The 5′-deoxyadenosyl radical (5′-dAdo·) abstracts a substrate H atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S-adenosyl-L-methionine (RS) enzymes. Notwithstanding its central biological role, 5′-dAdo· has eluded characterization despite efforts spanning more than a half-century. Here, we report generation of 5′-dAdo· in a RS enzyme active site at 12 K using a novel approach involving cryogenic photoinduced electron transfer from the [4Fe–4S]+ cluster to the coordinated S-adenosylmethionine (SAM) to induce homolytic S–C5′ bond cleavage. We unequivocally reveal the structure of this long-sought radical species through the use of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies with isotopic labeling, complemented by density-functional computations: a planar C5′ (2pπ) radical (~70% spin occupancy); the C5′(H)2 plane is rotated by ~37° (experiment)/39° (DFT) relative to the C5′–C4′–(C4′–H) plane, placing a C5′–H antiperiplanar to the ribose-ring oxygen, which helps stabilize the radical against elimination of the 4′–H. The agreement between φ from experiment and in vacuo DFT indicates that the conformation is intrinsic to 5-dAdo· itself, and not determined by its environment.

Significance Methanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.

In nature, methane is oxidized to methanol by two enzymes, the iron-dependent soluble methane monooxygenase (sMMO) and the copper-dependent particulate MMO (pMMO). While sMMO's diiron metal active site is spectroscopically and structurally well-characterized, pMMO's copper sites are not. Recent EPR and ENDOR studies have established the presence of two monocopper sites, but the coordination environment of only one has been determined, that within the PmoB subunit and denoted Cu B . Moreover, this recent work only focused on a type I methanotrophic pMMO, while previous observations of the type II enzyme were interpreted in terms of the presence of a dicopper site. First, this report shows that the type II Methylocystis species strain Rockwell pMMO, like the type I pMMOs, contains two monocopper sites and that its Cu B site has a coordination environment identical to that of type I enzymes. As such, for the full range of pMMOs this report completes the refutation of prior and ongoing suggestions of multicopper sites. Second, and of primary importance, EPR/ENDOR measurements (a) for the first time establish the coordination environment of the spectroscopically observed site, provisionally denoted Cu C , in both types of pMMO, thereby (b) establishing the assignment of this site observed by EPR to the crystallographically observed metal-binding site in the PmoC subunit. Finally, these results further indicate that Cu C is the likely site of biological methane oxidation by pMMO, a conclusion that will serve as a foundation for proposals regarding the mechanism of this reaction.

Radical S-adenosyl-l-methionine (SAM) enzymes employ a [4Fe–4S] cluster and SAM to initiate diverse radical reactions via either H-atom abstraction or substrate adenosylation. Here we use freeze-quench techniques together with electron paramagnetic resonance (EPR) spectroscopy to provide snapshots of the reaction pathway in an adenosylation reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme on a peptide substrate containing a dehydroalanine residue in place of the target glycine. The reaction proceeds via the initial formation of the organometallic intermediate Ω, as evidenced by the characteristic EPR signal with g ∥ = 2.035 and g ⊥ = 2.004 observed when the reaction is freeze-quenched at 500 ms. Thermal annealing of frozen Ω converts it into a second paramagnetic species centered at g iso = 2.004; this second species was generated directly using freeze-quench at intermediate times (∼8 s) and unequivocally identified via isotopic labeling and EPR spectroscopy as the tertiary peptide radical resulting from adenosylation of the peptide substrate. An additional paramagnetic species observed in samples quenched at intermediate times was revealed through thermal annealing while frozen and spectral subtraction as the SAM-derived 5′-deoxyadenosyl radical (5′-dAdo•). The time course of the 5′-dAdo• and tertiary peptide radical EPR signals reveals that the former generates the latter. These results thus support a mechanism in which Ω liberates 5′-dAdo• by Fe–C5′ bond homolysis, and the 5′-dAdo• attacks the dehydroalanine residue of the peptide substrate to form the adenosylated peptide radical species. The results thus provide a picture of a catalytically competent 5′-dAdo• intermediate trapped just prior to reaction with the substrate.

Second harmonic generation amplitude and phase measurements are acquired in real time from fused silica:water interfaces that are subjected to ionic strength transitions conducted at pH 5.8. In conjunction with atomistic modeling, we identify correlations between structure in the Stern layer, encoded in the total second-order nonlinear susceptibility, 𝜒 !"! ($) , and in the diffuse layer, encoded in the product of 𝜒 !"! ($) and the total interfacial potential, 𝛷(0) !"! .𝜒 !"! ($) : 𝛷(0) !"! correlation plots indicate that the dynamics in the Stern and diffuse layers are decoupled from one another under some conditions (large change in ionic strength), while they change in lockstep under others (smaller change in ionic strength) as the ionic strength in the aqueous bulk solution varies. The quantitative structural and electrostatic information obtained also informs on the molecular origin of hysteresis in ionic strength cycling over fused silica.Atomistic simulations suggest a prominent role of contact ion pairs (as opposed to solventseparated ion pairs) in the Stern layer. Those simulations also indicate that net water alignment is limited to the first 2 nm from the interface, even at 0 M ionic strength, highlighting water's polarization as an important contributor to nonlinear optical signal generation.