In silico studies of the mechanism of methanol oxidation by quinoprotein methanol dehydrogenase

The mechanism of bacterial methanol dehydrogenase involves hydride equivalent transfer from substrate to the ortho-quinone PQQ to provide a C5-reduced intermediate that subsequently rearranges to the hydroquinone PQQH(2). We have studied the PQQ reduction by molecular dynamic (MD) simulations in aqueous solution. Among the five simulated structures, either Asp297 or Glu171 or both are ionized. Reasonable structures are obtained only when both carboxyl groups are ionized. This is not unexpected since the kinetic pH optimum is 9.0. In the structure of the enzyme.PQQ.HOCH(3) complex, the hydrogen bonded Glu171-CO(2)(-).H-OCH(3) is in a position to act as a general base catalyst for hydride equivalent transfer to C5 of PQQ. We thus suggest that Glu171 plays the role of general base catalyst in PQQ reduction rather than Asp297 as previously suggested. The reduction is assisted by Arg324, which hydrogen bonds to the ortho-quinone moiety of PQQ. The rearrangement of the C5-reduced intermediate to provide hydroquinone PQQH(2) is also assisted by proton abstraction by Glu171-CO(2)(-) and the continuous hydrogen bonding of Arg324 throughout the entire reaction. These features as well as the mapping of the channel for substrate and water into the active site entrance are the observations of major importance.     

Mechanism of glucose oxidation by quinoprotein soluble glucose dehydrogenase: insights from molecular dynamics studies

We have generated 3 ns molecular dynamic (MD) simulations, in aqueous solution, of the bacterial soluble glucose dehydrogenase enzyme.PQQ.glucose complex and intermediates formed in PQQ reduction. In the MD structure of enzyme.PQQ.glucose complex the imidazole of His144 is hydrogen bonded to the hydroxyl hydrogen of H[bond]OC1(H) of glucose. The tightly hydrogen-bonded triad Asp163-His144-glucose (2.70 and 2.91 A) is involved in proton abstraction from glucose concerted with the hydride transfer from the C1[bond]H of glucose to the >C5[double bond]O quinone carbon of PQQ. The reaction is assisted by Arg228 hydrogen bonding to the carbonyl oxygen of >C5[double bond]O. The rearrangement of [bond](H)C5(O-)[bond]C4([double bond]O)[bond] of II to [bond]C5(OH)[double bond]C4(OH)[bond] of PQQH(2) hydroquinone is assisted by general acid protonatation of the >C4[double bond]O oxygen by protonated His144 and hydrogen bonds of Arg228 to the oxyanion O5. The continuous hydrogen bonding of the amide side chain of Asn229 to >C4[double bond]O4 oxygen and that of the O5 oxygen of the cofactor to Wat89 is observed throughout the entire reaction.     

Unexpected Direct Hydride Transfer Mechanism for the Hydrogenation of Ethyl Acetate to Ethanol Catalyzed by SNS Pincer Ruthenium Complexes

The hydrogenation of ethyl acetate to ethanol catalyzed by SNS pincer ruthenium complexes was computationally investigated by using DFT. Different from a previously proposed mechanism with fac‐[(SNS)Ru(PPh₃)(H)₂] ( 5′ ) as the catalyst, an unexpected direct hydride transfer mechanism with a mer‐SNS ruthenium complex as the catalyst, and two cascade catalytic cycles for hydrogenations of ethyl acetate to aldehyde and aldehyde to ethanol, is proposed base on our calculations. The new mechanism features ethanol‐assisted proton transfer for H₂ cleavage, direct hydride transfer from ruthenium to the carbonyl carbon, and C?OEt bond cleavage. Calculation results indicate that the rate‐determining step in the whole catalytic reaction is the transfer of a hydride from ruthenium to the carbonyl carbon of ethyl acetate, with a total free energy barrier of only 26.9 kcal mol^?1, which is consistent with experimental observations and significantly lower than the relative free energy of an intermediate in a previously postulated mechanism with 5′ as the catalyst.     

