Generation of hydrogen sulfide (HS) is challenging and few methods are capable of localized delivery of this gas. Here, a boron dipyrromethene-based carbamothioate (BDP-HS) that is uncaged by visible light of 470 nm to generate carbonyl sulfide (COS), which is rapidly hydrolyzed to HS in the presence of carbonic anhydrase, a widely prevalent enzyme, is reported.
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.
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 binding of L-Lys or an analog 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-[2H]-L-Lys, imply that the peroxide intermediate or a successor complex with which it reversibly interconverts initiates the oxidative fragmentation by abstracting hydrogen from C4. Surprisingly, the sluggish substrate L-Lys can dissociate after triggering the intermediate to form, thereby allowing one of the better substrates to bind and react. Observed linkage between Fe(II) and substrate binding suggests that the triggering event involves a previously documented (in SznF) ordering of the dynamic HDO architecture that contributes one of the iron sites, a hypothesis consistent with the observation that the diiron(III) product cluster produced upon decay of the intermediate spontaneously degrades, as it has been shown to do in all other HDOs studied to date.
Persulfides and polysulfides, collectively known as the sulfane sulfur pool along with hydrogen sulfide (H2S), play a central role in cellular physiology and disease. Exogenously enhancing these species in cells...
An aliphatic halogenase requires four substrates: 2-oxoglutarate
(2OG), halide (Cl– or Br–), the
halogenation target (“prime substrate”), and dioxygen.
In well-studied cases, the three nongaseous substrates must bind to
activate the enzyme’s Fe(II) cofactor for efficient capture
of O2. Halide, 2OG, and (lastly) O2 all coordinate
directly to the cofactor to initiate its conversion to a cis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen
(H•) from the non-coordinating prime substrate to
enable radicaloid carbon–halogen coupling. We dissected the
kinetic pathway and thermodynamic linkage in binding of the first
three substrates of the l-lysine 4-chlorinase, BesD. After
addition of 2OG, subsequent coordination of the halide to the cofactor
and binding of cationic l-Lys near the cofactor are associated
with strong heterotropic cooperativity. Progression to the haloferryl
intermediate upon the addition of O2 does not trap the
substrates in the active site and, in fact, markedly diminishes cooperativity
between halide and l-Lys. The surprising lability of the
BesD•[Fe(IV)=O]•Cl•succinate•l-Lys complex engenders pathways for decay of the haloferryl intermediate
that do not result in l-Lys chlorination, especially at low
chloride concentrations; one identified pathway involves oxidation
of glycerol. The mechanistic data imply (i) that BesD may have evolved
from a hydroxylase ancestor either relatively recently or under weak
selective pressure for efficient chlorination and (ii) that acquisition
of its activity may have involved the emergence of linkage between l-Lys binding and chloride coordination following the loss of
the anionic protein-carboxylate iron ligand present in extant hydroxylases.
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