The role of radicals in enzymatic processes

Research on the mechanism of action of coenzyme B12, adenosylcobalamin, as a graduate student introduced the author to the field of organic free radicals in enzymology. Twenty years later, related work on S-adenosylmethionine (SAM) as a "poor man's coenzyme B12" was initiated in a detailed analysis of the mechanism of action of lysine 2,3-aminomutase (LAM). The interconversion of L-lysine and L-beta-lysine is catalyzed by LAM, which requires SAM, pyridoxal-5'-phosphate (PLP), and a [4Fe-4S] cluster as coenzymes. The mechanism of this reaction has been delineated as a radical isomerization, in which radical formation is initiated by the [4Fe-4S]-dependent cleavage of the SAM into methionine and the 5'-deoxyadenosyl radical. The mechanism of this process is discussed, together with the role of this radical in hydrogen abstraction from lysine to initiate the substrate radical isomerization. The chemistry underlying the functions of SAM, PLP, and [4Fe-4S] in the action of LAM is novel in all respects, except for the formation of a lysine-PLP aldimine at the active site. Of the four free radicals in the mechanism, three have been characterized by EPR spectroscopy. In the suicide inactivation of adenosylcobalamin-dependent dioldehydrase (DDH) by glycolaldehyde, the formation of cob(II)alamin and 5'-deoxyadenosine is accompanied by the conversion of glycolaldehyde to cis-ethanesemidione radical at the active site. The cis-ethanesemidione radical has been characterized by EPR spectroscopy. Its exceptional stability at the active site is the basis for the inactivation of DDH by glycolaldehyde.     

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.     

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.     

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.     

A nucleosidase required for in vivo function of the S-adenosyl-L-methionine radical enzyme, biotin synthase

Biotin synthase is an S-adenosyl-L-methionine (SAM) radical enzyme that inserts sulfur into dethiobiotin to produce biotin. The reaction proceeds through 5'-deoxyadenosyl radical intermediates that become reduced during the sulfur insertion step to give another product of the reaction, 5'-deoxyadenosine. We report that Escherichia coli strains lacking the 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase encoded by the pfs gene are deficient in biotin synthase activity due to accumulation of 5'-deoxyadenosine, a new substrate of the pfs-encoded nucleosidase. Physiological experiments indicate that lipoic acid synthase, another SAM radical enzyme, is also inhibited by 5'-deoxyadenosine accumulation.     

The photo-dissociation of the pyrrole-ammonia complex--the role of hydrogen bonding in Rydberg states photochemistry

The photochemistry of the pyrrole-ammonia cluster is analyzed theoretically. Whereas in neat pyrrole the dominant photochemical reaction is H-atom cleavage, recent experiments show that in pyrrole-ammonia clusters the major reaction is H-transfer to form the NH(4) radical (solvated by ammonia molecules in the case of large clusters) and the pyrrolyl radical. A mechanism involving the hydrogen-bonded Rydberg state is offered to account for these results and verified computationally. Two minima are located on the lowest excited singlet PES. Both of them are Rydberg states, one leads to the formation of NH(4) and pyrrolyl radicals, the other is connected to the πσ* state through a relatively high barrier, leading to a 3-body dissociation reaction to form a pyrrolyl radical, ammonia and an H-atom. The former is the energetically and statistically preferred one.     

S K-edge XAS and DFT calculations on SAM dependent pyruvate formate-lyase activating enzyme: nature of interaction between the Fe4S4 cluster and SAM and its role in reactivity

S K-edge X-ray absorption spectroscopy on the resting oxidized and the S-adenosyl-l-methionine (SAM) bound forms of pyruvate formate-lyase activating enzyme are reported. The data show an increase in pre-edge intensity, which is due to additional contributions from sulfide and thiolate of the Fe(4)S(4) cluster into the C-S σ* orbital. This experimentally demonstrates that there is a backbonding interaction between the Fe(4)S(4) cluster and C-S σ* orbitals of SAM in this inner sphere complex. DFT calculations that reproduce the data indicate that this backbonding is enhanced in the reduced form and that this configurational interaction between the donor and acceptor orbitals facilitates the electron transfer from the cluster to the SAM, which otherwise has a large outer sphere electron transfer barrier. The energy of the reductive cleavage of the C-S bond is sensitive to the dielectric of the protein in the immediate vicinity of the site as a high dielectric stabilizes the more charge separated reactant increasing the reaction barrier. This may provide a mechanism for generation of the 5'-deoxyadenosyl radical upon substrate binding.     

