How an enzyme tames reactive intermediates: positioning of the active-site components of lysine 2,3-aminomutase during enzymatic turnover as determined by ENDOR spectroscopy

Lysine 2,3-aminomutase (LAM) utilizes a [4Fe-4S] cluster, S-adenosyl-L-methionine (SAM), and pyridoxal 5'-phosphate (PLP) to isomerize L-alpha-lysine to L-beta-lysine. LAM is a member of the radical-SAM enzyme superfamily in which a [4Fe-4S]+ cluster reductively cleaves SAM to produce the 5'-deoxyadenosyl radical, which abstracts an H-atom from substrate to form 5'-deoxyadenosine (5'-Ado) and the alpha-Lys* radical (state 3 (Lys*)). This radical isomerizes to the beta-Lys* radical (state 4(Lys*)), which then abstracts an H-atom from 5'-Ado to form beta-lysine and the 5'-deoxyadenosyl radical; the latter then regenerates SAM. We use 13C, 1,2H, 31P, and 14N ENDOR to characterize the active site of LAM in intermediate states that contain the isomeric substrate radicals or analogues. With L-alpha-lysine as substrate, we monitor the state with beta-Lys*. In parallel, we use two substrate analogues that generate stable analogues of the alpha-Lys* radical: trans-4,5-dehydro-L-lysine (DHLys) and 4-thia-L-lysine (SLys). This first glimpse of the motions of active-site components during catalytic turnover suggests a possible major movement of PLP during catalysis. However, the principal focus of this work is on the relative positions of the carbons involved in H-atom transfer. We conclude that the active site facilitates hydrogen atom transfer by enforcing van der Waals contact between radicals and their reacting partners. This constraint enables the enzyme to minimize and even eliminate side reactions of highly reactive species such as the 5'-deoxyadensosyl radical.     

Suicide inactivation of dioldehydratase by glycolaldehyde and chloroacetaldehyde: an examination of the reaction mechanism

High-level ab initio calculations have been used to study the mechanism for the inactivation of diol dehydratase (DDH) by glycolaldehyde or 2-chloroacetaldehyde. As in the case of the catalytic substrates of DDH, e.g., ethane-1,2-diol, the 5'-deoxyadenosyl radical (Ado*) is able to abstract a hydrogen atom from both substrate analogues in the initial step on the reaction pathway, as evidenced by comparable energy barriers. However, in subsequent step(s), each substrate analogue produces the highly stable glycolaldehyde radical. The barrier for hydrogen atom reabstraction by the glycolaldehyde radical is calculated to be too high ( approximately 110 kJ mol-1) to allow Ado* to be regenerated and recombine with the cob(II)alamin radical, the latter therefore remaining tightly bound to DDH. As a consequence, the catalytic pathway is disrupted, and DDH becomes an impotent enzyme. Interconversion of equivalent structures of the glycolaldehyde radical via the symmetrical cis-ethanesemidione radical is calculated to require 38 kJ mol-1. EPR indications of a symmetrical cis-ethanesemidione structure are likely to be the result of formation of an equilibrium mixture of glycolaldehyde radical structures, this equilibration being facilitated by partial deprotonation of the glycolaldehyde radical by the carboxylate of an amino acid residue within the active site of DDH.     

Coordination and mechanism of reversible cleavage of S-adenosylmethionine by the [4Fe-4S] center in lysine 2,3-aminomutase

