Substrate-derived Radical Research Articles

Radical-Mediated Photocatalysis for Lignocellulosic Biomass Conversion into Value-Added Chemicals and Hydrogen: Facts, Opportunities and Challenges.

Photocatalytic biomass conversion into high-value chemicals and fuels is considered one of the hottest ongoing research and industrial topics toward sustainable development. In short, this process can cleave Cβ -O/Cα -Cβ bonds in lignin to aromatic platform chemicals, and further conversion of the polysaccharides to other platform chemicals and H2 . From the chemistry point of view, the optimization of the unique cooperative interplay of radical oxidation species (which are activated via molecular oxygen species, ROSs) and substrate-derived radical intermediates by appropriate control of their type and/or yield is key to the selective production of desired products. Technically, several challenges have been raised that face successful real-world applications. This review aims to discuss the recently reported mechanistic pathways toward selective biomass conversion through the optimization of ROSs behavior and materials/system design. On top of that, through a SWOT analysis, we critically discussed this technology from both chemistry and technological viewpoints to help the scientists and engineers bridge the gap between lab-scale and large-scale production.

Radical Reaction Control in the AdoMet Radical Enzyme CDG Synthase (QueE): Consolidate, Destabilize, Accelerate.

Controlling radical intermediates and thus catalysing and directing complex radical reactions is a central feature of S‐adensosylmethionine (SAM)‐dependent radical enzymes. We report ab initio and DFT calculations highlighting the specific influence of ion complexation, including Mg2+, identified as a key catalytic component on radical stability and reaction control in 7‐carboxy‐7‐deazaguanine synthase (QueE). Radical stabilisation energies (RSEs) of key intermediates and radical clock‐like model systems of the enzyme‐catalysed rearrangement of 6‐carboxytetrahydropterin (CPH4), reveals a directing role of Mg2+ in destabilising both the substrate‐derived radical and corresponding side reactions, with the effect that the experimentally‐observed rearrangement becomes dominant over possible alternatives. Importantly, this is achieved with minimal disruption of the thermodynamics of the substrate itself, affording a novel mechanism for an enzyme to both maintain binding potential and accelerate the rearrangement step. Other mono and divalent ions were probed with only dicationic species achieving the necessary radical conformation to facilitate the reaction.

Substrate and Inhibitor Spectra of Ethylbenzene Dehydrogenase: Perspectives on Application Potential and Catalytic Mechanism

Ethylbenzene dehydrogenase (EbDH) catalyzes the initial step in anaerobic degradation of ethylbenzene in denitrifying bacteria, namely, the oxygen-independent hydroxylation of ethylbenzene to (S)-1-phenylethanol. In our study we investigate the kinetic properties of 46 substrate analogs acting as substrates or inhibitors of the enzyme. The apparent kinetic parameters of these compounds give important insights into the function of the enzyme and are consistent with the predicted catalytic mechanism based on a quantum chemical calculation model. In particular, the existence of the proposed substrate-derived radical and carbocation intermediates is substantiated by the formation of alternative dehydrogenated and hydroxylated products from some substrates, which can be regarded as mechanistic models. In addition, these results also show the surprisingly high diversity of EbDH in hydroxylating different kinds of alkylaromatic and heterocyclic compounds to the respective alcohols. This may lead to attractive industrial applications of ethylbenzene dehydrogenase for a new process of producing alcohols via hydroxylation of the corresponding aromatic hydrocarbons rather than the customary procedure of reducing the corresponding ketones.

Tangled Up in Knots: Structures of Inactivated Forms of E. coli Class Ia Ribonucleotide Reductase

Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4β4 rings. Here, we present the first X-ray structure of a gemcitabine-inhibited E. coli RNR and show that the previously described α4β4 rings can interlock to form an unprecedented (α4β4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNRs as well as present a mechanism for the formation of these unusual structures.

