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

Abstract

The enzyme BesC from the β-ethynyl-l-serine biosynthetic pathway in Streptomyces cattleya fragments 4-chloro-l-lysine (produced from l-Lysine by BesD) to ammonia, formaldehyde, and 4-chloro-l-allylglycine and can analogously fragment l-Lys itself. BesC belongs to the emerging family of O2-activating non-heme-diiron enzymes with the “heme-oxygenase-like” protein fold (HDOs). Here, we show that the binding of l-Lys or an analogue triggers capture of O2 by the protein’s diiron­(II) cofactor to form a blue μ-peroxodiiron­(III) intermediate analogous to those previously characterized in two other HDOs, the olefin-installing fatty acid decarboxylase, UndA, and the guanidino-N-oxygenase domain of SznF. The ∼5- and ∼30-fold faster decay of the intermediate in reactions with 4-thia-l-Lys and (4RS)-chloro-dl-lysine than in the reaction with l-Lys itself and the primary deuterium kinetic isotope effects (D-KIEs) on decay of the intermediate and production of l-allylglycine in the reaction with 4,4,5,5-[2H4]-l-Lys suggest that the peroxide intermediate or a reversibly connected successor complex abstracts a hydrogen atom from C4 to enable olefin formation. Surprisingly, the sluggish substrate l-Lys can dissociate after triggering intermediate formation, thereby allowing one of the better substrates to bind and react. The structure of apo BesC and the demonstrated linkage between Fe­(II) and substrate binding suggest that the triggering event involves an induced ordering of ligand-providing helix 3 (α3) of the conditionally stable HDO core. As previously suggested for SznF, the dynamic α3 also likely initiates the spontaneous degradation of the diiron­(III) product cluster after decay of the peroxide intermediate, a trait emerging as characteristic of the nascent HDO family.

Synergistic Binding of the Halide and Cationic Prime Substrate of l-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States

“…Assembly of the O 2 -reactive BesD•Fe II •2OG• l -Lys•Cl complex (hereafter the quinary complex) could have a preferred or even obligatory binding order. We probed the formation of the initial binary complex, BesD•Fe II , by ferrozine-chelation experiments , to find a K D of 3.7 ± 0.7 μM (Figure S5). Next, in titrations monitoring the ∼500 nm MLCT band, we found that binding of 2OG to the BesD•Fe II binary complex is associated with a K D , K 2OG , of 14 ± 5 μM.…”

“…Although we viewed the idea of reversible binding of substrates in an intermediate state of sufficient potency to cleave an unactivated C−H bond as surprising, we did recently demonstrate such lability in the substrate-triggered peroxodiiron(III) intermediate state in the diiron enzyme BesC, which fragments 4-Cl-L-Lys and L-Lys�putatively by abstracting hydrogen from C4�in the β-ethynylserine biosynthetic pathway. 30 Synergistic Binding of Cl − and L-Lys in the Chloroferryl Intermediate State. In light of the demonstrated binding synergy between the anion and prime substrate in the Fe II state of BesD, we further evaluated the possibility of reversible substrate association in the intermediate state by testing for an interplay between the Cl − and L-Lys concentrations in the kinetics of decay of the chloroferryl intermediate and its partitioning between productive and unproductive pathways.…”

Peroxo-Diiron(III/III) as the Reactive Intermediate for N-Hydroxylation Reactions in the Multidomain Metalloenzyme SznF: Evidence from Molecular Dynamics and Quantum Mechanical/Molecular Mechanical Calculations

“…30,37 Indeed, HDOs have emerged as a superfamily of non-heme diiron enzymes, with over >10 000 HDO sequences identified bioinformatically. 38 In addition to SznF, a growing member of HDOs has been characterized recently, including fatty acid oxidase UndA, 17−19 the 4-chloro-L-lysine-fragmenting BesC, 15,16 and other N-oxygenases RohS, 46 HrmI, 47 and BelL. 48 In the HDO domain of SznF, two irons are bridged by Glu215; one iron (donated as Fe2) is ligated by Glu189, His225, and His311, while the other iron (donated as Fe1) is ligated by Glu281, His318, and Asp315.…”

“…One superfamily of metalloenzymes can utilize non-heme diiron active sites for O 2 activation and reductions . These non-heme diiron enzymes catalyze many types of reactions, such as the challenging C–H activations occurring in sMMO, − BesC, , UndA, − PtmU3, and Δ9 Desaturase, − epoxidation of double bond occurring in BoxB, , N-oxygenation occurring in AurF − and CmlI, − as well as N-hydroxylation occurring in SznF. − …”

“…The X-ray structure of SznF reveals a heme-oxygenase-like (HO-like) HDO domain, in which the diiron cofactors have a flexible coordination architecture that differs from the ferritin-like diiron oxidase and oxygenase (FDO) family, such as Δ9 desaturase, − sMMO, , toluene/o-xylene monooxygenase, , and arylamine N-oxygenases AurF and CmlI. , Indeed, HDOs have emerged as a superfamily of non-heme diiron enzymes, with over >10 000 HDO sequences identified bioinformatically . In addition to SznF, a growing member of HDOs has been characterized recently, including fatty acid oxidase UndA, − the 4-chloro- l -lysine-fragmenting BesC, , and other N-oxygenases RohS, HrmI, and BelL . In the HDO domain of SznF, two irons are bridged by Glu215; one iron (donated as Fe2) is ligated by Glu189, His225, and His311, while the other iron (donated as Fe1) is ligated by Glu281, His318, and Asp315.…”