Expanding Radical SAM Chemistry by Using Radical Addition Reactions and SAM Analogues

Radical S‐adenosyl‐l ‐methionine (SAM) enzymes utilize a [4Fe‐4S] cluster to bind SAM and reductively cleave its carbon–sulfur bond to produce a highly reactive 5′‐deoxyadenosyl (dAdo) radical. In almost all cases, the dAdo radical abstracts a hydrogen atom from the substrates or from enzymes, thereby initiating a highly diverse array of reactions. Herein, we report a change of the dAdo radical‐based chemistry from hydrogen abstraction to radical addition in the reaction of the radical SAM enzyme NosL. This change was achieved by using a substrate analogue containing an olefin moiety. We also showed that two SAM analogues containing different nucleoside functionalities initiate the radical‐based reactions with high efficiencies. The radical adduct with the olefin produced in the reaction was found to undergo two divergent reactions, and the mechanistic insights into this process were investigated in detail. Our study demonstrates a promising strategy in expanding radical SAM chemistry, providing an effective way to access nucleoside‐containing compounds by using radical SAM‐dependent reactions.     

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.     

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.     

Hydrogen Transfer in SAM‐Mediated Enzymatic Radical Reactions

S‐Adenosylmethionine (SAM) plays an essential role in a variety of enzyme‐mediated radical reactions. One‐electron reduction of SAM is currently believed to generate the C5′‐desoxyadenosyl radical, which subsequently abstracts a hydrogen atom from the actual substrate in a catalytic or a non‐catalytic fashion. Using a combination of theoretical and experimental bond dissociation energy (BDE) data, the energetics of these radical processes have now been quantified. SAM‐derived radicals are found to react with their respective substrates in an exothermic fashion in enzymes using SAM in a stoichiometric (non‐catalytic) way. In contrast, the catalytic use of SAM appears to be linked to a sequence of moderately endothermic and exothermic reaction steps. The use of SAM in spore photoproduct lyase (SPL) appears to fit neither of these general categories and appears to constitute the first example of a SAM‐initiated radical reaction propagated independently of the cofactor.     

Probing the reaction mechanism of spore photoproduct lyase (SPL) via diastereoselectively labeled dinucleotide SP TpT substrates

5-Thyminyl-5,6-dihydrothymine (commonly called spore photoproduct or SP) is the exclusive DNA photodamage product in bacterial endospores. It is generated in the bacterial sporulation phase and repaired by a radical SAM enzyme, spore photoproduct lyase (SPL), at the early germination phase. SPL utilizes a special [4Fe-4S] cluster to reductively cleave S-adenosylmethionine (SAM) to generate a reactive 5'-dA radical. The 5'-dA radical is proposed to abstract one of the two H-atoms at the C6 carbon of SP to initiate the repair process. Via organic synthesis and DNA photochemistry, we selectively labeled the 6-H(proS) or 6-H(proR) position with a deuterium in a dinucleotide SP TpT substrate. Monitoring the deuterium migration in enzyme catalysis (employing Bacillus subtilis SPL) revealed that it is the 6-H(proR) atom of SP that is abstracted by the 5'-dA radical. Surprisingly, the abstracted deuterium was not returned to the resulting TpT after enzymatic catalysis; an H-atom from the aqueous buffer was incorporated into TpT instead. This result questions the currently hypothesized SPL mechanism which excludes the involvement of protein residue(s) in SPL reaction, suggesting that some protein residue(s), which is capable of exchanging a proton with the aqueous buffer, is involved in the enzyme catalysis. Moreover, evidence has been obtained for a possible SAM regeneration after each catalytic cycle; however, such a regeneration process is more complex than currently thought, with one or even more protein residues involved as well. These observations have enabled us to propose a modified reaction mechanism for this intriguing DNA repair enzyme.     

Stabilisation of methylene radicals by cob(II)alamin in coenzyme B12 dependent mutases

Coenzyme B12 initiates radical chemistry in two types of enzymatic reactions, the irreversible eliminases (e.g., diol dehydratases) and the reversible mutases (e.g., methylmalonyl-CoA mutase). Whereas eliminases that use radical generators other than coenzyme B12 are known, no alternative coenzyme B12 independent mutases have been detected for substrates in which a methyl group is reversibly converted to a methylene radical. We predict that such mutases do not exist. However, coenzyme B12 independent pathways have been detected that circumvent the need for glutamate, beta-lysine or methylmalonyl-CoA mutases by proceeding via different intermediates. In humans the methylcitrate cycle, which is ostensibly an alternative to the coenzyme B12 dependent methylmalonyl-CoA pathway for propionate oxidation, is not used because it would interfere with the Krebs cycle and thereby compromise the high-energy requirement of the nervous system. In the diol dehydratases the 5'-deoxyadenosyl radical generated by homolysis of the carbon-cobalt bond of coenzyme B12 moves about 10 A away from the cobalt atom in cob(II)alamin. The substrate and product radicals are generated at a similar distance from cob(II)alamin, which acts solely as spectator of the catalysis. In glutamate and methylmalonyl-CoA mutases the 5'-deoxyadenosyl radical remains within 3-4 A of the cobalt atom, with the substrate and product radicals approximately 3 A further away. It is suggested that cob(II)alamin acts as a conductor by stabilising both the 5'-deoxyadenosyl radical and the product-related methylene radicals.     