On the Reaction of Glycerol Dehydratase with But‐3‐ene‐1,2‐diol

High-level conventional ab initio and density functional theory (DFT) calculations have been performed to examine the fate of the native substrate glycerol (1) and its analogue but-3-ene-1,2-diol (7) in the coenzyme B(12)-dependent enzyme glycerol dehydratase (GDH). Experimental studies find that 7 irreversibly inactivates GDH, though the mechanism for the inactivation remains unknown. Interestingly, the EPR data suggest that the spin density for an observed radical is located in the vicinity of the C1 atom, which has been interpreted to indicate termination of the pathway at the substrate-derived radical 8. Our calculations show that if analogue 7 were to follow a similar mechanistic pathway to that followed by 1, then the reaction would be unlikely to stop at 8 but would rather proceed to the highly stabilized product-related radical 9. However, the EPR characteristics of 9 would not be consistent with the observed EPR data. Calculations involving an initial H-atom abstraction from the C2 position of 7 identify alternative radicals that might account for the EPR data, though they are of relatively high energy. A proposal that could explain the experimental observations is that the enzyme binds 7 in such a manner as to prevent the efficient transformation of 8. Recent work with the related enzyme diol dehydratase suggests that a common, but as yet unexplained, inactivation mechanism may be operative for both enzymes. Finally, we note the good overall performance of the MPWB1K, BMK, M05-2X, and B2-PLYP DFT procedures for these reactions, with the BMK method producing the best results.

The Carbon-Skeleton Rearrangement in Tropane Alkaloid Biosynthesis

High-level quantum chemistry calculations have been performed to examine the carbon-skeleton rearrangement of the tropane alkaloid littorine to hyoscyamine. Two pathways involving radical and carbocation intermediates have been investigated in this regard, namely, stepwise (or fragmentation-recombination) and concerted. The fragmentation products are calculated to be of high energy for both the radical- and carbocation-based mechanisms (136.3 and 170.9 kJ mol(-1), respectively). Similarly, the rearrangement barrier for the radical-based concerted pathway is calculated to be quite high (135.6 kJ mol(-1)). In contrast, the carbocation-based concerted pathway is found to be associated with a relatively low barrier (47.4 kJ mol(-1)). The ionization energy of the substrate-derived radical 3a is calculated to be 7.01 eV, suggesting that its oxidation to generate the substrate-derived carbocation 3b ought to be facile. In an attempt to investigate how an enzyme might modulate the rearrangement barriers, the separate and combined influences of partially protonating the migrating group and partially deprotonating the spectator OH group of the substrate were investigated. Such interactions can lead to significant reductions in the rearrangement barrier for both the radical- and carbocation-based concerted pathways, although the carbocation pathway continues to have significantly lower energy requirements. Also, the relatively high (gas-phase) acidity of the OH group of the product-related carbocation 4b indicates that the direct formation of hyoscyamine aldehyde (6) is a highly exothermic process. Although we would not wish to rule out alternative possibilities, our calculations suggest that a concerted rearrangement mechanism involving carbocations constitutes a viable low-energy pathway for the carbon-skeleton rearrangement in tropane alkaloid biosynthesis.

In Search of Radical Intermediates in the Reactions Catalyzed by Lysine 2,3-Aminomutase and Lysine 5,6-Aminomutase

High-level ab initio calculations have been used to study radical intermediates in the reactions catalyzed by lysine 2,3-aminomutase (2,3-LAM) and lysine 5,6-aminomutase (5,6-LAM). The reactions of these enzymes with the substrate analogues 4-oxalysine (4-OL), 4-thialysine (4-TL), or trans-4,5-dehydrolysine (t-4,5-DL) are rationalized in terms of stabilization provided by the substituent to the adjacent radical center. Large changes in the exothermicity accompanying the initial H-abstraction are observed relative to the lysine reference values that follow the series 4-OL < 4-TL < t-4,5-DL. These changes have the primary effect of increasing the endothermicity for subsequent ring-closure to form the putative aziridinylcarbinyl radical intermediate. Such stabilization is consistent with experimental observations of the substrate-derived radical (S*) in the reaction of 2,3-LAM with 4-TL as well as the ability of t-4,5-DL to act as an irreversible inhibitor of 2,3-LAM. Our calculations suggest that 4-TL and trans-3,4-dehydrolysine may also permit experimental characterization of S* radicals in the reactions catalyzed by 5,6-LAM. Strategies for modifying PLP are presented that might lead to the first observation of the aziridinylcarbinyl radical intermediate (I*) in the aminomutase-catalyzed reactions.