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of l-lysine and l-beta-lysine, by a radical mechanism initiated by the reversible, reductive homolytic scission of the C5'-S bond in S-adenosylmethionine (SAM) to form methionine and the 5'-deoxyadenosyl radical at the active site. LAM is a member of a superfamily of enzymes in which a [4Fe-4S]+ cluster with a unique, noncysteinyl coordinated Fe provides the electron required in the cleavage of SAM. Little is known of the mechanism by which the electron is inserted into SAM, and it is not known whether all enzymes of the family employ the same mechanism. Selenium X-ray absorption spectroscopy (XAS) in the reaction of Se-adenosyl-l-selenomethionine (SeSAM) in place of SAM shows that electron transfer occurs by an inner sphere mechanism culminating in direct ligation of selenomethionine to iron upon cleavage of SeSAM. Here, we report an electron nuclear double resonance (ENDOR) spectroscopic investigation of LAM to which has been bound 14N, 17O, 2H, or 13C labeled SAM. It is found that LAM exhibits the same motif for SAM binding to the [4Fe-4S]+,2+ clusters as does pyruvate formate lyase: chelation by the unique iron of the amino and carboxylato groups of SAM; close proximity of the methionine methyl group to the cluster. However, there appear to be significant, and possibly mechanistically important, differences in the details of the binding geometry of SAM. On the basis of the correlation of the ENDOR and XAS spectroscopic results, we postulate a mechanism by which LAM cleaves SAM to generate an intermediate where N, O, and S of the methionine product are bound to the octahedrally coordinated unique Fe of the [4Fe-4S] cluster.     

The [4Fe-4S](2+) cluster in reconstituted biotin synthase binds S-adenosyl-L-methionine

The combination of resonance Raman, electron paramagnetic resonance and M?ssbauer spectroscopies has been used to investigate the effect of S-adenosyl-l-methionine (SAM) on the spectroscopic properties of the [4Fe-4S]2+ cluster in biotin synthase. The results indicate that SAM interacts directly at a unique iron site of the [4Fe-4S]2+ cluster in BioB and support the hypothesis of a common inner-sphere mechanism for the reductive cleavage of SAM in the radical SAM family of Fe-S enzymes.     

Spectroscopic approaches to elucidating novel iron-sulfur chemistry in the "radical-Sam" protein superfamily

Electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and M?ssbauer spectroscopies and other physical methods have provided important new insights into the radical-SAM superfamily of proteins, which use iron-sulfur clusters and S-adenosylmethionine to initiate H atom abstraction reactions. This remarkable chemistry involves the generation of the extremely reactive 5'-deoxyadenosyl radical, the same radical intermediate utilized in B12-dependent reactions. Although early speculation focused on the possibility of an organometallic intermediate in radical-SAM reactions, current evidence points to novel chemistry involving a site-differentiated [4Fe-4S] cluster. The focus of this forum article is on one member of the radical-SAM superfamily, pyruvate formate-lyase activating enzyme, and how physical methods, primarily EPR and ENDOR spectroscopies, are contributing to our understanding of its structure and mechanism. New ENDOR data supporting coordination of the methionine moiety of SAM to the unique site of the [4Fe-4S]2+/+ cluster are presented.     

Spectroscopic evidence for site specific chemistry at a unique iron site of the [4Fe-4S] cluster in ferredoxin:thioredoxin reductase

Ferredoxin:thioredoxin reductase (FTR) catalyzes the reduction of the disulfide in thioredoxin in two one-electron steps using an active site comprising a [4Fe-4S] in close proximity to a redox active disulfide. M?ssbauer spectroscopy has been used to investigate the ligation and electronic properties of the [4Fe-4S] cluster in as-prepared FTR which has the active-site disulfide intact and in the N-ethylmaleimide (NEM)-modified form which provides a stable analogue of the one-electron-reduced heterodisulfide intermediate and has one of the cysteines of the active-site disulfide alkylated with NEM. The results reveal novel site-specific cluster chemistry involving weak interaction of the active-site disulfide with a unique Fe site of the [4Fe-4S]2+ cluster in the resting enzyme and cleavage of the active-site disulfide with concomitant coordination of one of the cysteines to yield a [4Fe-4S]3+ cluster with a five-coordinate Fe site ligated by two cysteine residues in the NEM-modified enzyme. The results provide molecular-level insight into the catalytic mechanism of FTR and other Fe-S-cluster-containing disulfide reductases, and suggest a possible mechanism for the reductive cleavage of S-adenosylmethionine by the radical SAM family of Fe-S enzymes.     

