The H-cluster of [FeFe]-hydrogenase consists of a [4Fe−4S] H -subcluster linked by a cysteinyl bridge to a unique organometallic [2Fe] H -subcluster assigned as the site of interconversion between protons and molecular hydrogen. This [2Fe] Hsubcluster is assembled by a set of Fe−S maturase enzymes HydG, HydE and HydF. Here we show that the HydG product [Fe II (Cys)(CO) 2 (CN)] synthon is the substrate of the radical SAM enzyme HydE, with the generated 5′-deoxyadenosyl radical attacking the cysteine S to form a C5′−S bond concomitant with reduction of the central low-spin Fe(II) to the Fe(I) oxidation state. This leads to the cleavage of the cysteine C3−S bond, producing a mononuclear [Fe I (CO) 2 (CN)S] species that serves as the precursor to the dinuclear Fe(I)Fe(I) center of the [2Fe] H -subcluster. This work unveils the role played by HydE in the enzymatic assembly of the H-cluster and expands the scope of radical SAM enzyme chemistry.
S-Adenosyl methionine (SAM) is employed as a [4Fe-4S]-bound
cofactor in the superfamily of radical SAM (rSAM) enzymes, in which
one-electron reduction of the [4Fe-4S]-SAM moiety leads to homolytic
cleavage of the S-adenosyl methionine to generate
the 5′-deoxyadenosyl radical (5′dAdo•), a potent H-atom abstractor. HydG, a member of this rSAM family,
uses the 5′dAdo• radical to lyse its substrate,
tyrosine, producing CO and CN that bind to a unique Fe site of a second
HydG Fe–S cluster, ultimately producing a mononuclear organometallic
Fe-l-cysteine-(CO)2CN complex as an intermediate
in the bioassembly of the catalytic H-cluster of [Fe–Fe] hydrogenase.
Here we report the use of non-native tyrosine substrate analogues
to further probe the initial radical chemistry of HydG. One such non-native
substrate is 4-hydroxy phenyl propanoic acid (HPPA) which lacks the
amino group of tyrosine, replacing the CαH-NH2 with a CH2 at the C2 position. Electron paramagnetic
resonance (EPR) studies show the generation of a strong and relatively
stable radical in the HydG reaction with natural abundance and 13C2-HPPA, with appreciable spin density localized at C2. These
results led us to try parallel experiments with the more oxidized
non-native substrate coumaric acid, which has a C2=C3 alkene substitution relative to HPPA’s single bond.
Interestingly, the HydG reaction with the cis-p-coumaric
acid isomer led to the trapping of a new radical EPR signal, and EPR
studies using cis-p-coumaric acid along with isotopically
labeled SAM reveal that we have for the first time trapped and characterized
the 5′dAdo• radical in an actual rSAM enzyme
reaction, here by using this specific non-native substrate cis-p-coumaric acid. Density functional theory energetics
calculations show that the cis-p-coumaric acid has
approximately the same C–H bond dissociation free energy as
5′dAdo•, providing a possible explanation
for our ability to trap an appreciable fraction of 5′dAdo• in this specific rSAM reaction. The radical’s
EPR line shape and its changes with SAM isotopic substitution are
nearly identical to those of a 5′dAdo• radical
recently generated by cryophotolysis of a prereduced [4Fe-4S]-SAM
center in another rSAM enzyme, pyruvate formate-lyase activating enzyme,
further supporting our assignment that we have indeed trapped and
characterized the 5′dAdo• radical in a radical
SAM enzymatic reaction by appropriate tuning of the relative radical
free energies via the judicious selection of a non-native substrate.
Aminofutalosine synthase (MqnE) is a radical SAM enzyme involved in the menaquinone biosynthetic pathway. In this communication, we propose a novel mechanism for this reaction involving the addition of the adenosyl radical to the substrate double bond to form a captodative radical followed by rearrangement and decarboxylation to form an aryl radical anion which is then oxidized by the [4Fe-4S] cluster. Consistent with this proposal, we describe the trapping of the captodative radical and the aryl radical anion using radical triggered C-Br fragmentation reactions. We also describe the trapping of the captodative radical by replacing the vinylic carboxylic acid with an amide.
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