DFT Study of the Active Site of the XoxF‐Type Natural,Cerium‐Dependent Methanol Dehydrogenase Enzyme

Rare‐earth metal cations have recently been demonstrated to be essential co‐factors for the growth of the methanotrophic bacterium Methylacidiphilum fumariolicum SolV. A crystal structure of the rare‐earth‐dependent methanol dehydrogenase (MDH) includes a cerium cation in the active site. Herein, the Ce–MDH active site has been analyzed through DFT calculations. The results show the stability of the Ce^III–pyrroloquinoline quinone (PQQ) semiquinone configuration. Calculations on the active oxidized form of this complex indicate a 0.81 eV stabilization of the PQQ⁰ LUMO at cerium versus calcium, supporting the observation that the cerium cation in the active site confers a competitive advantage to Methylacidiphilum fumariolicum SolV. Using reported aqueous electrochemical data, a semi‐empirical correlation was established based on cerium(IV/III) redox potentials. The correlation allowed estimation of the cerium oxidation potential of +1.35 V versus saturated calomel electrode (SCE) in the active site. The results are expected to guide the design of functional model complexes and alcohol‐oxidation catalysts based on lanthanide complexes of biologically relevant quinones.     

Computational study of ketosteroid isomerase: insights from molecular dynamics simulation of enzyme bound substrate and intermediate

Delta(5)-3-Ketosteroid Isomerase (KSI) catalyzes the isomerization of 5,6-unsaturated ketosteroids to their 4,5-unsaturated isomers at a rate approaching the diffusion limit. The isomerization reaction follows a two-step general acid-base mechanism starting with Asp38-CO(2)(-) mediated proton abstraction from a sp(3)-hybridized carbon atom, alpha to carbonyl group, providing a dienolate intermediate. In the second step, Asp38-CO(2)H protonates the C6 of the intermediate providing a 4,5-unsaturated ketosteroid. The details of the mechanism have been highly controversial despite several experimental and computational studies of this enzyme. The general acid-base catalysis has been proposed to involve either a catalytic diad or a cooperative hydrogen bond mechanism. In this paper, we report our results from the 1.5 nanosecond molecular dynamics (MD) simulation of enzyme bound natural substrate (E.S) and enzyme bound intermediate (E.In) solvated in a TIP3P water box. The final coordinates from our MD simulation strongly support the cooperative hydrogen bond mechanism. The MD simulation of E.S and E.In shows that both Tyr14 and Asp99 are hydrogen bonded to the O3 of the substrate or intermediate. The average hydrogen bonding distance between Tyr14-OH and O3 becomes shorter and exhibits less fluctuation on E.S --> E.In. We also observe dynamic motions of water moving in and out of the active site in the E.S structures. This free movement of water disappears in the E.In structures. The active site is shielded by hydrophobic residues, which come together and squeeze out the waters from the active site in the E.In complex.     

Identity hydride-ion transfer from C-H donors to C acceptor sites. Enthalpies of hydride addition and enthalpies of activation. Comparison with C...H...C proton transfer. An ab initio study

Enthalpies of addition of hydride ion to eleven carbonyl acceptors (X-CHO), two conjugate addition sites (X-CH=CH2; X = CHO, NO2), eight carbenium ion acceptors, fulvene, borane, and SiH3(+) were calculated at the MP2/6-311+G level. Correlation between calculated and experimental enthalpies of addition of hydride ion is excellent. Transition states (ts) for the identity hydride transfers between the acceptors and their corresponding hydride adducts (hydride donors) were also calculated. The carbonyl and fulvene reactions have transition states with one imaginary frequency: the hydrogen transfer coordinate. The carbenium ions, borane, and SiH3(+) gave not transition states but stable compounds upon addition of the hydride donor. Computational differences between these hydride transfers and previously reported proton transfers include shorter partial C...H bonds and a tendency toward bent C...H...C angles for the hydride transfer ts and addition compound structures, particularly when a bent geometry improves interactions elsewhere in the structure. These and other differences are explained by modeling the hydride transfer ts and addition compounds as two-electron, three-center systems involving the transfer termini and the shared hydrogen but the proton transfer ts structures as four-electron, three-center systems. Charge and geometry measures suggest transition states in which these features change synchronously, again in contrast to many proton transfer reactions. For the X-CHO set, polar effects dominate enthalpies of hydride addition, with resonance effects also important for resonance donors; these preferentially stabilize the acceptor, reducing its hydride ion affinity. Activation enthalpies are dominated by resonance stabilization of the acceptors, greatly attenuated in the transition states.     