Coordination of adenosylmethionine to a unique iron site of the [4Fe-4S] of pyruvate formate-lyase activating enzyme: a Mössbauer spectroscopic study

Pyruvate formate-lyase activating enzyme (PFL-AE) generates the catalytically essential glycyl radical of PFL. It is a member of the so-called "radical-SAM superfamily" of enzymes that use a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet or SAM) to catalyze diverse radical-mediated reactions. Evidence suggests that this class of enzymes operate by common initial steps involving the generation of an AdoMet-derived adenosyl radical intermediate, of which the mechanism remains unresolved. The three-cysteine CX3CX2C cluster-binding motif common to all members of this superfamily suggests a unique Fe site in the [4Fe-4S] cluster, which presumably interacts with AdoMet to effect the reductive cleavage and radical generation. Here we employ a dual-iron-isotope (56Fe/57Fe) approach to demonstrate the existence of a unique Fe site in the [4Fe-4S] cluster of PFL-AE by M?ssbauer spectroscopy. Coordination of AdoMet to this unique Fe site was made evident by the observation of a substantial increase in the isomer shift (delta) of the M?ssbauer spectrum associated with the unique Fe site: delta = 0.42 mm/s in the absence of AdoMet increases to delta = 0.72 mm/s in the presence of AdoMet. Further, the M?ssbauer data show that the binding of AdoMet to the unique Fe site occurs in the [4Fe-4S]2+ state, prior to the injection of the reducing equivalent required for catalysis. This observation indicates that AdoMet coordination is a necessary prerequisite to adenosyl radical generation.     

Cfr and RlmN contain a single [4Fe-4S] cluster, which directs two distinct reactivities for S-adenosylmethionine: methyl transfer by SN2 displacement and radical generation

The radical SAM (RS) proteins RlmN and Cfr catalyze methylation of carbons 2 and 8, respectively, of adenosine 2503 in 23S rRNA. Both reactions are similar in scope, entailing the synthesis of a methyl group partially derived from S-adenosylmethionine (SAM) onto electrophilic sp(2)-hybridized carbon atoms via the intermediacy of a protein S-methylcysteinyl (mCys) residue. Both proteins contain five conserved Cys residues, each required for turnover. Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe-4S]-cluster cofactor; the remaining two are at opposite ends of the polypeptide. Here we show that each protein contains only the one "radical SAM" [4Fe-4S] cluster and the two remaining conserved cysteines do not coordinate additional iron-containing species. In addition, we show that, while wild-type RlmN bears the C355 mCys residue in its as-isolated state, RlmN that is either engineered to lack the [4Fe-4S] cluster by substitution of the coordinating cysteines or isolated from Escherichia coli cultured under iron-limiting conditions does not bear a C355 mCys residue. Reconstitution of the [4Fe-4S] cluster on wild-type apo RlmN followed by addition of SAM results in rapid production of S-adenosylhomocysteine (SAH) and the mCys residue, while treatment of apo RlmN with SAM affords no observable reaction. These results indicate that in Cfr and RlmN, SAM bound to the unique iron of the [4Fe-4S] cluster displays two reactivities. It serves to methylate C355 of RlmN (C338 of Cfr), or to generate the 5'-deoxyadenosyl 5'-radical, required for substrate-dependent methyl synthase activity.     

Super-exchange and Exchange-Enhanced Reactivity in Fe4S4-Mediated Activation of SAM by Radical SAM Enzymes?

[4Fe-4S]-dependent radical S-adenosylmethionine (SAM) proteins are a superfamily of oxidoreductases that can catalyze a series of challenging transformations using the common 5-dAdo radical intermediate. Although the structures and functions of radical SAM enzymes have been extensively studied, the electronic state-dependent reactions of the [4Fe-4S] clusters in these enzymes are still elusive. Herein we performed QM/MM calculations to elucidate the electronic state-dependent reactivity of the [4Fe-4S] cluster in pyruvate-formate lyase activating enzyme. Our calculations show that the electronic state-dependent SAM activation by the [4Fe-4S] clusters in radical SAM enzyme is determined by both the super-exchange and exchange-enhanced reactivities. The super-exchange coupling in the [4Fe-4S] cluster favors the antiferromagnetic coupling between two neighbouring pairs, which results in the \begin{document}$\alpha$\end{document}-electron rather than the \begin{document}$\beta$\end{document}-electron donation from the [4Fe-4S]\begin{document}$^{1+}$\end{document} cluster toward the SAM activation. Meanwhile, in the most favorable electronic state for the reductive cleavage of S\begin{document}$-$\end{document}C5\begin{document}$'$\end{document}, Fe4 would donate its \begin{document}$\alpha$\end{document}-electron to gain the maximum exchange interactions in the Fe4-block. Such super-exchange and exchange-enhanced reactivity could be the general principles for reactivities of [4Fe-4S] cluster in RS enzymes.     