Identification of the 1,2-Propanediol-1-yl Radical as an Intermediate in Adenosylcobalamin-Dependent Diol Dehydratase Reaction

The reaction catalyzed by adenosylcobalamin-dependent diol dehydratase proceeds by a radical mechanism. A radical pair consisting of the Co(II) of cob(II)alamin and an organic radical intermediate formed during catalysis gives EPR spectra. The high-field doublet and the low-field broad signals arise from the weak interaction of an organic radical with the low-spin Co(II) of cob(II)alamin. To characterize the organic radical intermediate in the diol dehydratase reaction, several deuterated and (13)C-labeled 1,2-propanediols were synthesized, and the EPR spectra observed in the catalysis were measured using them as substrate. The EPR spectra with the substrates deuterated on C1 showed significant line width narrowing of the doublet signal. A distinct change in the hyperfine coupling was seen with [1-(13)C]-1,2-propanediol, but not with the [2-(13)C]-counterpart. Thus, the organic radical intermediate observed by EPR spectroscopy was identified as the 1,2-propanediol-1-yl radical, a C1-centered substrate-derived radical.

Spin–spin interaction in ethanolamine deaminase

The adenosylcobalamin coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium catalyzes the deamination of aminoethanol to acetaldehyde and ammonia. The radical intermediate observed during steady state turnover of substrate aminoethanol has been characterized by continuous wave electron paramagnetic resonance (EPR) spectroscopy [J. Am. Chem. Soc. 121 (1999) 10522]. This study presents simulations of EPR spectra of this radical intermediate. Quantitative fits to the EPR spectra are achieved with a model of isotropic exchange and magnetic dipolar interaction between the substrate-derived radical and the Co II in the corrin ring. The simulated parameters are compared with those of substrate analog 2-aminopropanol-derived radical in the same enzyme. The comparison confirms that the aminoethanol-derived product radical interacts more weakly with the Co II than the 2-aminopropanol-derived radical and suggests that the reduction of isotropic exchange between the aminoethanol-derived product radical and the Co II is probably due to orientational-dependent wave function overlap. Successful fits to the radical line shapes of different isotope substitutions unequivocally establish that the observed radical intermediate is an π-electron-based product radical. The derived principal hyperfine values for the 13C α and 1H α nucleus are consistent with previous electron nuclear double resonance (ENDOR) studies on similar radicals, thus providing reliable experimental hyperfine coupling constants for comparison with quantum mechanical-based calculations to gain further insight into the molecular structure of the observed radical.

Rotation of the exo-methylene group of (R)-3-methylitaconate catalyzed by coenzyme B(12)-dependent 2-methyleneglutarate mutase from Eubacterium barkeri.

