Thermal Methyl-Group Transfer between Methylcobalt(III) Corrinates and Cobalt(II) Corrinates. Equilibration Experiments with Heptamethyl Cobyrinates and Cobalamins

Separate neutral aqueous solutions of either (a) methylcob(III)alamin ( 2 ) and (heptamethyl cob(II)yrinate) perchlorate ( 3 ) or of b) cob(II)alamin ( = vitamin B_12r; ( 4 ) and [Coβ-methyl(heptamethyl cob(III)yrinate)] perchlorate ( 5 ) equilibrated thermally at r.t. according to 2 + 3 ? 4 + 5 . The corresponding equilibrium constant K_e was determined (K_e = 0.63 ± 0.15). This equilibration experiment indicates that the coordination of the nucleotide function in methylcob(II)alamin ( 2 ) hardly affects the thermodynamics of the Co? C bond homolysis in aqeous solution when compared to nucleotide-free methylcorrinoids such as 5 .     

Cob(I)alamin als Katalysator. 6. Mitteilung [1]. Bildung und Fragmentierung von Alkylcobalaminen,ein Gleichgewichtsprozess zwischen nukleophiler Addition und reduktiver Fragmentierung

Cob(I)alamin as Catalyst. 6. Communication [1]. Formation and Fragmentation of Alkylcobalamins: the Nucleophilic Addition – Reductive Fragmentation Equilibrium Isolated olefines can be saturated using catalytic amounts of cob(I)alamin in aqueous acetic acid; as electron source an excess of zinc dust is added to the solution containing the homogeneous catalyst. During this overall hydrogenation of isolated double bonds intermediate alkylcobalamins are formed (compare e.g. Schemes 2, 4, 5, 7 and 12). Clear evidence is presented that the nucleophilic attack on the isolated double bond is carried out by cob(I)alamin and not by cob(II)alamin also present in the system (see Scheme 3b and 3c). As this catalytic saturation of olefins depends on the pH of the solution, characterized by a slow reaction at pH = 7.0 compared to the same reduction in aqueous acetic acid (see Scheme 2, 2 → 4 , and Scheme 3a), it is reasonable to accept the participation of an electrophilic attack by a proton during the generation of alkylcobalamins. – We use the term nucleophilic addition to describe the formation of alkylcobalamins from a proton, an olefin and cob(I)alamin (compare Schemes 4–7 and 12). A special sequence of experiments showed the nucleophilic addition to be regioselective. Preferentially the higher substituted alkylcobalamin revealed to be produced. Therefore, the nucleophilic addition of cob(I)alamin follows the Markownikoff rule (compare chap. 4: formation and fragmentation of β-hydroxyalkylcobalamins). Under the reaction conditions applied the intermediate alkylcobalamins can be present in base-on and base-off forms. They are known to exist as octahedral complexes and might also be stable to some extent as tetragonal-pyramidal species. In addition the base-off forms can partially be protonated at the dimethylbenzimidazole moiety in aqueous acetic acid (compare Scheme 12). From this equilibrium of intermediate alkylcobalamins three modes of decay disclosed to be possible: (i) The reductive fragmentation leading to an olefin, a proton, and cob(I)alamin is the formal retro-reaction of the nucleophilic addition (see Schemes 2, 4 and 6–12). This equilibrium of an associated alkylcobalamin and the corresponding dissociation products revealed to be a fast process compared to the reductive cleavage of the Co, C-bond cited below (s. (iii)). (ii) As the second reaction pattern an oxidative fragmentation producing an olefin, a hydroxy anion (or water, respectively) and cob (III)alamin has been observed (see Schemes 7, 8, 10 and 12). (iii) The slow reductive cleavage of the Co, C-bond, initiated by addition of electrons (see [1a] [24]), was the third reaction path observed (see Schemes 2, 4–8 and 10–12). – The stereochemistry of the three transformations originating from the intermediate alkylcobalamins is unknown up to now. The antiperiplanar pattern of the fragmentation reactions presented in the Schemes has been chosen arbitrarily (see e.g. Scheme 12).     

