Catching a radical in action: Many enzymes catalyze reactions through the production of radical intermediates. Radical SAM enzymes, the largest superfamily of enzymes in nature, do this by using an iron-sulfur cluster to cleave S-adenosylmethionine and produce a radical intermediate. Using freeze quenching, Horitani et al. were able to trap a previously unseen radical intermediate from bacterial pyruvate formate-lyase activating enzyme. Spectroscopy revealed that the intermediate consists of a short-lived covalent bond between the terminal carbon of 5′-deoxyadenosyl and the single iron atom of the iron-sulfur cluster. Not only does the observation of this radical expand our mechanistic understanding of radical SAM enzymes, but it expands the range of enzyme active sites or cofactors that function through an organometallic center. Science , this issue p. 822

In enzymatic C–H activation by hydrogen tunneling, reduced barrier width is important for efficient hydrogen wave function overlap during catalysis. For native enzymes displaying nonadiabatic tunneling, the dominant reactive hydrogen donor–acceptor distance (DAD) is typically ca. 2.7 Å, considerably shorter than normal van der Waals distances. Without a ground state substrate-bound structure for the prototypical nonadiabatic tunneling system, soybean lipoxygenase (SLO), it has remained unclear whether the requisite close tunneling distance occurs through an unusual ground state active site arrangement or by thermally sampling conformational substates. Herein, we introduce Mn²⁺ as a spin-probe surrogate for the SLO Fe ion; X-ray diffraction shows Mn-SLO is structurally faithful to the native enzyme. ¹³C ENDOR then reveals the locations of ¹³C10 and reactive ¹³C11 of linoleic acid relative to the metal; ¹H ENDOR and molecular dynamics simulations of the fully solvated SLO model using ENDOR-derived restraints give additional metrical information. The resulting three-dimensional representation of the SLO active site ground state contains a reactive (a) conformer with hydrogen DAD of ∼3.1 Å, approximately van der Waals contact, plus an inactive (b) conformer with even longer DAD, establishing that stochastic conformational sampling is required to achieve reactive tunneling geometries. Tunneling-impaired SLO variants show increased DADs and variations in substrate positioning and rigidity, confirming previous kinetic and theoretical predictions of such behavior. Overall, this investigation highlights the (i) predictive power of nonadiabatic quantum treatments of proton-coupled electron transfer in SLO and (ii) sensitivity of ENDOR probes to test, detect, and corroborate kinetically predicted trends in active site reactivity and to reveal unexpected features of active site architecture.

Significance: Nitrogenase catalyzes N ₂ reduction to ammonia, the largest N input into the biogeochemical nitrogen cycle. This difficult reaction involves delivery of electrons from the Fe protein component to the catalytic MoFe protein component in a process that involves hydrolysis of two ATP per electron delivered. MoFe contains two catalytic halves, each of which binds an Fe protein. The prevailing picture has been that the two halves function independently. Here, it is demonstrated that electron transfer (ET) in the two halves exhibits negative cooperativity: Fe→MoFe ET in one-half partially suppresses ET in the other. These findings thus show that conformational coupling in nitrogenase not only gates ET within each half, as shown previously, but introduces negative cooperativity between the two halves.

Transfer RNA (tRNA) is an adaptor molecule indispensable for assigning amino acids to codons on mRNA during protein synthesis. 2-thiouridine (s²U) derivatives in the anticodons (position 34) of tRNAs for glutamate, glutamine, and lysine are post-transcriptional modifications essential for precise and efficient codon recognition in all organisms. s²U34 is introduced either by (i) bacterial MnmA/eukaryote mitochondrial Mtu1 or (ii) eukaryote cytosolic Ncs6/archaeal NcsA, and the latter enzymes possess iron-sulfur (Fe–S) cluster. Here, we report the identification of novel-type MnmA homologs containing three conserved Cys residues, which could support Fe–S cluster binding and catalysis, in a broad range of bacteria, including thermophiles, Cyanobacteria, Mycobacteria, Actinomyces, Clostridium, and Helicobacter. Using EPR spectroscopy, we revealed that Thermus thermophilus MnmA (TtMnmA) contains an oxygen-sensitive [4Fe–4S]-type cluster. Efficient in vitro formation of s²U34 in tRNA^Lys and tRNA^Gln by holo-TtMnmA occurred only under anaerobic conditions. Mutational analysis of TtMnmA suggested that the Fe–S cluster is coordinated by the three conserved Cys residues (Cys105, Cys108, and Cys200), and is essential for its activity. Evolutionary scenarios for the sulfurtransferases, including the Fe–S cluster containing Ncs6/NcsA s²U thiouridylases and several distantly related sulfurtransferases, are proposed.