Explore the vast range of titles available.

The Irving-Williams series refers to the predicted stabilities of transition metal complexes where the observed general stability for divalent first-row transition metal complexes increase across the row. Acetone carboxylases (ACs) use a coordinated divalent metal at their active site in the catalytic conversion of bicarbonate and acetone to form acetoacetate. Highly homologous ACs discriminate among different divalent metals at their active sites such that variations of the enzyme prefer Mn(II) over Fe(II), defying Irving-Williams-predicted behavior. Defining the determinants that promote metal discrimination within the first-row transition metals is of broad fundamental importance in understanding metal-mediated catalysis and metal catalyst design.

Acetone carboxylase (AC) from Xanthobacter autotrophicus is a 360 KDa α2β2γ2 heterohexamer that catalyzes the ATP-dependent formation of phosphorylated acetone and bicarbonate intermediates that react at Mn(II) metal active sites to form acetoacetate. Structural models of X. autotrophicus AC (XaAC) with and without nucleotides reveal that the binding and phosphorylation of the two substrates occurs ~40 Å from the Mn(II) active sites where acetoacetate is formed. Based on the crystal structures, a significant conformational change was proposed to open and close a tunnel that facilitates the passage of reaction intermediates between the sites for nucleotide binding and phosphorylation of substrates and Mn(II) sites of acetoacetate formation. We have employed electron paramagnetic resonance (EPR), kinetic assays, and hydrogen/deuterium exchange mass spectrometry (HDX-MS) of poised ligand-bound states and site-specific amino acid variants to complete an in-depth analysis of Mn(II) coordination and allosteric communication throughout the catalytic cycle. In contrast with the established paradigms for carboxylation, our analyses of XaAC suggested a carboxylate shift that couples both local and long-range structural transitions. Shifts in the coordination mode of a single carboxylic acid residue (αE89) mediate both catalysis proximal to a Mn(II) center and communication with an ATP active site in a separate subunit of a 180 kDa α2β2γ2 complex at a distance of 40 Å. This work demonstrates the power of combining structural models from X-ray crystallography with solution-phase spectroscopy and biophysical techniques to elucidate functional aspects of a multi-subunit enzyme.

Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to cleave SAM to initiate diverse radical reactions. These reactions are thought to involve the 5′-deoxyadenosyl radical intermediate, which has not yet been detected. We used rapid freeze-quenching to trap a catalytically competent intermediate in the reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme. Characterization of the intermediate by electron paramagnetic resonance and ¹³C, ⁵⁷Fe electron nuclear double-resonance spectroscopies reveals that it contains an organometallic center in which the 5′ carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster. Discovery of this intermediate extends the list of enzymatic bioorganometallic centers to the radical SAM enzymes, the largest enzyme superfamily known, and reveals intriguing parallels to B₁₂ radical enzymes.

Many enzymes catalyze reactions through the production of radical intermediates. Radical SAM enzymes, the largest superfamily of enzymes in nature, do this by using an iron-sulfur cluster to cleave S-adenosylmethionine and produce a radical intermediate. Using freeze quenching, Horitani et al. were able to trap a previously unseen radical intermediate from bacterial pyruvate formate-lyase activating enzyme. Spectroscopy revealed that the intermediate consists of a short-lived covalent bond between the terminal carbon of 5[variant prime]-deoxyadenosyl and the single iron atom of the iron-sulfur cluster. Not only does the observation of this radical expand our mechanistic understanding of radical SAM enzymes, but it expands the range of enzyme active sites or cofactors that function through an organometallic center. Science, this issue p. 822 Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to cleave SAM to initiate diverse radical reactions. These reactions are thought to involve the 5[variant prime]-deoxyadenosyl radical intermediate, which has not yet been detected. We used rapid freeze-quenching to trap a catalytically competent intermediate in the reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme. Characterization of the intermediate by electron paramagnetic resonance and 13C, 57Fe electron nuclear double-resonance spectroscopies reveals that it contains an organometallic center in which the 5[variant prime] carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster. Discovery of this intermediate extends the list of enzymatic bioorganometallic centers to the radical SAM enzymes, the largest enzyme superfamily known, and reveals intriguing parallels to B12 radical enzymes.

The radical S-adenosyl-L-methionine (SAM) superfamily of enzymes carry out diverse and complex reactions through generation of a 5’-deoxyadenosyl (5’-dAdo•) radical followed by transfer to substrate. These enzymes contain a [4Fe-4S] cluster which binds and transfers an electron to SAM. The exact mechanism of 5’-dAdo• generation is unknown and the studies herein provide further investigation into pyruvate formate lyase activating enzyme (PFL-AE) and lysine 2,3-aminomutase (LAM) pre and post SAM cleavage. To understand the active site of PFL-AE prior to SAM cleavage, cation and small molecule effects were examined by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. Previously, PFL-AE had been observed to contain a valence localized cluster in the presence of small molecules and this work used EPR and ENDOR spectroscopy to further probe the effects of these molecules. These studies determined that these molecules do not directly bind the cluster but rather an HxO species occupies the unique Fe site. The crystal structure of PFL-AE revealed a cation site and to probe this site, EPR and ENDOR spectroscopies were employed. Monovalent cations stimulated PFL-AE activity, with the greatest activity in the presence of potassium. The identity of the cation perturbed the EPR signal of PFL-AE which was more pronounced in the presence of SAM. ENDOR spectroscopy determined that SAM coordination differed depending on the monovalent cation. Due to its high reactivity, 5’-dAdo• has never been spectroscopically observed. In order to examine any intermediate states, a SAM analog and rapid freeze quench (RFQ) techniques were employed in conjunction with EPR and ENDOR spectroscopies. LAM can cleave the SAM analog, S-3’,4’-anhydroadenosyl-L-methionine, to produce a stable allylic radical which was coupled with isotopically labeled lysine for ENDOR analysis. It was determined that radical generation is highly controlled with little movement towards its substrate upon 5’-dAdo• production. During RFQ techniques on PFL-AE, an organometallic intermediate species was observed. To probe this intermediate, isotopically labeled SAM and an 57Fe labeled cluster were coupled with the unknown paramagnetic species. It was determined that this intermediate was an unprecedented organometallic Fe-adenosyl bound species post SAM cleavage.

Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to cleave SAM to initiate diverse radical reactions. These reactions are thought to involve the 5'-deoxyadenosyl radical intermediate, which has not yet been detected. We used rapid freeze-quenching to trap a catalytically competent intermediate in the reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme. Characterization of the intermediate by electron paramagnetic resonance and (13)C, (57)Fe electron nuclear double-resonance spectroscopies reveals that it contains an organometallic center in which the 5' carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster. Discovery of this intermediate extends the list of enzymatic bioorganometallic centers to the radical SAM enzymes, the largest enzyme superfamily known, and reveals intriguing parallels to B12 radical enzymes.Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to cleave SAM to initiate diverse radical reactions. These reactions are thought to involve the 5'-deoxyadenosyl radical intermediate, which has not yet been detected. We used rapid freeze-quenching to trap a catalytically competent intermediate in the reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme. Characterization of the intermediate by electron paramagnetic resonance and (13)C, (57)Fe electron nuclear double-resonance spectroscopies reveals that it contains an organometallic center in which the 5' carbon of a SAM-derived deoxyadenosyl moiety forms a bond with the unique iron site of the [4Fe-4S] cluster. Discovery of this intermediate extends the list of enzymatic bioorganometallic centers to the radical SAM enzymes, the largest enzyme superfamily known, and reveals intriguing parallels to B12 radical enzymes.