products.

Keywords: 2-oxoglutarate; enzyme mechanism; ferryl; fumitremorgin B endoperoxidase; hydrogen atom transfer; iron; oxygenase; tyrosyl radical.

Figure 1.. Structure and function of verruculogen synthase (FtmOx1).

(A) Reaction(s) catalyzed by FtmOx1 and its Y68F variant, in which partitioning depends on [O₂] and the presence or absence of reductant (ascorbate). (B) Blow-up of the active site in the FtmOx1 reactant complex (PDB entry: 7ETK). The facial triad is shown in green, iron(II) and 2OG are shown in orange, fumitremorgin B (1) is shown in magenta, the two contentious Tyr residues are highlighted in yellow,^, and distances relevant to the three HAT steps are shown with salmon dashed lines. The structure of the ternary complex is shown in Figure S1. (C) General fates of carbon-centered radicals of relevance to the FtmOx1 reactions. (D) Mechanisms proposed in reference and this work for substrate endoperoxidation (orange arrows) and hydroxylation (green arrow) by FtmOx1. The key HAT step (Tyr68 → C26•) of interest is highlighted by a red box. A complete scheme relating the CarC-like and COX-like catalytic cycles is shown in Figure S2.

Figure 2.. Experimental strategies to characterize the key HAT step (Tyr68 → C26•) in the presence of the H• donor and the alternative product 5 that can form when this the key HAT step is impeded.

(A) Wild-type (wt) enzyme predominantly generates 2 accompanying Tyr68• under single-turnover conditions. (B) Replacement of Tyr68 with Phe diverts the reaction toward an alternative product 5 with no transient Tyr• detected. (C) Replacement of Tyr68 or Tyr224 with non-canonical tyrosine analogues (3-FY, 3,5-F₂Y, 2,3-F₂Y, 3-ClY, and 4-NH₂F; only ring fluorination is shown in the scheme) allows for unambiguous identification of the radical harboring tyrosine (Tyr68). This strategy can also perturb the H•-donation ability of the residue and the partition between 2 and 5. (D and E) D₂O leads to deuterium on the phenol, slowing donation. The partition ratio between 2 and 5 reflects competition between the HAT step (dominant in wt) and the second O₂ capture (dominant in the Y68F variant). The isotopic distribution of 2 and 5 reinforces Tyr68 as the key donor and reveals the lack of newly formed C–D bond in 5. The solvent exchangeable sites in the products are shown with protia, as detected with LC-MS, in panels D and E. (F) Reaction of the Y68F variant with ¹⁸O₂ generates 5 with three ¹⁸O atoms, and the LC-MS² analysis reveals the identity of 5.

Figure 3.. LC-MS chromatograms from the reactions of wild-type (wt) FtmOx1 and its (A) Tyr68- and (B) Tyr224-substituted variants.

The traces are color-coded as in Figure S3 according to the tyrosine analog at position 68 or 224. The reactions contained 10 μM enzyme, 10 μM Fe(II), 1 mM 2OG, 1 mM ascorbate, 0.50 mM 1, and ~ 1.8 mM O₂, pH 8.0 and were carried out at room temperature as described in Materials and Methods (Supporting Information).

Figure 4.. SF-abs (left) and X-band FQ-EPR spectra (30 K, right) of the early radical intermediates in the reactions of wild-type (wt) FtmOx1 and variants that support their accumulation.

(A, B) Spectra from the reactions of the wt, Y68(3-FY), and Y68(3,5-F₂Y) proteins, all at 350 ms. (C, D) Spectra from the reactions of the wt protein at 350 ms, the Y68(4-NH₂F) variant at 270 ms, and the Y68(3-ClY) variant at 420 ms. (E, F) Spectra from the reactions of the wt protein at 350 ms, the Y224(4-NH₂F) variant at 640 ms, and the Y224(3-ClY) variant at 750 ms. The reaction times for variants were selected to coincide with the time of maximal accumulation. The spectra from the wt FtmOx1 reaction are reproduced in each panel for ease of comparison. The absorption spectra have been scaled to coincide at the peak maxima, and the EPR spectra have been scaled to have equal double-integrated intensity; the scaling factors with respect to the wt spectra are noted in each panel. Quantification of the radical in the FQ samples from the reaction of wt FtmOx1 revealed that it accumulates to 25 (±1) % of the iron concentration at 350 ms (assuming a packing factor of 0.52 – 0.55). The absorption was directly extracted from the same SF-abs data sets used in preparing Figure S5, and the reaction conditions in the FQ-EPR experiments were the same as in the stopped-flow experiments (see Figure S5 caption).

Figure 5.. LC-MS characterization of products derived from 1 in reactions of wild-type FtmOx1 and its Y68F variant in H₂O (pH 8.0) and D₂O (pD 8.0).

(A) Total-ion chromatograms (TICs) of products from the reactions of wt FtmOx1 in H₂O (solid blue trace), wt enzymes in D₂O (dashed cyan trace), the Y68F variant in H₂O (solid red trace), and the Y68F variant in D₂O (dashed orange trace). The color code also applies to panels B and C. (B) Isotope analysis from EICs of 1, 2, and 5 formed in the reactions of panel A. Traces of 2 from the reaction of the Y68F variant are magnified to better illustrate the isotopic distributions. (C) Relative abundances of the isotopologues of 1, 2, and 5 extracted from panel B. The reactions contained 10 μM enzyme, 10 μM Fe(II), 1 mM 2OG, 1 mM ascorbate, 0.50 mM 1, and ~ 1.8 mM O₂, pH/pD 8.0 at room temperature.

Figure 6.. LC-MS characterization of products derived from 1 in reactions of wild-type FtmOx1 and its Y68F variant with ¹⁸O₂.

(A) TICs from the reaction of the wt (blue trace) and Y68F (red trace) enzymes. The same color code applies to panels B and C. (B) Isotope analysis from the EICs in A of 1, 2, and 5 produced in these reactions. Traces for 2 from the Y68F variant are magnified to better illustrate the isotope distributions. (C) Relative abundances for isotopologues of 1, 2, and 5 extracted from panel B. The reactions contained 510 μM enzyme, 510 μM Fe(II), 2.5 mM 2OG, 250 μM ascorbate, 0.25 mM 1, and ~ 1.0 mM ¹⁸O₂, pH 8.0 at room temperature.

Figure 7.. Origins of the dominant 1-derived products (except for 3, which is shown in Figure 1D) and the fate of the substrate radical.

The rate constants and equilibrium constant are labeled at each critical step, and the predicted product ratio is governed by the oxygen concentration [O₂], the rebound rate constant (k_rebound), the HAT rate constant (k_HAT), and various rate constants for oxygen addition, dissociation, and cyclization. The equation assumes rapid equilibria among the radical intermediates (see text).