Photoinduced Electron Transfer in a Radical SAM Enzyme Generates an S-Adenosylmethionine Derived Methyl Radical

Abstract

Radical SAM (RS) enzymes use S-adenosyl-l-methionine (SAM) and a [4Fe-4S] cluster to initiate a broad spectrum of radical transformations throughout all kingdoms of life. We report here that low-temperature photoinduced electron transfer from the [4Fe-4S]¹⁺ cluster to bound SAM in the active site of the hydrogenase maturase RS enzyme, HydG, results in specific homolytic cleavage of the S-CH₃ bond of SAM, rather than the S-C5' bond as in the enzyme-catalyzed (thermal) HydG reaction. This result is in stark contrast to a recent report in which photoinduced ET in the RS enzyme pyruvate formate-lyase activating enzyme cleaved the S-C5' bond to generate a 5'-deoxyadenosyl radical, and provides the first direct evidence for homolytic S-CH₃ bond cleavage in a RS enzyme. Photoinduced ET in HydG generates a trapped ^•CH₃ radical, as well as a small population of an organometallic species with an Fe-CH₃ bond, denoted Ω_M. The ^•CH₃ radical is surprisingly found to exhibit rotational diffusion in the HydG active site at temperatures as low as 40 K, and is rapidly quenched: whereas 5'-dAdo^• is stable indefinitely at 77 K, ^•CH₃ quenches with a half-time of ∼2 min at this temperature. The rapid quenching and rotational/translational freedom of ^•CH₃ shows that enzymes would be unable to harness this radical as a regio- and stereospecific H atom abstractor during catalysis, in contrast to the exquisite control achieved with the enzymatically generated 5'-dAdo^•.

Figure 1.

Reductive cleavage of SAM and the structure of HydG. (A) The SAM-cluster interaction in RS enzymes (left), and the three different radical products that can result from reductive S–C bond cleavage in SAM (right). Canonical RS enzymes cleave the S–C5′ bond (red) to generate the 5′-dAdo^• radical intermediate, while the noncanonical RS enzyme Dph2 cleaves the S–C(γ) bond (green) to yield an ACP radical fragment that adds to substrate. (B) Overview of the structure of HydG (PDB 4WCX), with the radical SAM cluster (top) and the auxiliary cluster (bottom) connected by a long active site tunnel (purple mesh).

Figure 2.

X-band EPR spectra of (A) ([4Fe–4S]⁺ + SAM) HydG complex before and (B) after photolysis at 12 K with 450 nm LED for 1 h. (C) Time course for decay of ([4Fe–4S]⁺ + SAM) (■) upon photolysis monitored at 3640 G, and increase of ^•CH₃ (●) monitored at 3306 G. The photo reaction appears to be complete at ~90 m, as the EPR spectrum does not change upon further photolysis. The two progress curves are then normalized and fit to stretched-exponential decay, I = exp(−[t/τ]ⁿ), and rise, I = 1 − exp(−[t/τ]ⁿ), functions with the same parameters, τ = 7 ± 1 min and n = 0.52 ± 0.02. In (B), the unchanged signal from [4Fe–4S]_AUX[(Cys)Fe] cluster and that from ^•CH₃ are both identified. EPR conditions: microwave frequency, 9.38 GHz; modulation 10 G; T = 12 K.

Figure 3.

EPR spectra (black) and simulations (red). (A) Spectrum of rapidly tumbling ^•CH₃ generated with natural abundance SAM, obtained by subtraction of the properly scaled signal from the persistent signal (see text). (B) ^•CD₃ generated with methyl-d₃-SAM. (C) ^•13CH₃ generated with ¹³C-methyl-SAM. Conditions: T = 40 K, modulation amplitude, 5 G; see Figure 4 for spectra with lower modulation. EasySpin simulation parameters: function, chili; g = [2.0015, 2.0015, 2.0055,], (g_iso = 2.003). For spectra (A) and (C), A(¹Ha,b,c) = −[86, 44, 62] MHz (a_iso(¹Ha,b,c) = −63.3 MHz), (α,β,γ) = (120, 0, 0) for 1Ha, (α,β,γ) = (−120, 0, 0) for 1Hb, (α,β,γ) = (0, 0, 0) for 1Hc; for spectrum (C), A(¹³C) = [−10, −10, 260] MHz. τ_C = 3.2 ns; uniform Gaussian line widths 21 MHz.

Figure 4.

X-band EPR spectra. (A) Ω_M (¹²CH₃); (B) Ω_M (¹³CH₃); (C) 35 GHz ¹³C CW ENDOR Spectra of Ω_M (¹³CH₃). Inverted triangle, ¹³C Larmor frequency; “goalpost”, half the hyperfine coupling, a_iso/2. EPR conditions: T = 40 K; modulation amplitude, 5 G; microwave frequency, 9.38 GHz. ENDOR conditions: T = 2 K; modulation amplitude, 4 G; scan speed, 3 MHz/s; scan direction, forward; microwave frequency, 34.8 GHz. EasySpin EPR simulations: function, pepper; g = [2.021, 2.0047,1.987]; Gaussian line widths 30 MHz; for spectrum (B), A(¹³C) = [20, 30, 20] MHz; H_strain = [60, 30, 60] MHz.

Figure 5.

Temperature dependence of the ^•CH₃ signal. Conditions: modulation amplitude, 2G to avoid modulation broadening in the higher-T spectra; power adjusted at each temperature to avoid saturation; because of this adjustment, the intensities are simply normalized to equal height.