Reported here are the competitive 18 O/ 16 O kinetic isotope effects ( 18 O KIEs) on k cat /K m (O 2 ) for three non-heme iron enzymes that activate O 2 at an iron center coordinated by a 2-His-1-carboxylate facial triad: taurine dioxygenase (TauD), S-(2)-hydroxypropylphosphonic acid epoxidase (HppE), and 1-aminocyclopropyl-1-carboxylic acid oxidase (ACCO). The comparison of the measured 18 O KIEs with calculated 18 O equilibrium isotope effects ( 18 O EIEs) reveals an excellent correlation with the proposed mechanisms for these enzymes. 18 O KIEs of 1.0104 ± 0.0002 (TauD), 1.0120 ± 0.0002 (HppE), and 1.0215 ± 0.0005 (ACCO) suggest the formation in the rate limiting step of O 2 activation of an Fe III -alkylperoxo, Fe III -OOH, and Fe IV =O species, respectively. By probing only the steps from initial O 2 binding up to and including the first irreversible step of O 2 activation, the measured 18 O KIEs can be a valuable companion to pre-steady state kinetic analyses in studying the complex catalytic mechanisms of non-heme iron enzymes.The O 2 -activating, non-heme iron enzymes catalyze a wide range of oxygenation and oxidation reactions with important biological implications, such as DNA repair, hypoxic response, collagen biosynthesis, and histone demethylation. 1 Most of these enzymes contain a single iron center coordinated by two His and one Asp/Glu residues in a tridentate binding motif referred to as "2-His-1-carboxylate facial triad". Understanding the O 2 -activation process for these enzymes may provide key insights into the source of their divergent substrate specificity despite similarly coordinated active site metal centers. HppE is a reductase-dependent non-heme iron enzyme that catalyzes the epoxidation of S-HPP, the last step in the biosynthesis of the antibiotic fosfomycin. 9 The mechanism of HppE is not as well known as for TauD, formation of an Fe III -OOH species being proposed to involve either a hydrogen atom transfer (HAT) from S-HPP or proton-coupled electron transfer (PCET) from the reductant. 10 The measured 18 O KIE for HppE is 1.0120 ± 0.0002 at 25 °C, using FMN in the presence of NADH as the reductant (Figure 1

Ferroptosis is a form of programmed cell death associated with inflammation, neurodegeneration, and ischemia. Vitamin E (alpha-tocopherol) has been reported to prevent ferroptosis, but the mechanism by which this occurs is controversial. To elucidate the biochemical mechanism of vitamin E activity, we systematically investigated the effects of its major vitamers and metabolites on lipid oxidation and ferroptosis in a striatal cell model. We found that a specific endogenous metabolite of vitamin E, alpha-tocopherol hydroquinone, was a dramatically more potent inhibitor of ferroptosis than its parent compound, and inhibits 15-lipoxygenase via reduction of the enzyme’s non-heme iron from its active Fe3+ state to an inactive Fe2+ state. Furthermore, a non-metabolizable isosteric analog of vitamin E which retains antioxidant activity neither inhibited 15-lipoxygenase nor prevented ferroptosis. These results call into question the prevailing model that vitamin E acts predominantly as a non-specific lipophilic antioxidant. We propose that, similar to the other lipophilic vitamins A, D and K, vitamin E is instead a pro-vitamin, with its quinone/hydroquinone metabolites responsible for its anti-ferroptotic cytoprotective activity.

Taurine ␣-ketoglutarate dioxygenase (tauD) is one of the beststudied ␣-ketoglutarate (␣KG)-dependent nonheme iron oxygenases. As with all oxygenases, a fine balance must be struck between generating a species sufficiently reactive for the required chemistry and controlling that species to prevent undesirable side reactions

The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical SAM (S-adenosyl-L-methionine) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center (PTC) of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr proceed by a shared radical-mediated mechanism. However, in addition to the radical SAM CX3CX2C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function. These conserved cysteine residues are putatively involved in a covalent intermediate employed by RlmN and Cfr in order to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further elucidate the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species formed between RlmN and its rRNA substrate and the roles of the conserved cysteine residues in catalysis.