BesC Initiates C–C Cleavage through a Substrate-Triggered and Reactive Diferric-Peroxo Intermediate

Abstract

BesC catalyzes the iron- and O2-dependent cleavage of 4-chloro-l-lysine to form 4-chloro-l-allylglycine, formaldehyde, and ammonia. This process is a critical step for a biosynthetic pathway that generates a terminal alkyne amino acid which can be leveraged as a useful bio-orthogonal handle for protein labeling. As a member of an emerging family of diiron enzymes that are typified by their heme oxygenase-like fold and a very similar set of coordinating ligands, recently termed HDOs, BesC performs an unusual type of carbon–carbon cleavage reaction that is a significant departure from reactions catalyzed by canonical dinuclear-iron enzymes. Here, we show that BesC activates O2 in a substrate-gated manner to generate a diferric-peroxo intermediate. Examination of the reactivity of the peroxo intermediate with a series of lysine derivatives demonstrates that BesC initiates this unique reaction trajectory via cleavage of the C4–H bond; this process represents the rate-limiting step in a single turnover reaction. The observed reactivity of BesC represents the first example of a dinuclear-iron enzyme that utilizes a diferric-peroxo intermediate to capably cleave a C–H bond as part of its native function, thus circumventing the formation of a high-valent intermediate more commonly associated with substrate monooxygenations.

Substrate-Triggered μ-Peroxodiiron(III) Intermediate in the 4-Chloro-l-Lysine-Fragmenting Heme-Oxygenase-like Diiron Oxidase (HDO) BesC: Substrate Dissociation from, and C4 Targeting by, the Intermediate

“…The data are thus consistent with a reaction pathway in which C4 of the substrate donates H • directly to the μperoxodiiron(III) complex (Scheme 2B, lower black arrow), as recently suggested by Manley et al 36 From the resultant intermediate state, pathways involving either (1) an (protoncoupled) electron transfer [(PC)ET] from the C4 radical to the Fe 2 (III/IV) complex followed by a polar fragmentation of the C4 carbocation (blue arrows) or (2) direct radical C5−C6 scission followed by a (PC)ET step from the resultant C6 radical to the Fe 2 (III/IV) cluster (red arrows) can be envisaged. On the basis of their detection of 4-hydroxylysine in the BesC reaction with L-Lys and claim that the solvent is the sole source of the incorporated oxygen in that abortive product, Manley et al weighed in favor of a polar pathway via C4 carbocation intermediate.…”

“…yield k H = 0.00075 s −1 , k D = 0.000031 s −1 , k unc = 0.0060 s −1 , CE H = 0.55 (55% coupled to L-allylglycine production), and CE D = 0.049 (4.9% coupled). As noted above, decay of the intermediate in the reaction with 4-Cl-Lys is faster than in the reaction with L-Lys (∼30-fold) and was found by Manley et al 36 to be more faithfully coupled to the olefin-installing fragmentation. Regardless, the analysis yields a resolved D-KIE (k H /k D ) of 24 for production of L-allyglycine, which should be dominated by the primary effect on H/DAT from C4 to the μperoxodiiron(III) intermediate or the hypothetical successor with which it reversibly interconverts.…”

“…On the basis of similar evidence (an estimated D-KIE of 2.7), Manley et al also recently surmised that the μ-peroxodiiron(III) complex in BesC abstracts H • from C4 of the substrate. 36 In their study, the authentic 4-Cl-L-Lys substrate was seen to afford faster decay and more efficient coupling to the olefinic product but an identical D-KIE on decay of the intermediate. Although it has been proposed in computational studies, 46 little experimental evidence for HAT to a peroxodiiron(III) complex has been reported.…”

“…4,6 The markedly faster decay of the complex in the presence of L-Lys analogues modified at C4 (by H → Cl or CH 2 → S) and the normal primary C4 deuterium kinetic isotope effect (D-KIE) on its decay in the reaction with L-lysine imply that the complex (1) is an authentic intermediate and (2) either is, or reversibly interconverts with, the species that abstracts hydrogen from C4 to initiate the oxidative fragmentation, conclusions that comport with those reached in another study that appeared while this work was in review (addressed in more detail below). 36 Remarkably, once the substrate has promoted diiron(II) cofactor assembly and O 2 capture, it can dissociate from the intermediate complex to allow a different (i.e., more reactive) substrate to bind and react. Decay of the μperoxodiiron(III) intermediate is, as previously shown for UndA and SznF, 4,6 followed by spontaneous disintegration of the product diiron(III) cluster.…”

CADD from Chlamydia trachomatis is a manganese-dependent oxygenase that employs a self-sacrificing reaction for the synthesis of p-aminobenzoate

“…Thus, CADD is a member of the emerging h eme-oxygenase-like d iiron o xidase (HDO) superfamily, of which ~10,000 members are bioinformatically proposed, but only a handful have defined enzymatic activities (6). Characterized HDOs catalyze diverse reactions including N -oxygenation (SznF (6–9), RohS (10), and FlcE (11)), oxidative C-C bond cleavage (UndA (12–14), BesC (15–17), and FlcE (11)), and methylene excision (FlcD) (11).…”