Structural basis for a Kolbe-type decarboxylation catalyzed by a glycyl radical enzyme

4-Hydroxyphenylacetate decarboxylase is a [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in clostridia. The decarboxylation product p-cresol (4-methylphenol) is a virulence factor of the human pathogen Clostridium difficile . Here we describe the crystal structures at 1.75 and 1.81 ? resolution of substrate-free and substrate-bound 4-hydroxyphenylacetate decarboxylase from the related Clostridium scatologenes . The structures show a (βγ)(4) tetramer of heterodimers composed of a catalytic β-subunit harboring the putative glycyl/thiyl dyad and a distinct small γ-subunit with two [4Fe-4S] clusters at 40 ? distance from the active site. The γ-subunit comprises two domains displaying pseudo-2-fold symmetry that are structurally related to the [4Fe-4S] cluster-binding scaffold of high-potential iron-sulfur proteins. The N-terminal domain coordinates one cluster with one histidine and three cysteines, and the C-terminal domain coordinates the second cluster with four cysteines. Whereas the C-terminal cluster is buried in the βγ heterodimer interface, the N-terminal cluster is not part of the interface. The previously postulated decarboxylation mechanism required the substrate's hydroxyl group in the vicinity of the active cysteine residue. In contrast to expectation, the substrate-bound state shows a direct interaction between the substrate's carboxyl group and the active site Cys503, while His536 and Glu637 at the opposite side of the active site pocket anchor the hydroxyl group. This state captures a possible catalytically competent complex and suggests a Kolbe-type decarboxylation for p-cresol formation.     

Structural basis for reductive radical formation and electron recycling in (R)-2-hydroxyisocaproyl-CoA dehydratase

The radical enzyme (R)-2-hydroxyisocaproyl-CoA dehydratase catalyzes the dehydration of (R)-2-hydroxyisocaproyl-CoA in the fermentation of l-leucine by the human pathogenic bacterium Clostridium difficile. In contrast to other radical enzymes, such as bacterial class II ribonucleotide reductase or biotin synthase, the Fe/S cluster containing (R)-2-hydroxyisocaproyl-CoA dehydratase requires no special cofactors such as coenzyme B(12) or S-adenosylmethionine for radical generation. Instead it uses a single high-energy electron that is recycled after each turnover. The catalyzed reaction, an atypical α/β-dehydration, depends on the reductive formation of ketyl radicals on the substrate generated by injection of a single electron from the ATP-dependent activator protein. So far, it is unknown how the active electron is recycled and how unwanted side reactions are prevented, allowing for up to 10,000 turnovers. The crystal structure reveals that the heterodimeric protein contains two [4Fe-4S] clusters at a distance of 12 ?, each coordinated by three cysteines and one terminal ligand. The cluster in the α-subunit is part of the active site. In the absence of substrate, a water/hydroxide ion acts as the fourth ligand. The substrate replaces this ligand and coordinates the cluster via the carbonyl-oxygen of the thioester group. The cluster in the β-subunit has a terminal sulfhydryl/sulfido ligand and can act as a reservoir to protect the electron from unwanted side reactions via a recycling mechanism. The crystal structure of (R)-2-hydroxyisocaproyl-CoA dehydratase serves as a model for the reductively radical-generating metalloenzymes of the (R)-2-hydroxyacyl-CoA dehydratase and benzoyl-CoA reductase families.     