POSSIBLE HYDRIDE AND METHIDE TRANSFER REACTIONS: REACTIONS OF Fe(CO)4R−(R = H,CH3) AND W(CO)5R− (R = H,CH3, Cl,Br, I) WITH METAL CARBONYL CATIONS

Abstract

Reactions of metal carbonyl cations (M(CO)₆ ⁺, M = Mn, Re) with hydride-, methide- or halide-containing metal carbonyl anions (Fe(CO)₄R^?, R = H, Me; W(CO)₅R^?, R = H, Me, Cl, Br, I) produce products that indicate several mechanisms are operative. Reactions of the halo-tungsten complexes produce neutral, solvated tungsten complexes, W(CO)₅(CH₃CN) and W(CO)₄(CH₃CN)₂ and M(CO)₅X in a reaction that appears to be initiated by decomposition of W(CO)₅X^?. In contrast, the tungsten hydride and methide complexes react, predominantly, by transfer of the hydride or methide to a carbonyl of the cation at a much faster rate. The iron hydride and methide complexes react by iron-based nucleophilicity involving a two-electron process.     

Catalytic Electrophilic CH Silylation of Pyridines Enabled by Temporary Dearomatization

A C? H silylation of pyridines that seemingly proceeds through electrophilic aromatic substitution (S_EAr) is reported. Reactions of 2‐ and 3‐substituted pyridines with hydrosilanes in the presence of a catalyst that splits the Si? H bond into a hydride and a silicon electrophile yield the corresponding 5‐silylated pyridines. This formal silylation of an aromatic C? H bond is the result of a three‐step sequence, consisting of a pyridine hydrosilylation, a dehydrogenative C? H silylation of the intermediate enamine, and a 1,4‐dihydropyridine retro‐hydrosilylation. The key intermediates were detected by ¹H NMR spectroscopy and prepared through the individual steps. This complex interplay of electrophilic silylation, hydride transfer, and proton abstraction is promoted by a single catalyst.     

B(C6F5)3‐Catalyzed Transfer of Dihydrogen from One Unsaturated Hydrocarbon to Another

A transition‐metal‐free transfer hydrogenation of 1,1‐disubstituted alkenes with cyclohexa‐1,4‐dienes as the formal source of dihydrogen is reported. The process is initiated by B(C₆F₅)₃‐mediated hydride abstraction from the dihydrogen surrogate, forming a Brønsted acidic Wheland complex and [HB(C₆F₅)₃]^?. A sequence of proton and hydride transfers onto the alkene substrate then yields the alkane. Although several carbenium ion intermediates are involved, competing reaction channels, such as dihydrogen release and cationic dimerization of reactants, are largely suppressed by the use of a cyclohexa‐1,4‐diene with methyl groups at the C1 and C5 as well as at the C3 position, the site of hydride abstraction. The alkene concentration is another crucial factor. The various reaction pathways were computationally analyzed, leading to a mechanistic picture that is in full agreement with the experimental observations.     

Dynamic structures of horse liver alcohol dehydrogenase (HLADH): results of molecular dynamics simulations of HLADH-NAD(+)-PhCH(2)OH, HLADH-NAD(+)-PhCH(2)O(-), and HLADH-NADH-PhCHO.