Coα‐(1H‐Imidazolyl)‐Coβ‐methylcob(III)amide: Model for Protein‐Bound Corrinoid Cofactors

Coα‐(1H‐Imidazol‐1‐yl)‐Coβ‐methylcob(III)amide ( 4 ) was synthesized by methylation with methyl iodide of (1H‐imidazol‐1‐yl)cob(I)amide, obtained by electrochemical reduction of Coα‐(1H‐imidazol‐1‐yl)‐Coβ‐cyanocob(III)amide ( 5 ). The spectroscopic data and a single‐crystal X‐ray structure analysis indicated 4 to exhibit a base‐on constitution in solution and in the crystal. The crucial lengths of the axial Co−N and Co−CH₃ bonds also emerged from the crystallographic data and were found to be smaller by 0.1 and 0.02 Å, respectively, than those in methylcob(III)alamin ( 2 ). The data of 4 support the view, that the `long' axial Co−N bonds as determined by X‐ray crystallography for the B<sub>12</sub>‐dependent methionine synthase, for methylmalonyl‐CoA mutase, and for glutamate mutase represent stretched Co−N bonds. The thermodynamic effect (the `trans influence') of the 1H‐imidazole base in 4 on the organometallic reactivity of this model for protein‐bound organometallic B₁₂ cofactors was examined by studying Me‐group‐transfer equilibria in aqueous solution and using (5′,6′‐dimethyl‐1H‐benzimidazol‐1‐yl)cobamides (cobalamins) as reaction partners (Schemes 2 – 5, Table). In comparison with methylcob(III)alamin ( 2 ), 4 was found to be destabilized for an abstraction of the Co‐bound Me group by a Co^III electrophile. In contrast, the abstraction of the Co‐bound Me group by a radical(oid) Co^II species was not significantly influenced thermodynamically by the exchange of the nucleotide base. Likewise, exploratory Me‐group‐transfer experiments with Me−Co^III and nucleophilic Co^I corrinoids at pH 6.8 provided an apparent equilibrium constant near unity. However, this finding also was consistent with partial protonation of the imidazolylcob(I)amide at pH 6.8, suggesting an interesting pH dependence of the Megroup‐transfer equilibrium near neutral pH. Therefore, the replacement of the 5′,6′‐dimethyl‐1H‐benzimidazole base by an 1H‐imidazole moiety, as observed in methyl transferases and in C‐skeleton mutases, does not by itself strongly alter the inherent reactivity of the B₁₂ cofactors in the crucial homolytic and nucleophilic‐heterolytic reactions involving the organometallic bond, but may help to enhance the control of the organometallic reactivity by protonation/deprotonation of the axial base.     

Cob(I)alamin als Katalysator, 5. Mitteilung [1]. Enantioselektive Reduktion α,β-ungesättigter Carbonylderivate

Cob(I)alamin as Catalyst. 5. Communication [1]. Enantioselective Reduction of α,β-Unsaturated Carbonyl Derivatives The cob(I)alamin-catalyzed reduction of an α,β-unsaturated ethyl ester in aqueous acetic acid produced the (S)-configurated saturated derivative 2 with an enantiomeric excess of 21%. The starting material 1 is not reduced at pH = 7.0 in the presence of catalytic amounts of cob(I)alamin (see Scheme 2). It is shown that the attack of cob(I)alamin and not of cob(II)alamin, also present in Zn/CH₃COOH/H₂O, accounts for the enantioselective reduction observed. All the (Z)-configurated starting materials 1 , 3 , 5 , 7 , 9 and 11 have been transformed to the corresponding (S)-configurated saturated derivatives 2 , 4 , 6 , 8 , 10 and 12 , respectively. The highest enantiomeric excess revealed to be present in the saturated product 12 (32,7%, S) derived from the (Z)-configurated methyl ketone 11 (see Scheme 3 and Table 1). The reduction of the (E)-configurated starting materials led mainly to racemic products. A saturated product having the (R)-configuration with a rather weak enantiomeric excess (5.9%) has been obtained starting from the (E)-configurated methyl ketone 23 (see Scheme 5 and Table 2). The allylic alcohols 16 and 24 have been reduced to the saturated racemic derivative 17 .     