2-Methyleneglutarate mutase from the anaerobe Eubacterium (Clostridium) barkeri is an adenosylcobalamin (coenzyme B(12))-dependent enzyme that catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Two possibilities for the mechanism of the carbon skeleton rearrangement of the substrate-derived radical to the product-related radical are considered. In both mechanisms an acrylate group migrates from C-3 of 2-methyleneglutarate to C-4. In the "addition-elimination" mechanism this 1,2-shift occurs via an intermediate, a 1-methylenecyclopropane-1,2-dicarboxylate radical, in which the migrating acrylate is simultaneously attached to both C-3 and C-4. In the "fragmentation-recombination" mechanism the migrating group, a 2-acrylyl radical, becomes detached from C-3 before it starts bonding to C-4. In an attempt to distinguish between these two possibilities we have investigated the action of 2-methyleneglutarate mutase on the stereospecifically deuterated substrates (Z)-3-methyl[2'-(2)H(1)]itaconate and (Z)-3-[2'-(2)H(1),methyl-(2)H(3)]methylitaconate. The enzyme catalyzes the equilibration of both compounds with their corresponding E-isomers and with a 1:1 mixture of the corresponding (E)- and (Z)-2-methylene[2'-(2)H(1)]glutarates, as shown by monitoring of the reactions with (1)H and (2)H NMR. In the initial phase of the enzyme-catalyzed equilibration a significant excess (8-11%) of (E)-3-methyl[2'-(2)H(1)]itaconate over its equilibrium value was observed ("E-overshoot"). The E-overshoot was only 3-4% with (Z)-3-[2'-(2)H(1),methyl-(2)H(3)]methylitaconate because the presence of the deuterated methyl group raises the energy barrier from 3-methylitaconate to the corresponding radical. The overshoot is explained by postulating that the migrating acrylate group has to overcome an additional energy barrier from the state leading back to the substrate-derived radical to the state leading forward to the product-related radical. It is concluded that the fragmentation-recombination mechanism can provide an explanation for the results in terms of an additional energy barrier, despite the higher calculated activation energy for this pathway.

Oxygenases without requirement for cofactors or metal ions.

Mono- and dioxygenases usually depend on a transition metal or an organic cofactor to activate dioxygen, or their organic substrate, or both. This review points out that there are at least two separate families of oxygenases without any apparent requirement for cofactors or metal ions: the quinone-forming monooxygenases which are important 'tailoring enzymes' in the biosynthesis of several types of aromatic polyketide antibiotics, and the bacterial dioxygenases involved in the degradation of distinct quinoline derivatives, catalyzing the 2,4-dioxygenolytic cleavage of 3-hydroxy-4-quinolones with concomitant release of carbon monoxide. The quinone-forming monooxygenases might be useful for the modification of polyketide structures, either by using them as biocatalysts, or by employing combinatorial biosynthesis approaches. Cofactor-less oxygenases present the mechanistically intriguing problem of how dioxygen is activated for catalysis. However, the reactions catalyzed by these enzymes are poorly understood in mechanistic terms. Formation of a protein radical and a substrate-derived radical, or direct electron transfer from a deprotonated substrate to molecular oxygen to form a caged radical pair may be discussed as hypothetical mechanisms. The latter reaction route is expected for substrates that can easily donate an electron to dioxygen, and requires the ability of the enzyme to stabilize anionic intermediates. Histidine residues found to be catalytically relevant in both types of cofactor-less oxygenases might be involved in substrate deprotonation and/or electrostatic stabilization.

Enzymatic radical catalysis: coenzyme B12-dependent diol dehydratase.

A peculiar function resides in a peculiar structure. Coenzyme B(12) or adenosylcobalamin, a naturally occurring organometallic compound, serves as a cofactor for enzymatic radical reactions. How do the enzymes form catalytic radicals at the active sites? How do the enzymes utilize and control the high reactivity of the radicals for catalysis? Recently, three-dimensional structures of several radical-containing or radical-forming enzymes including B(12) enzymes have been reported, enabling the analysis of the fine mechanisms of the action of these interesting enzymes. Our biochemical, mutational, and crystallographic studies as well as theoretical calculations on diol dehydratase, an adenosylcobalamin-dependent enzyme, revealed that its structure is adapted for its function-that is, activation of the Cobond;C bond toward homolysis, abstraction of a specific hydrogen atom from the substrate and its recombination to a particular product, and transition state stabilization in the hydroxyl group migration of a substrate-derived radical. The functions of K(+) and the active-site amino acid residues in enzyme catalysis are also investigated. Based on the results, the fine mechanism of the enzyme and the energetic feasibility of enzymatic radical catalysis are described here.

