Radical SAM catalysis via an organometallic intermediate with an Fe-[5'-C]-deoxyadenosyl bond

Abstract

Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to cleave SAM to initiate diverse radical reactions. These reactions are thought to involve the 5'-deoxyadenosyl radical intermediate, which has not yet been detected. We used rapid freeze-quenching to trap a catalytically competent intermediate in the reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme. Characterization of the intermediate by electron paramagnetic resonance and (13)C, (57)Fe electron nuclear double-resonance spectroscopies reveals that it contains an organometallic center in which the 5' carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster. Discovery of this intermediate extends the list of enzymatic bioorganometallic centers to the radical SAM enzymes, the largest enzyme superfamily known, and reveals intriguing parallels to B12 radical enzymes.

Fig. 1

Activation of PFL by PFL-AE, with concomitant cleavage of SAM to methionine and 5′-deoxyadenosine.

Fig. 2

EPR spectra showing the formation of the PFL glycyl radical (G•) from Ω. (A, upper) EPR spectra of mixture of photoreduced PFL-AE and PFL/SAM freeze-quenched at ∼77K (500 ms) and stored at 77 K, and then annealed at progressively higher T for indicated times (See SI). At 12 K, the spectrum of G• radical is highly saturated and its amplitude diminished; at 40 K, signal from rapidly relaxing Ω is correspondingly diminished. The spectra here have had the residual intensities at both temperatures subtracted out (See SI, Fig. S2), with one exception. Ω is completely lost after annealing at 220 K; the dashed curve shows the residual signal from saturated G• radical. (A, lower) Populations of Ω and G• radical relative to the final (220 K) G• radical concentration taken as 100%, as derived from EPR spectra (See SI). (B) X-band EPR spectra for photoreduced PFL-AE freeze-quenched (77K) 500 ms after mixing with PFL G734A/SAM, with spectra collected at 12 and 40 K. Conditions: microwave frequency = 9.23 GHz, microwave power = 1 mW, 100 kHz modulation amplitude = 8 G, T, as indicated; the gain at a given T is fixed.

Fig. 3

35 GHz ENDOR spectra at g_⊥ for photoreduced PFL-AE freeze-quenched with PFL/SAM (See SI for details). To first order, an ENDOR spectrum of an I = 1/2 nucleus (N) in a frozen solution comprises a superposition of signals from different orientations, each signal a doublet at frequencies, ν_± = |ν(N) ± A/2|, where ν(N) is the nuclear larmor frequency and A is the orientation-dependent hyperfine coupling.(23) For ¹³C, A/2 ≪ ν(¹³C) and it is convenient to plot spectra vs ν - ν(¹³C). For ⁵⁷Fe, ν(⁵⁷Fe) ≪ A/2 and spectra are plotted vs ν.(A) ¹³C CW ENDOR for [adenosyl-¹³C₁₀] SAM. Best match simulation to axial hyperfine tensor (See SI), green dash line. Simulation parameters, a_iso = 9.4 MHz, 2T = 5.3 MHz and β = 90°. Conditions: microwave frequency = 35.39 GHz, microwave power = 1 mW, 100 kHz modulation amplitude = 1.3 G, rf sweep rate = 1 MHz/s and T = 2 K. Inset: Mims ENDOR spectrum. Conditions: microwave frequency = 35.20 GHz, MW pulse length, (π/2) = 50 ns, τ = 500 ns and T = 2 K. (B) Mims ENDOR spectrum from [methyl-¹³C] SAM. Conditions: microwave frequency = 35.08 GHz, MW pulse length, (π/2) = 50 ns, τ = 500 ns and T = 2 K. (C) ⁵⁷Fe CW ENDOR for ⁵⁷Fe enriched Ω (prepared using [adenosyl-¹³C₁₀] SAM) and photoreduced PFL-AE. Upper: CW ENDOR spectra for ⁵⁷Fe-enriched (red) and natural abundance (gray) rfq samples. Lower: Frequency sweep and randomly hopped stochastic CW ENDOR spectra (23) for ⁵⁷Fe-enriched reduced PFL-AE. Conditions: microwave frequency = 35.45 GHz and 35.07GHz for rfq and ⁵⁷Fe-enriched reduced PFL-AE, respectively, microwave power = 1 mW, 100 kHz modulation amplitude = 1.3 G, rf sweep rate = 1 MHz/s, stochastic CW ENDOR cycle; rf-on = 3 ms, rf-off = 1 ms, sample collection time = 3 ms, and T = 2 K.

Fig. 4

Model for bio-organometallic intermediate, Ω. Whether methionine remains coordinated to the unique iron is not currently known.