Cob (I)alamin als Katalysator 4. Mitteilung. Reduktion von α,β-ungesättigten Nitrilen

Cob(I)alamin as Catalyst. 4. Communication. Reduction of α,β-Unsaturated Nitriles Using catalytic amounts of cob (I)alamin and an excess of metallic zinc as source of electrons 1-naphthonitril ( 5 ) has been reduced to (1-naphthyl)methylamin ( 6 ) and in small amounts to (1-naphthyl)methanol ( 7 ) and (1,2,3,4-tetrahydro-1-naphthyl)methanol ( 8 ) (5 ½ h, CH₃COOH/H₂O; s. Scheme 3). Starting from cyclododecylideneacetonitrile ( 15 ) similar conditions (68 h, CH₃COOH/H₂O) produced the amines 16–19 as well as the nitrogen free saturated aldehyde 20 , the corresponding allylic alcohol 21 and the saturated derivative 22 (s. Scheme 6). It is deduced that the first attack of cob (I)alamin on an α,β-unsaturated nitrile might occur on both the nitrile dipole as well as on the carbon atom in β-position. Cob (I)alamin in aqueous acetic acid saturates the isolated double bonds in allylic alcohols and amines. In a slow reaction the two different aromatic rings of (1-naphthyl)methanol ( 7 ) have been reduced giving the corresponding tetrahydronaphthalene derivatives 8 and 12 , and in one case the production of the octahydroderivative 14 has been observed in a low yield (s. Scheme 5).     

the Mechanism of the Oxidation of Cob(I)alamins

Abstract

Spectroelectrochemical investigations of the reoxidation sequence of the reduced cob(I)alamin to the oxidized cob(III)alamin show that two different cob(II)alamin intermediates are formed during the processes which appear to correlate to base-on and base-off cob(II)-alamin species.     

Unprecedented Mechanism Employed by the Salmonella enterica EutT ATP:CoIrrinoid Adenosyltransferase Precludes Adenosylation of Incomplete CoIIrrinoids

Three distinct families of ATP:corrinoid adenosyltransferases (ACATs) exist that are capable of converting vitamin B₁₂ derivatives into coenzyme B₁₂ by catalyzing the thermodynamically challenging reduction of Co^IIrrinoids to form “supernucleophilic” Co^I intermediates. While the structures and mechanisms of two of the ACAT families have been studied extensively, little is known about the EutT enzymes beyond the fact that they exhibit a unique requirement for a divalent metal cofactor for enzymatic activity. In this study we have obtained compelling evidence that EutT converts cob(II)alamin into an effectively four‐coordinate Co^II species so as to facilitate Co^II→Co^I reduction. Intriguingly, EutT fails to promote axial ligand dissociation from the substrate analogue cob(II)inamide, a natural precursor of cob(II)alamin. This unique substrate specificity of EutT has important physiological implications.     

Reduction‐Labile Organo‐cob(III)alamins via Cob(II)alamin: Efficient Synthesis and Solution and Crystal Structures of [(Methoxycarbonyl)methyl]cob(III)alamin

An efficient synthesis of Coβ‐[(methoxycarbonyl)methyl]cob(III)alamin ( 6 ) is reported as an example of a new method for the preparation of some easily reducible organo‐cob(III)alamins via the alkylation of cob(II)alamin. The procedure represents a considerable improvement compared to earlier methods that were based on an alkylation of cob(I)alamin. Thus, aquacob(III)alamin chloride ( 5 ⁺?Cl) was reduced to cob(II)alamin ( 4 ), either by controlled potential electrolytic reduction or with an excess of sodium formate as reducing agent. The solution of 4 was then treated with an excess of methyl bromoacetate while being reductively poised potentiostatically or kept reduced by the formate, to give crystalline 6 in a yield of up to 91%. The structure of 6 in aqueous solution was mainly established by the completely assigned ¹H‐ and ¹³CNMR spectra (Table 1). The NOE data (Table 2) were best rationalized by the presence of a single main conformation of the (methoxycarbonyl)methyl ligand. Single crystals of 6 were obtained by crystallization from an aqueous solution, and the crystal structure was determined by X‐ray analysis at cryotemperatures. The NMR and crystallographic data of 6 indicated similar structures in aqueous solution and in the crystal with the (methoxycarbonyl)methyl ligand preferring a ‘southern' orientation in each case.     