Pyrylium salt-photosensitized degradation of phenolic contaminants present in olive oil wastewater with solar light: Part III. Tyrosol and p-hydroxyphenylacetic acid

Photodegradation of tyrosol and p-hydroxyphenylacetic acid, present in olive oil wastewater, can be achieved using 2,4,6-triphenylpyrylium salt as solar photocatalyst. Fluorescence quenching measurements support the involvement of electron transfer as the key step in this process, with formation of substrate-derived radical cations. Important degradation yields are achieved. Other advanced oxidation methods (ozone and/or UV-radiation) have been tested as well; as expected, tyrosol and p-hydroxyphenylacetic acid abatement is faster, but ozone and UV are dangerous and expensive for industrial uses. However, ozone and UV do not show an important synergistic effect in tyrosol and p-hydroxyphenylacetic acid oxidation. p-Hydroxymandelic acid and p-hydroxybenzaldehyde have been detected and identified by HPLC as intermediates; on the basis of these data, a reaction mechanism involving oxidation at the benzylic position is proposed.

Direct participation of potassium ion in the catalysis of coenzyme B(12)-dependent diol dehydratase.

The direct ion-dipolar interactions between potassium ion (K(+)) and the two hydroxyl groups of the substrate are the most striking feature of the crystal structure of coenzyme B(12)-dependent diol dehydratase. We carried out density-functional-theory computations to determine whether K(+) can assist the 1,2-shift of the hydroxyl group in the substrate-derived radical. Between a stepwise abstraction/recombination reaction proceeding via a direct hydroxide abstraction by K(+) and a concerted hydroxyl group migration assisted by K(+), only a transition state for the latter concerted mechanism was found from our computations. The barrier height for the transition state from the complexed radical decreases by only 2.3 kcal/mol upon coordination of the migrating hydroxyl group to K(+), which corresponds to a 42-fold rate acceleration at 37 degrees C. The net binding energy upon replacement of the K(+)-bound water for substrate was calculated to be 10.7 kcal/mol. It can be considered that such a large binding energy is at least partly used for the substrate-induced conformational changes in the enzyme that trigger the homolytic cleavage of the Co-C bond of the coenzyme and the subsequent catalysis by a radical mechanism. We propose here a new mechanism for diol dehydratase in which K(+) plays a direct role in the catalysis.

Understanding the Mechanism of B12-Dependent Methylmalonyl-CoA Mutase: Partial Proton Transfer in Action

Ab initio molecular orbital theory is used to investigate several possible mechanisms involving free radical intermediates for the coenzyme-B12-dependent rearrangement catalyzed by methylmalonyl-CoA mutase. Our calculations suggest that an intermolecular pathway in which transient fragmentation of the substrate-derived radical is followed by recombination of the fragments (“fragmentation−recombination”) is unlikely, but not out of the question. An alternative intramolecular pathway (“addition−elimination”) is found to be energetically more favorable. Protonation of the species involved in this latter pathway is found to further reduce the barrier for rearrangement. Examination of the middle ground between the protonated and unprotonated intramolecular mechanisms reveals a continuous spectrum of behavior and demonstrates the potential importance of partial proton transfer. Support for this proposal is obtained from the X-ray crystal structure of the protein. The stereochemistry of the rearrangement has also been examined and leads to a new proposal consistent with experiment.

Detection of a new substrate-derived radical during inactivation of ribonucleotide reductase from Escherichia coli by gemcitabine 5'-diphosphate.