Suicide Inactivation of Dioldehydrase by 2‐Chloroacetaldehyde: Formation of the ‘cis‐Ethanesemidione’ Radical,and the Role of a Monovalent Cation

Dioldehydrase is an adenosylcobalamin‐dependent enzyme that catalyzes the dehydration of (R)‐ or (S)‐propane‐1,2‐diol to propanal. The reaction proceeds by a radical mechanism initiated by the homolytic scission of the covalent Co? C(5′) bond in the coenzyme to form cob(II)alamin and the 5‐deoxyadenosyl radical as transient intermediates. Dioldehydrase is subject to ‘suicide inactivation’ by substrate/product analogs. Inactivation by 2‐chloroacetaldehyde converts the inactivator into the ‘cis‐ethanesemidione’ radical. A mechanism for this process includes reaction of chloroacetaldehyde in the reverse of the normal catalytic process to a rearranged radical that eliminates HCl. K⁺ and other monovalent cations of similar size, including Tl⁺, are required for dioldehydrase activity and for suicide inactivation by glycolaldehyde or 2‐chloroacetaldehyde. A K⁺ ion is bound to propane‐1,2‐diol in dioldehydrase. Both EPR and pulsed‐EPR experiments show that the magnetic nuclei of thallous ions (²⁰³Tl⁺, ²⁰⁵Tl⁺) do not interact with the unpaired electron in the cis‐ethanesemidione radical at the active site of dioldehydrase. Pulsed‐EPR experiments implicate a ¹⁴NH group, possibly of His¹⁴³, interacting with the radical at the active site.     

Evidence from Mössbauer spectroscopy for distinct [2Fe-2S](2+) and [4Fe-4S](2+) cluster binding sites in biotin synthase from Escherichia coli

Biotin synthase is an AdoMet-dependent radical enzyme that catalyzes the insertion of an FeS cluster-derived sulfur atom into dethiobiotin. The dimeric enzyme is purified containing one [2Fe-2S]2+ cluster per monomer, but it is most active when reconstituted with an additional [4Fe-4S]2+ cluster per monomer. Using M?ssbauer spectroscopy coupled with differential reconstitution of each cluster with 57Fe, we show that the reconstituted enzyme has approximately 1:1 [2Fe-2S]2+ and [4Fe-4S]2+ clusters and that the [4Fe-4S]2+ cluster is assembled at an alternate site not previously occupied by the [2Fe-2S]2+ cluster. These data suggest that biotin synthase is evolved to simultaneously accommodate two different clusters with unique roles in catalysis.     

Are free radicals involved in IspH catalysis? An EPR and crystallographic investigation

The [4Fe-4S] protein IspH in the methylerythritol phosphate isoprenoid biosynthesis pathway is an important anti-infective drug target, but its mechanism of action is still the subject of debate. Here, by using electron paramagnetic resonance (EPR) spectroscopy and (2)H, (17)O, and (57)Fe isotopic labeling, we have characterized and assigned two key reaction intermediates in IspH catalysis. The results are consistent with the bioorganometallic mechanism proposed earlier, and the mechanism is proposed to have similarities to that of ferredoxin, thioredoxin reductase, in that one electron is transferred to the [4Fe-4S](2+) cluster, which then performs a formal two-electron reduction of its substrate, generating an oxidized high potential iron-sulfur protein (HiPIP)-like intermediate. The two paramagnetic reaction intermediates observed correspond to the two intermediates proposed in the bioorganometallic mechanism: the early π-complex in which the substrate's 3-CH(2)OH group has rotated away from the reduced iron-sulfur cluster, and the next, η(3)-allyl complex formed after dehydroxylation. No free radical intermediates are observed, and the two paramagnetic intermediates observed do not fit in a Birch reduction-like or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy and X-ray crystallography that two substrate analogues (4 and 5) follow the same reaction mechanism.     

9-Mercaptodethiobiotin is generated as a ligand to the [2Fe-2S]+ cluster during the reaction catalyzed by biotin synthase from Escherichia coli

