The iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases catalyze an array of challenging transformations, but how individual members of the enzyme family direct different outcomes is poorly understood. The Fe/2OG halogenase, SyrB2, chlorinates C4 of its native substrate, L-threonine appended to the carrier protein, SyrB1, but hydroxylates C5 of L-norvaline and, to a lesser extent, C4 of L-aminobutyric acid when SyrB1 presents these non-native amino acids. To test the hypothesis that positioning of the targeted carbon dictates the outcome, we defined the positions of these three substrates by measuring hyperfine couplings between substrate deuterium atoms and the stable, EPR-active iron-nitrosyl adduct, a surrogate for reaction intermediates. The Fe-2H distances and N-Fe-2H angles, which vary from 4.2 Å and 85° for threonine to 3.4 Å and 65° for norvaline, rationalize the trends in reactivity. This experimental correlation of position to outcome should aid in judging from structural data on other Fe/2OG enzymes whether they suppress hydroxylation or form hydroxylated intermediates on the pathways to other outcomes.

Hydroxylation of aliphatic carbons by nonheme Fe(IV)-oxo (ferryl) complexes proceeds by hydrogen-atom (H•) transfer (HAT) to the ferryl and subsequent coupling between the carbon radical and Fe(III)-coordinated oxygen (termed rebound). Enzymes that use H•-abstracting ferryl complexes for other transformations must either suppress rebound or further process hydroxylated intermediates. For olefin-installing C-C desaturations, it has been proposed that a second HAT to the Fe(III)-OH complex from the carbon α to the radical preempts rebound. Deuterium (H) at the second site should slow this step, potentially making rebound competitive. Desaturations mediated by two related l-arginine-modifying iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases behave oppositely in this key test, implicating different mechanisms. NapI, the l-Arg 4,5-desaturase from the naphthyridinomycin biosynthetic pathway, abstracts H• first from C5 but hydroxylates this site (leading to guanidine release) to the same modest extent whether C4 harbors H orH. By contrast, an unexpected 3,4-desaturation of l-homoarginine (l-hArg) by VioC, the l-Arg 3-hydroxylase from the viomycin biosynthetic pathway, is markedly disfavored relative to C4 hydroxylation when C3 (the second hydrogen donor) harbors H. Anchimeric assistance by N6 permits removal of the C4-H as a proton in the NapI reaction, but, with no such assistance possible in the VioC desaturation, a second HAT step (from C3) is required. The close proximity (≤3.5 Å) of both l-hArg carbons to the oxygen ligand in an X-ray crystal structure of VioC harboring a vanadium-based ferryl mimic supports and rationalizes the sequential-HAT mechanism. The results suggest that, although the sequential-HAT mechanism is feasible, its geometric requirements may make competing hydroxylation unavoidable, thus explaining the presence of α-heteroatoms in nearly all native substrates for Fe/2OG desaturases.

Iron(II)- and 2-(oxo)-glutarate-dependent oxygenases catalyze diverse oxidative transformations that are often initiated by abstraction of hydrogen from carbon by iron(IV)-oxo (ferryl) complexes. Control of the relative orientation of the substrate C–H and ferryl Fe–O bonds, primarily by direction of the oxo group into one of two cis-related coordination sites (termed inline and offline), may be generally important for control of the reaction outcome. Neither the ferryl complexes nor their fleeting precursors have been crystallographically characterized, hindering direct experimental validation of the offline hypothesis and elucidation of the means by which the protein might dictate an alternative oxo position. Comparison of high-resolution x-ray crystal structures of the substrate complex, an Fe(II)-peroxysuccinate ferryl precursor, and a vanadium(IV)-oxo mimic of the ferryl intermediate in the L-arginine 3-hydroxylase, VioC, reveals coordinated motions of active site residues that appear to control the intermediate geometries to determine reaction outcome.

The bicyclic β-lactam/2-pyrrolidine precursor to all carbapenem antibiotics is biosynthesized by attachment of a carboxymethylene unit to C5 of L-proline followed by β-lactam ring closure. Carbapenem synthase (CarC), an Fe(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenase, then inverts the C5 configuration. Here we report the structure of CarC in complex with its substrate and biophysical dissection of its reaction to reveal the stereoinversion mechanism. An Fe(IV)-oxo intermediate abstracts the hydrogen (H•) from C5, and tyrosine 165, a residue not visualized in the published structures of CarC lacking bound substrate, donates H• to the opposite face of the resultant radical. The reaction oxidizes the Fe(II) cofactor to Fe(III), limiting wild-type CarC to one turnover, but substitution of the H•-donating tyrosine disables stereoinversion and confers to CarC the capacity for catalytic substrate oxidation.

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