Ribonucleotide reductases (RNRs) play a central role in replication and repair by catalyzing the conversion of nucleotides to deoxynucleotides. Gemcitabine 5'-diphosphate (F2CDP), the nucleoside of which was recently approved by the FDA for treatment of pancreatic cancer, is a potent mechanism-based inhibitor of class I and II RNRs. Inactivation of the Eschericia coli class I RNR is accompanied by loss of two fluorides and one cytosine. This RNR is composed of two homodimeric subunits: R1 and R2. R1 is the site of nucleotide reduction, and R2 contains the essential diferric-tyrosyl radical cofactor. The mechanism of inactivation depends on the availability of reductant. In the presence of reductant [thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH or dithiothreitol], inhibition results from R1 inactivation. In the absence of reductant with prereduced R1 and R2, inhibition results from loss of the essential tyrosyl radical in R2. The same result is obtained with C754S/C759S-R1 in the presence of TR/TRR/NADPH. In both cases, tyrosyl radical loss is accompanied by formation of a new stable radical (0.15-0.25 equiv/RNR). EPR studies in 2H2O, with [U-2H]R1, and examination of the microwave power saturation of the observed signal, indicate by process of elimination that this new radical is nucleotide-based. In contrast to all previously investigated 2'-substituted nucleotide inhibitors of RNR, inactivation is not accompanied by formation of a new protein-associated chromophore under any conditions. The requirement for reductant in the R1 inactivation pathway, the lack of chromophore on the protein, the loss of two fluoride ions, and the stoichiometry of the inactivation all suggest a unique mechanism of RNR inactivation not previously observed with other 2'-substituted nucleotide inhibitors of RNR. This unique mode of inactivation is proposed to be responsible for its observed clinical efficacy.

Characterization of a Substrate-Derived Radical Detected during the Inactivation of Ribonucleotide Reductase fromEscherichia coliby 2‘-Fluoromethylene-2‘-deoxycytidine 5‘-Diphosphate

Ribonucleotide reductase (RDPR) from E. coli catalyzes the conversion of nucleotides to deoxynucleotides and contains an unusual tyrosyl radical−diferric cluster cofactor. (E)-2‘-Fluoromethylene-2‘-deoxycytidine 5‘-diphosphate [(E)-1] obtained by phosphorylation of the clinically promising antitumor agent MDL 101,731, is a potent time-dependent inactivator of this protein. Electron paramagnetic resonance (EPR) spectroscopy reveals that inactivation is accompanied by loss of the essential tyrosyl radical cofactor and formation of a new radical species. The 9.4-GHz EPR spectrum of this new radical reveals two hyperfine splittings of approximately 1.5 mT producing a triplet-like signal. Incubation of the enzyme with [6‘-2H]-(E)-1 alters this EPR spectrum, providing the first evidence for a nucleotide-based radical species generated by RDPR. The observed spectrum is a 1:1 composite of a doublet and a triplet signal, the latter being identical to that obtained with unlabeled (E)-1. Studies with (E)-1 in 2H2O also produce a 1:1 mixture of these two radical signals. The results of these isotope labeling experiments suggest wash-out or wash-in of ∼0.5 equiv of deuterium at the 6‘ position, respectively. Incubation of the enzyme with [6‘-2H]-(E)-1 in 2H2O produced only the doublet as would be expected on the basis of this hypothesis. EPR (139.5 GHz) spectra established the principal g values of the new radical species. Simulation of the 9.4- and 139.5-GHz EPR spectra yield a self-consistent set of principal hyperfine values. A structure is proposed for the radical intermediate that is consistent with the EPR data and kinetic data on release of the fluoride ion that accompanies inactivation. The proposed structure and a postulated mechanism for its formation provide further support for the hypothesis that catalysis is initiated by 3‘-hydrogen atom abstraction from the nucleotide substrate.

Mechanism of the Clostridium thermoaceticum pyruvate:ferredoxin oxidoreductase: evidence for the common catalytic intermediacy of the hydroxyethylthiamine pyropyrosphate radical.