Molecular dynamics simulations of the oxidation of benzyl alcohol by horse liver alcohol dehydrogenase (HLADH) have been carried out. The following three states have been studied: HLADH.PhCH(2)OH.NAD(+) (MD1), HLADH.PhCH(2)O(-).NAD(+) (MD2), and HLADH.PhCHO.NADH (MD3). MD1, MD2, and MD3 simulations were carried out on one of the subunits of the dimeric enzyme covered in a 32-A-radius sphere of TIP3P water centered on the active site. The proton produced on ionization of the alcohol when HLADH.PhCH(2)OH.NAD(+) --> HLADH.PhCH(2)O(-).NAD(+) is transferred from the active site to solvent water via a hydrogen bonding network consisting of serine48 hydroxyl, ribose 2'- and 3'-hydroxyl groups, and Hist51. Hydrogen bonding of the 3'OH of ribose to Ile269 carbonyl maintains this proton in position to be transferred to water. Molecular dynamic simulations have been employed to track water1287 from the TIP3 water pool to the active site, thus exhibiting the mode of entrance of water to the active site. With time the water1287 accumulates in two different positions in order to accept the proton from the ribose 3'-OH and from His51. There can be identified two structural substates for proton passage. In the first substate the imidazole Ne2 of His51 is adjacent to the nicotinamide ribose C2'-OH and hydrogen bonding distances for proton transfer through the hydrogen bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...His51...OH(2) (path 1) average 2.0, 2.0, and 2.1 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A. The structure for path 1 is present 20% of the time span. And in the second substate, there are two possible proton passages: path 1 as before and path 2. Path 2 involves the hydrogen-bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...Ribose3'-OH...His51.OH(2) with the average bonding distances being 2.0, 2.0, 2.1, and 2.0 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A (20% probability of the time span), respectively. During the molecular dynamics simulation the NAD(+) ribose conformations have stabilized at the C2'-endo-C3'-exo or the C2'-endo conformations. With the C2'-endo conformation the first and second substates are able to persist for different time spans, while with the C2'-endo-C3'-exo conformation the only possible pathway involves the first substate. For both first and second substates the fluctuation of the distances between the ribose-OH protons and N epsilon 2 of His51 imidazole ring is partially contributed by the "windshield wiper" motion of the His51 imidazole ring. Since the imidazole of His-51 contributes only about 10-fold to activity, as estimated from the decrease in activity upon substitution with a Gln, there must be an alternate route for the proton to pass to solvent without going through this histidine. A third pathway involves ribose C3'-OH and Ile-269. In MD2, near attack conformers (NACs) for hydride transfer from PhCH(2)O(-) to NAD(+) represent approximately 60% of E.S conformers. The molecular dynamic study of MD3 at mildly basic pH reveals that reactive ground state conformers (NACs) for hydride transfer from NADH to PhCHO amount to 12 mol % of conformers. In MD3, anisotropic bending of the dihydronicotinamide ring of NADH (average value of alpha(c) = 4.0 degrees and alpha(n) = 0.5 degrees, respectively) is observed.     

Stepwise Versus Concerted Mechanisms in General‐Base Catalysis by Serine Proteases

General‐base catalysis in serine proteases still poses mechanistic challenges despite decades of research. Whether proton transfer from the catalytic Ser to His and nucleophilic attack on the substrate are concerted or stepwise is still under debate, even for the classical Asp‐His‐Ser catalytic triad. To address these key catalytic steps, the transformation of the Michaelis complex to tetrahedral complex in the covalent inhibition of two prototype serine proteases was studied: chymotrypsin (with the catalytic triad) inhibition by a peptidyl trifluoromethane and GlpG rhomboid (with Ser‐His dyad) inhibition by an isocoumarin derivative. The sampled MD trajectories of averaged pK_a values of catalytic residues were QM calculated by the MD‐QM/SCRF(VS) method on molecular clusters simulating the active site. Differences between concerted and stepwise mechanisms are controlled by the dynamically changing pK_a values of the catalytic residues as a function of their progressively reduced water exposure, caused by the incoming ligand.     

Catalytic and stoichiometric reduction of ketones and aldehydes by the hydridotetracarbonyl ferrate anion

Acetone is catalytically reduced to isopropyl alcohol by carbon monoxide and water in the presence of iron carbonyls and triethylamine at 100°C and 100 bar. Use of NaOH in place of triethylamine gives a much less efficient catalyst system. The Et₃NH·HFe(CO)₄ system also catalyses the reduction of n-butyraldehyde to n-butyl alcohol at room temperature in a fast stoichiometric reaction, whereas NaHFe(CO)₄ is inactive under the same conditions. The Et₃NH⁺ cation is necessary for the transfer of a proton to the carbonyl group, while the HFe(CO)₄^? anion carries out nucleophilic attack on carbonyl group and supplies the hydride ion.     