Experimental Study of Hydrogen Bonding Potentially Stabilizing the 5′‐Deoxyadenosyl Radical from Coenzyme B12

Coenzyme B₁₂ can assist radical enzymes that accomplish the vicinal interchange of a hydrogen atom with a functional group. It has been proposed that the Co? C bond homolysis of coenzyme B₁₂ to cob(II)alamin and the 5′‐deoxyadenosyl radical is aided by hydrogen bonding of the corrin C19? H to the 3′‐O of the ribose moiety of the incipient 5′‐deoxyadenosyl radical, which is stabilized by 30 kJ mol^?1 (B. Durbeej et al., Chem. Eur. J. 2009 , 15, 8578–8585). The diastereoisomers (R)‐ and (S)‐2,3‐dihydroxypropylcobalamin were used as models for coenzyme B₁₂. A downfield shift of the NMR signal for the C19? H proton was observed for the (R)‐isomer (δ=4.45 versus 4.01 ppm for the (S)‐isomer) and can be ascribed to an intramolecular hydrogen bond between the C19? H and the oxygen of CHOH. Crystal structures of (R)‐ and (S)‐2,3‐dihydroxypropylcobalamin showed C19? H???O distances of 3.214(7) Å (R‐isomer) and 3.281(11) Å (S‐isomer), which suggest weak hydrogen‐bond interactions (?ΔG<6 kJ mol^?1) between the CHOH of the dihydroxypropyl ligand and the C19? H. Exchange of the C19? H, which is dependent on the cobalt redox state, was investigated with cob(I)alamin, cob(II)alamin, and cob(III)alamin by using NMR spectroscopy to monitor the uptake of deuterium from deuterated water in the pH range 3–11. No exchange was found for any of the cobalt oxidation states. 3′,5′‐Dideoxyadenosylcobalamin, but not the 2′,5′‐isomer, was found to act as a coenzyme for glutamate mutase, with a 15‐fold lower k_cat/K_M than 5′‐deoxyadenosylcobalamin. This indicates that stabilization of the 5′‐deoxyadenosyl radical by a hydrogen bond that involves the C19? H and the 3′‐OH group of the cofactor is, at most, 7 kJ mol^?1 (?ΔG). Examination of the crystal structure of glutamate mutase revealed additional stabilizing factors: hydrogen bonds between both the 2′‐OH and 3′‐OH groups and glutamate 330. The actual strength of a hydrogen bond between the C19? H and the 3′‐O of the ribose moiety of the 5′‐deoxyadenosyl group is concluded not to exceed 6 kJ mol^?1 (?ΔG).     

Cob(I)alamin Differentiating Alkenes During Saturation

The olefins 2, 7, 11 , and 19 , have been reduced using catalytic amounts of cob(I)alamin( I(I) ). During a slow saturation, the catalyst is able to differentiate the two diastereotopic faces of the endocyclic double bonds in 11 (t_1/2 4 h,. cf. Scheme 3) are reduced much faster. A rationalization of the data can be obtained formulating tertiary alkylcobalamins as intermediates. Of the oxime 6 (cf. Scheme 2) and the p- bromobenzoate 23 (cf. Scheme 5) the structures have been determined by X-ray analysis.     

Influence of the Axial Alkyl Ligand on the Reduction Potential of Alkylcob(III)alamins and Alkylcob(III)yrinates

The irreversible-reduction potentials of 26 alkylcob(III)alamins (RCbl^III 1a – z ) and 26 alkylcob(III)yrinates (R‘Cby’^III; 2a – z ) (E_p 1a – z and E_p 2a – z , resp.) have been measured in situ by single-scan voltammetry of hydroxocob(III)alamin hydrochloride (vitamin B_12b- HCl; 1 ) or heptamethyl cob(II)yrinate perchlorate (ClO₄‘Cby’^II; 2 ) in presence of the corresponding alkyl halides (RX; 3a – z ) in DMF. The reduction potentials of alkylcobalt complexes exhibiting half-life times as short as a few seconds become measurable by this technique. Thermodynamic cycles prove that the observed reduction potentials are closely related to the standard reduction potentials E°(R? Co^III + e^??R? + Co^I). Electron-withdrawing groups and/or an increased degree of substitution at the Co-bound C-atom in RCbl^III and, R‘Cby’^III shift E_p( 1a – z ) and E_p ( 2a – z ) towards positive potentials. Linear correlations have been found between E_p( 1a – z ) (E_p( 2a – z )) of RCbl^III (R‘Cby’^III) and the pK_a of RH (or the Taft σ*- or the Hammett σ-values of R) within each class of R, i. e. MeCbl^III (Me‘Cby’^III), primary RCbl^III (R‘Cby’^III) and secondary RCbl^III (R‘Cby’^III). The correlations allow to distinguish between electronic effects of the Co-bound alkyl residues and their steric interactions with the corrin side chains. The correlations have further been used to visualize the light-induced formal insertion of an olefin into the Co, C-bond of an alkylcobalamin (Scheme 2, 1a → 1u ), a key step in the vitamin-B₁₂-catalized C, C-bond formation.     