Biotin synthase catalyzes formation of the thiophane ring through stepwise substitution of a sulfur atom for hydrogen atoms at the C9 and C6 positions of dethiobiotin. Biotin synthase is a radical S-adenosylmethionine (SAM) enzyme that reductively cleaves S-adenosylmethionine, generating 5'-deoxyadenosyl radicals that initially abstract a hydrogen atom from the C9 position of dethiobiotin. We have proposed that the resulting dethiobiotinyl radical is quenched by the μ-sulfide of the nearby [2Fe-2S](2+) cluster, resulting in coupled formation of 9-mercaptodethiobiotin and a reduced [2Fe-2S](+) cluster. This reduced FeS cluster is observed by electron paramagnetic resonance spectroscopy as a mixture of two orthorhombic spin systems. In the present work, we use isotopically labeled 9-mercaptodethiobiotin and enzyme to probe the ligand environment of the [2Fe-2S](+) cluster in this reaction intermediate. Hyperfine sublevel correlation spectroscopy (HYSCORE) spectra exhibit strong cross-peaks demonstrating strong isotropic coupling of the nuclear spin with the paramagnetic center. The hyperfine coupling constants are consistent with a structural model for the reaction intermediate in which 9-mercaptodethiobiotin is covalently coordinated to the remnant [2Fe-2S](+) cluster.     

Pulsed electron paramagnetic resonance experiments identify the paramagnetic intermediates in the pyruvate ferredoxin oxidoreductase catalytic cycle

Pyruvate ferredoxin oxidoreductase (PFOR) is central to the anaerobic metabolism of many bacteria and amitochondriate eukaryotes. PFOR contains thiamine pyrophosphate (TPP) and three [4Fe-4S] clusters, which link pyruvate oxidation to reduction of ferredoxin. In the PFOR reaction, TPP reacts with pyruvate to form lactyl-TPP, which undergoes decarboxylation to form a hydroxyethyl-TPP (HE-TPP) intermediate. One electron is then transferred from HE-TPP to one of the three [4Fe-4S] clusters to form an HE-TPP radical and a [4Fe-4S]1+ intermediate. Pulsed EPR methods have been used to measure the distance between the HE-TPP radical and the [4Fe-4S]1+ cluster to which it is coupled. Computational analysis including the PFOR crystal structure and the spin distribution in the HE-TPP radical and in the reduced [4Fe-4S] cluster demonstrates that the distance between the HE-TPP radical and the medial cluster B matches the experimentally determined dipolar interaction, while one of the other two clusters is too close and the other is too far away. These results clearly demonstrate that it is the medial cluster (cluster B) that is reduced. Thus, rapid electron transfer occurs through the electron-transfer chain, which leaves an oxidized proximal cluster poised to accept an electron from the HE-TPP radical in the subsequent reaction step.     

EPR and infrared spectroscopic evidence that a kinetically competent paramagnetic intermediate is formed when acetyl-coenzyme A synthase reacts with CO

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme which enables archaea and bacteria to grow autotrophically on CO and hydrogen/carbon dioxide using the Wood-Ljundahl pathway. CO produced from reduction of carbon dioxide by CODH is transferred to the active site of ACS through an intramolecular tunnel, where it combines with Coenzyme A and a methyl cation to produce acetyl-CoA. The active site of ACS contains a single [4Fe-4S] cluster bridged by a cysteine sulfur atom to a binuclear center. The binuclear center is composed of two Ni atoms bridged by two separate cysteine sulfurs. The Ni site attached to the [4Fe-4S] is referred to as proximal Ni, while the other Ni atom, which assumes a square-planar geometry, is referred to as the distal site. We report the characterization of the carbonylated form of highly active (0.67 spins/mol) heterologously expressed monomeric ACS from C. hydrogenoformans in E. coli by rapid-freeze quench EPR (RFQ-EPR) and stopped-flow infrared (SF-IR) spectroscopies. The reaction of ACS with CO produces a single metal-carbonyl species whose formation rate, measured by SF-IR, correlates with the rate of formation, measured by RFQ-EPR, of the paramagnetic state of the enzyme (NiFeC species). These results indicate that the NiFeC species is the predominant form observed in solution when ACS reacts with CO. The NiFeC species contains the proximal Ni in the +1 redox state and the [4Fe-4S] cluster in the 2+ state, thus there is no evidence for either a Ni(0) or a Ni(II) state in the active carbonylated form of the enzyme.     