The cofactor content and mechanism of pyruvate:ferredoxin oxidoreductase (PFOR) are controversial. By using rapid freeze-quench EPR and stopped-flow spectroscopy, the elementary steps that constitute the first half-reaction of the Clostridiumthermoaceticum PFOR mechanism were elucidated. A hydroxyethyl-TPP (HE-TPP) radical was identified and characterized as a transient intermediate, and for the first time, the kinetic competence of this substrate-derived radical was demonstrated. When the C. thermoaceticum PFOR was reacted with pyruvate and CoA, it had a lifetime of only approximately 100 ms. The results described here suggest that this radical intermediate is often not detected in studies of alpha-ketoacid oxidoreductases because it rapidly decays. It is postulated here that the HE-TPP radical is an intermediate in the mechanism of all PFORs irrespective of the number of 4Fe-4S clusters and will be detected in all PFORs when rapid mixing methods are used. The C. thermoaceticum PFOR was shown to contain two 4Fe-4S clusters, as concluded earlier [Wahl, R. C., & Orme-Johnson, W. H. (1987) J. Biol. Chem. 262, 10489-10496]. The first reductive half-reaction was shown to involve the following steps: (i) reaction with pyruvate with PFOR to form the hydroxyethylidene-TPP intermediate; (ii) one-electron transfer to reduce one of the two Fe4S4 clusters and yield the HE-TPP radical; and, (iii) reaction with CoA resulting in formation of acetyl-CoA, rapid decay of the HE-TPP radical intermediate, and reduction of the second Fe4S4 cluster. Thus, at the end of the first half-reaction, the two Fe4S4 clusters are fully reduced. The rate of the third step was found to depend on the CoA concentration (k = 35 per s at saturating concentrations of CoA); however, in its absence, this step was slower by approximately 4400-fold.

Cytochrome P-450-catalyzed rearrangement of a peroxyquinol derived from butylated hydroxytoluene. Involvement of radical and cationic intermediates.

The p-peroxyquinol derived from butylated hydroxytoluene, 2,6-di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone, was degraded by the ferric form of rat liver cytochrome P-450, and the resulting products and their mechanisms of formation were investigated. Quinoxy radical BO. from homolysis of the O-O bond reacted by competing pathways; beta-scission yielded 2,6-di-t-butyl-p-benzoquinone, and rearrangement with ring-expansion produced an oxacycloheptadienone free radical (X(.)). This rearranged radical was stabilized by the captodative effect that facilitated competitive interactions with the P-450 iron-oxo complexes formed during O-O bond scission. Approximately 15% of X(.) was captured by oxygen rebound with a hydroxyl radical from the P-450 complex (FeOH)3+ to form a hemiketal, that led to the ring-contracted product 2,5-di-t-butyl-5-(2'-oxopropyl)-4-oxa-2-cyclopentenone by spontaneous rearrangement. The major fraction of X(.), however, underwent electron transfer oxidation to form the corresponding cation. Hydration of this cation produced the ring-contracted product, and proton elimination (or, alternatively, direct H(.) removal from X(.) led to the product 2,7-di-t-butyl-4-methylene-5-oxacyclohepta-2,6-dienone. The findings indicate that cytochrome P-450 intermediate complexes are mainly responsible for oxidation of X(.). The results complement our previous study with 2,6-di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (Thompson, J. A., and Wand, M. D. (1985) J. Biol. Chem. 260, 10637-10644), demonstrating competitive heterolytic and homolytic mechanisms of O-O bond cleavage, and competitive rebound and oxidation processes when a substrate-derived radical interacts with P-450 complexes.

Electron spin-echo studies of the composition of the paramagnetic intermediate formed during the deamination of propanolamine by ethanolamine ammonia-lyase, and AdoCbl-dependent enzyme.

During the deamination of S-2-aminopropanol by the AdoCbl-dependent ethanolamine ammonia-lyase of Clostridia sp., a catalytic intermediate accumulates whose active site contains two paramagnetic species: cob(II)alamin and a free radical derived from the substrate molecule. Spin-echo spectroscopy has revealed that the unpaired electron on the substrate-derived radical is delocalized over a nitrogen atom that from its quadrupole splittings is probably a component of a secondary amide group. Experiments with 15N- and deuterium-labeled propanolamine gave no evidence of an interaction between this unpaired electron and the nitrogen originally attached to the substrate molecule. These results strongly suggest that the substrate-derived radical in this intermediate has already lost its nitrogen, and that this radical is stabilized by delocalization of the unpaired electron onto a nitrogen most likely situated in one of the peptide bonds of the enzyme backbone.