Iridium α-Carboxyimine Complexes Hyperpolarized with para-Hydrogen Exist in Nuclear Singlet States before Conversion into Iridium Carbonates

The formation and hyperpolarization of an [Ir(H)₂(amine)(IMes)(η²-imine)]Cl complex that can be created in a hyperpolarized nuclear singlet state is reported. These complexes are formed when an equilibrium mixture of pyruvate, amine (benzylamine or phenylethylamine), and the corresponding imine condensation product, react with preformed [Ir(H)₂(amine)₃(IMes)]Cl. These iridium α-carboxyimine complexes exist as two regioisomers differentiated by the position of amine. When examined with para-hydrogen the hydride resonances of the isomer with amine trans to hydride become strongly hyperpolarized. The initial hydride singlet states readily transfer to the corresponding ¹³C₂ state in the labelled imine and exhibit magnetic state lifetimes of up to 11 seconds. Their ¹³C signals have been detected with up to 420 fold signal gains at 9.4 T. On a longer timescale, and in the absence of H₂, further reaction leads to the formation of neutral carbonate containing [Ir(amine)(η²-CO₃)(IMes)(η²-imine)]. Complexes are characterized by, IR, MS, NMR and X-ray diffraction.     

Chemoselective Electrochemical Hydrogenation of Ketones and Aldehydes with a Well-Defined Base-Metal Catalyst

Hydrogenation reactions are fundamental functional group transformations in chemical synthesis. Here, we introduce an electrochemical method for the hydrogenation of ketones and aldehydes by in situ formation of a Mn-H species. We utilise protons and electric current as surrogate for H₂ and a base-metal complex to form selectively the alcohols. The method is chemoselective for the hydrogenation of C=O bonds over C=C bonds. Mechanistic studies revealed initial 3 e⁻ reduction of the catalyst forming the steady state species [Mn₂(H₋₁L)(CO)₆]⁻. Subsequently, we assume protonation, reduction and internal proton shift forming the hydride species. Finally, the transfer of the hydride and a proton to the ketone yields the alcohol and the steady state species is regenerated via reduction. The interplay of two manganese centres and the internal proton relay represent the key features for ketone and aldehyde reduction as the respective mononuclear complex and the complex without the proton relay are barely active.     

A Reversible Proton Relay Process Mediated by Hydrogen‐Bonding Interactions in [FeFe]Hydrogenase Modeling

A reversible and temperature‐dependent proton‐relay process is demonstrated for a Fe₂ complex possessing a terminal thiolate in the presence of nitrogen‐based acids. The terminal sulfur site (S^t) of the complex forms a hydrogen‐bond interaction with N,N‐dimethylanilinium acid at 183 K. The Fe₂ core, instead, is protonated to generate a bridging hydride at 298 K. Reversibility is observed for the tautomerization between the hydrogen‐bonded pair and the Fe–hydride species. X‐ray structural analysis of the hydrogen‐bonded species at 193 K reveals a short N(H)???S^t contact. Employment of pyridinium acid also results in similar behavior, with reversible proton–hydride interconversion. DFT investigation of the proton‐transfer pathways indicates that the pK_a value of the hydrogen‐bonded species is enhanced by 3.2 pK_a units when the temperature is decreased from 298 K to 183 K.     

The Earlier the Better: Structural Analysis and Separation of Lanthanides with Pyrroloquinoline Quinone

Lanthanides (Ln) are critical raw materials, however, their mining and purification have a considerable negative environmental impact and sustainable recycling and separation strategies for these elements are needed. In this study, the precipitation and solubility behavior of Ln complexes with pyrroloquinoline quinone (PQQ), the cofactor of recently discovered lanthanide (Ln) dependent methanol dehydrogenase (MDH) enzymes, is presented. In this context, the molecular structure of a biorelevant europium PQQ complex was for the first time elucidated outside a protein environment. The complex crystallizes as an inversion symmetric dimer, Eu₂PQQ₂, with binding of Eu in the biologically relevant pocket of PQQ. LnPQQ and Ln1Ln2PQQ complexes were characterized by using inductively coupled plasma mass spectrometry (ICP-MS), infrared (IR) spectroscopy, ¹⁵¹Eu-Mössbauer spectroscopy, X-ray total scattering, and extended X-ray absorption fine structure (EXAFS). It is shown that a natural enzymatic cofactor is capable to achieve separation by precipitation of the notoriously similar, and thus difficult to separate, lanthanides to some extent.     