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.     

A QM/MM simulation study of transamination reaction at the active site of aspartate aminotransferase: Free energy landscape and proton transfer pathways

Transaminase is a key enzyme for amino acid metabolism, which reversibly catalyzes the transamination reaction with the help of PLP (pyridoxal 5^' -phosphate) as its cofactor. Here we have investigated the mechanism and free energy landscape of the transamination reaction involving the aspartate transaminase (AspTase) enzyme and aspartate-PLP (Asp-PLP) complex using QM/MM simulation and metadynamics methods. The reaction is found to follow a stepwise mechanism where the active site residue Lys258 acts as a base to shuttle a proton from α -carbon (CA) to imine carbon (C4A) of the PLP-Asp Schiff base. In the first step, the Lys258 abstracts the CA proton of the substrate leading to the formation of a carbanionic intermediate which is followed by the reprotonation of the Asp-PLP Schiff base at C4A atom by Lys258. It is found that the free energy barrier for the proton abstraction by Lys258 and that for the reprotonation are 17.85 and 3.57 kcal/mol, respectively. The carbanionic intermediate is 7.14 kcal/mol higher in energy than the reactant. Hence, the first step acts as the rate limiting step. The present calculations also show that the Lys258 residue undergoes a conformational change after the first step of transamination reaction and becomes proximal to C4A atom of the Asp-PLP Schiff base to favor the second step. The active site residues Tyr70* and Gly38 anchor the Lys258 in proper position and orientation during the first step of the reaction and stabilize the positive charge over Lys258 generated at the intermediate step.     

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.     

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.     

Mechanisms of reaction of aminoxyl (nitroxide), iminoxyl, and imidoxyl radicals with alkenes and evidence that in the presence of lead tetraacetate, N-hydroxyphthalimide reacts with alkenes by both radical and nonradical mechanisms

1,2-dideuterio-cyclohexene, 1,2-dideuterio-cyclooctene, and trans-3,4-dideuterio-hex-3-ene were reacted with three >NO* radicals: 4-hydroxyTempo, di-tert-butyliminoxyl, both used as the actual radicals, and phthalimide-N-oxyl (PINO) generated from N-hydroxyphthalimide (NHPI) by its reaction with tert-alkoxyl radicals (t-RO*) and with lead tetraacetate. In all cases, except the NHPI/Pb(OAc)4 system, only mono >NO-substituted alkenes were produced. The 2H NMR spectra imply that 88-92% of monoadducts were formed by the initial abstraction of an allylic H-atom, followed by capture of the allylic radical by a second >NO*, while the remaining 12-8% appear to be formed by an initial addition of >NO* to the double bond followed by H-atom abstraction by a second >NO*. A substantial and sometimes the major product formed with the NHPI/Pb(OAc)4 system has two PINO moieties added across the double bond. Since such diadducts are not formed with the NHPI/t-RO* system, a heterolytic mechanism is proposed, analogous to that known for the Pb(OAc)4-induced acetoxylation of alkenes. A detailed analysis of the NHPI/Pb(OAc)4/alkene products indicates that monosubstitution occurs by both homolytic and heterolytic processes.     

Computational insights into the mechanism of radical generation in B12-dependent methylmalonyl-CoA mutase

ONIOM calculations have provided novel insights into the mechanism of homolytic Co-C5' bond cleavage in the 5'-deoxyadenosylcobalamin cofactor catalyzed by methylmalonyl-CoA mutase. We have shown that it is a stepwise process in which conformational changes in the 5'-deoxyadenosine moiety precede the actual homolysis step. In the transition state structure for homolysis, the Co-C5' bond elongates by approximately 0.5 Angstroms from the value found in the substrate-bound reactant complex. The overall barrier to homolysis is approximately 10 kcal/mol, and the radical products are approximately 2.5 kcal/mol less stable than the initial ternary complex of enzyme, substrate, and cofactor. The movement of the deoxyadenosine moiety during the homolysis step positions the resulting 5'-deoxyadenosyl radical for the subsequent hydrogen atom transfer from the substrate, methylmalonyl-CoA.     

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.