Studies of inhibitor binding to the [4Fe-4S] cluster of quinolinate synthase

Stop for NadA! A [4Fe-4S] enzyme, NadA, catalyzes the formation of quinolinic acid in de?novo nicotinamide adenine dinucleotide (NAD) biosynthesis. A structural analogue of an intermediate, 4,5-dithiohydroxyphthalic acid (DTHPA), has an in?vivo NAD biosynthesis inhibiting activity in E. coli. The inhibitory effect can be explained by the coordination of DTHPA thiolate groups to a unique Fe site of the NadA [4Fe-4S] cluster.     

Revisiting the Mechanism of the Anaerobic Coproporphyrinogen III Oxidase HemN

HemN is a radical S‐adenosyl‐l ‐methionine (SAM) enzyme that catalyzes the oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, an intermediate in heme biosynthesis. HemN binds two SAM molecules in the active site, but how these two SAMs are utilized for the sequential decarboxylation of the two propionate groups of coproporphyrinogen III remains largely elusive. Provided here is evidence showing that in HemN catalysis a SAM serves as a hydrogen relay which mediates a radical‐based hydrogen transfer from the propionate to the 5′‐deoxyadenosyl (dAdo) radical generated from another SAM in the active site. Also observed was an unexpected shunt product resulting from trapping of the SAM‐based methylene radical by the vinyl moiety of the mono‐decarboxylated intermediate, harderoporphyrinogen. These results suggest a major revision of the HemN mechanism and reveal a new paradigm of the radical‐mediated hydrogen transfer in radical SAM enzymology.     

Electron-nuclear double resonance spectroscopic evidence that S-adenosylmethionine binds in contact with the catalytically active [4Fe-4S](+) cluster of pyruvate formate-lyase activating enzyme

Pyruvate formate-lyase activating enzyme (PFL-AE) is a representative member of an emerging family of enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. Although these enzymes have diverse functions, evidence is emerging that they operate by a common mechanism in which a [4Fe-4S](+) interacts with AdoMet to generate a 5'-deoxyadenosyl radical intermediate. To date, however, it has been unclear whether the iron-sulfur cluster is a simple electron-transfer center or whether it participates directly in the radical generation chemistry. Here we utilize electron paramagnetic resonance (EPR) and pulsed 35 GHz electron-nuclear double resonance (ENDOR) spectroscopy to address this question. EPR spectroscopy reveals a dramatic effect of AdoMet on the EPR spectrum of the [4Fe-4S](+) of PFL-AE, changing it from rhombic (g = 2.02, 1.94, 1.88) to nearly axial (g = 2.01, 1.88, 1.87). (2)H and (13)C ENDOR spectroscopy was performed on [4Fe-4S](+)-PFL-AE (S = (1)/(2)) in the presence of AdoMet labeled at the methyl position with either (2)H or (13)C (denoted [1+/AdoMet]). The observation of a substantial (2)H coupling of approximately 1 MHz ( approximately 6-7 MHz for (1)H), as well as hyperfine-split signals from the (13)C, manifestly require that AdoMet lie close to the cluster. (2)H and (13)C ENDOR data were also obtained for the interaction of AdoMet with the diamagnetic [4Fe-4S](2+) state of PFL-AE, which is visualized through cryoreduction of the frozen [4Fe-4S](2+)/AdoMet complex to form the reduced state (denoted [2+/AdoMet](red)) trapped in the structure of the oxidized state. (2)H and (13)C ENDOR spectra for [2+/AdoMet](red) are essentially identical to those obtained for the [1+/AdoMet] samples, showing that the cofactor binds in the same geometry to both the 1+ and 2+ states of PFL-AE. Analysis of 2D field-frequency (13)C ENDOR data reveals an isotropic hyperfine contribution, which requires that AdoMet lie in contact with the cluster, weakly interacting with it through an incipient bond/antibond. From the anisotropic hyperfine contributions for the (2)H and (13)C ENDOR, we have estimated the distance from the closest methyl proton of AdoMet to the closest iron of the cluster to be approximately 3.0-3.8 A, while the distance from the methyl carbon to the nearest iron is approximately 4-5 A. We have used this information to construct a model for the interaction of AdoMet with the [4Fe-4S](2+/+) cluster of PFL-AE and have proposed a mechanism for radical generation that is consistent with these results.