DFT-based prediction of reactivity of short-chain alcohol dehydrogenase

The reaction mechanism of ketone reduction by short chain dehydrogenase/reductase, (S)-1-phenylethanol dehydrogenase from Aromatoleum aromaticum, was studied with DFT methods using cluster model approach. The characteristics of the hydride transfer process were investigated based on reaction of acetophenone and its eight structural analogues. The results confirmed previously suggested concomitant transfer of hydride from NADH to carbonyl C atom of the substrate with proton transfer from Tyr to carbonyl O atom. However, additional coupled motion of the next proton in the proton-relay system, between O2′ ribose hydroxyl and Tyr154 was observed. The protonation of Lys158 seems not to affect the pKa of Tyr154, as the stable tyrosyl anion was observed only for a neutral Lys158 in the high pH model. The calculated reaction energies and reaction barriers were calibrated by calorimetric and kinetic methods. This allowed an excellent prediction of the reaction enthalpies (R²?=?0.93) and a good prediction of the reaction kinetics (R²?=?0.89). The observed relations were validated in prediction of log K _eq obtained for real whole-cell reactor systems that modelled industrial synthesis of S-alcohols.     

Photoinduced Decomposition of Formaldehyde on Rutile TiO2(100)-(1×1)?

We have investigated the photoinduced decomposition of formaldehyde (CH₂O) on a rutile TiO₂(100)-(1×1) surface at 355 nm using temperature-programmed desorption. Products, formate (HCOO^-), methyl radical (CH₃·), ethylene (C₂H₄), and methanol (CH₃OH) have been detected. The initial step in the decomposition of CH₂O on the rutile TiO₂(100)-(1×1) surface is the formation of a dioxymethylene intermediate in which the carbonyl O atom of CH₂O is bound to a Ti atom at the five-fold-coordinated Ti⁴⁺ (Ti_5c) site and its carbonyl C atom bound to a nearby bridge-bonded oxygen (O_b) atom, respectively. During 355 nm irradiation, the dioxymethylene intermediate can transfer an H atom to the O_b atom, thus forming HCOO^- directly, which is considered as the main reaction channel. In addition, the dioxymethylene intermediate can also transfer methylene to the O_b row and break the C-O bond, thus leaving the original carbonyl O atom at the Ti_5c site. After the transfer of methylene, several pathways to products are available. Thus, we have found that O_b atoms are intimately involved in the photoinduced decomposition of CH₂O on the rutile TiO₂(100)-(1×1) surface.     

Hydrogen and dihydrogen bonding of transition metal hydrides

Intermolecular interactions between a prototypical transition metal hydride WH(CO)₂NO(PH₃)₂ and a small proton donor H₂O have been studied using DFT methodology. The hydride, nitrosyl and carbonyl ligand have been considered as site of protonation. Further, DFT-D calculations in which empirical corrections for the dispersion energy are included, have been carried out. A variety of pure and hybrid density functionals (BP86, PW91, PBE, BLYP, OLYP, B3LYP, B1PW91, PBE0, X3LYP) have been considered, and our calculations indicate the PBE functional and its hybrid variation are well suited for the calculation of transition metal hydride hydrogen and dihydrogen bonding. Dispersive interactions make up for a sizeable portion of the intermolecular interaction, and amount to 20–30% of the bond energy and to 30–40% of the bond enthalpy. An energy decomposition analysis reveals that the H?H bond of transition metal hydrides contains both covalent and electrostatic contributions.     

Stepwise Hydride Transfer in a Biological System: Insights into the Reaction Mechanism of the Light‐Dependent Protochlorophyllide Oxidoreductase

Hydride transfer plays a crucial role in a wide range of biological systems. However, its mode of action (concerted or stepwise) is still under debate. Light‐dependent NADPH: protochlorophyllide oxidoreductase (POR) catalyzes the stereospecific trans addition of a hydride anion and a proton across the C₁₇?C₁₈ double bond of protochlorophyllide. Time‐resolved absorption and emission spectroscopy were used to investigate the hydride transfer mechanism in POR. Apart from excited states of protochlorophyllide, three discrete intermediates were resolved, consistent with a stepwise mechanism that involves an initial electron transfer from NADPH. A subsequent proton‐coupled electron transfer followed by a proton transfer yield distinct different intermediates for wild type and the C226S variant, that is, initial hydride attaches to either C₁₇ or C₁₈, but ends in the same chlorophyllide stereoisomer. This work provides the first evidence of a stepwise hydride transfer in a biological system.