Radical S-adenosyl-l-methionine (SAM)
enzymes comprise a vast superfamily catalyzing diverse reactions essential
to all life through homolytic SAM cleavage to liberate the highly
reactive 5′-deoxyadenosyl radical (5′-dAdo·). Our
recent observation of a catalytically competent organometallic intermediate
Ω that forms during reaction of the radical SAM (RS) enzyme
pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite
surprising, and led to the question of its broad relevance in the
superfamily. We now show that Ω in PFL-AE forms as an intermediate
under a variety of mixing order conditions, suggesting it is central
to catalysis in this enzyme. We further demonstrate that Ω forms
in a suite of RS enzymes chosen to span the totality of superfamily
reaction types, implicating Ω as essential in catalysis across
the RS superfamily. Finally, EPR and electron nuclear double resonance
spectroscopy establish that Ω involves an Fe–C5′
bond between 5′-dAdo· and the [4Fe–4S] cluster.
An analogous organometallic bond is found in the well-known adenosylcobalamin
(coenzyme B12) cofactor used to initiate radical reactions
via a 5′-dAdo· intermediate. Liberation of a reactive
5′-dAdo· intermediate via homolytic metal–carbon
bond cleavage thus appears to be similar for Ω and coenzyme
B12. However, coenzyme B12 is involved in enzymes
catalyzing only a small number (∼12) of distinct reactions,
whereas the RS superfamily has more than 100 000 distinct sequences
and over 80 reaction types characterized to date. The appearance of
Ω across the RS superfamily therefore dramatically enlarges
the sphere of bio-organometallic chemistry in Nature.
Diphthamide biosynthesis involves a carbon-carbon bond-forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a carbon-sulfur (C-S) bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. Using rapid freezing, we have captured an organometallic intermediate with an iron-carbon (Fe-C) bond between ACP and the enzyme's [4Fe-4S] cluster. In the presence of the substrate protein, elongation factor 2, this intermediate converts to an organic radical, formed by addition of the ACP radical to a histidine side chain. Crystal structures of archaeal diphthamide biosynthetic radical SAM enzymes reveal that the carbon of the SAM C-S bond being cleaved is positioned near the unique cluster Fe, able to react with the cluster. Our results explain how selective C-S bond cleavage is achieved in this radical SAM enzyme.
Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy remains challenging up-to-date. We here demonstrate an efficient and generally applicable posttranscriptional SDSL method for large RNAs...
We report a rare example of a mixed-valence iron compound with an FeNNFe core, which gives insight into the structural, spectroscopic, and magnetic influences of single-electron reductions and oxidations. In the new compound, the odd electron is localized as judged from Mössbauer spectra at 80 K and infrared spectra at room temperature, and the backbonding into the N2 unit is intermediate between diiron(I) and diiron(0) congeners. Magnetic susceptibility and relaxation studies on the series of FeNNFe compounds show significant magnetic anisotropy, but through-barrier pathways enable fairly rapid magnetic relaxation.
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