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, β‐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 Å 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 Å of the cobalt atom, with the substrate and product radicals approximately 3 Å 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.     

Theoretical investigations into the intermediacy of chlorinated vinylcobalamins in the reductive dehalogenation of chlorinated ethylenes

The reductive dehalogenation of perchloroethylene and trichloroethylene by vitamin B(12) produces approximately 95% (Z)-dichloroethylene (DCE) and small amounts of (E)-DCE and 1,1-DCE, which are further reduced to ethylene and ethane. Chloroacetylene and acetylene have been detected as intermediates, but not dichloroacetylene. Organocobalamins (RCbls) have been proposed to be intermediates in this process. Density functional theory based approaches were employed to investigate the properties of chlorinated vinylcobalamins and chlorinated vinyl radicals. They reveal that all vinyl radicals studied have reduction potentials more positive (E degrees >or= -0.49) than that of the Co(II)/Co(I) couple of B(12) (E degrees = -0.61 V), indicating that any (chlorinated) vinyl radicals formed in the reductive dehalogenation process should be reduced to the corresponding anions by cob(I)alamin in competition with their combination with Co(II) to yield the corresponding vinylcobalamins. The computed Co-C homolytic bond dissociation enthalpies (BDEs) of the latter complexes range from 33.4 to 45.8 kcal/mol. The substituent effects on the BDEs are affected by the stabilities of the vinyl radicals as well as steric interactions between (Z)-chloro substituents and the corrin ring. The calculated E degrees values of the cobalamin models were within approximately 200 mV of one another since electron attachment is to a corrin ring pi-orbital, whose energy is relatively unaffected by chloride substitution of the vinyl ligand, and all were >500 mV more negative than that of the Co(II)/Co(I) couple of B(12). Reduction of the base-off forms of vinyl- and chlorovinylcobalamin models also involves the corrin pi* orbital, but reduction of the base-off dichlorovinyl- and trichlorovinylcobalamin models occurs with electron attachment to the sigma(Co)(-)(C*) orbital, yielding calculated E degrees values more positive than that of the calculated Co(II)/Co(I) couple of B(12). Thus, cob(I)alamin is expected to reduce these base-off vinyl-Cbls. Heterolytic cleavage of the Co-C bonds is much more favorable than homolysis (>21 kcal/mol) and is significantly more exergonic when coupled to chloride elimination.     

Skeletal Migrations Observed during the Cob(I)alamin-Catalyzed Reduction of 4β-(tert-Butyl)-1β-(1-methylvinyl)cyclohexanecarbaldehyde

During the cob(I)alamin( 1(I) )-catalyzed reduction of 3 , intermediate formation of 2 and final generation of 4–10 was observed (see Scheme 1, cf. Tables 1 and 2). Identical products in similar ratios were generated starting from either 2 or 3 . Accepting the intermediate formation of six interconnected cobalt complexes, i.e. A–F (cf. Scheme 2), the generation of all the products observed can be explained.     

Aspects of the Reduction of Double Bonds Using Cob (I)alamin as Catalys

The olefinic system in 3β-methoxy-4-cholesten-6 a-ol ( 2 ) is reduced using cob (I)alamin ( 1 ( I ); see Scheme 1) as catalyst, aqueous acetic acid as solvent and metallic zinc as electron source (cf. Schemes 2 and 3). Experimental evidence for an attack of 1 ( I ) on both faces of the double bond is presented. By the same catalyst (1 R)-10, 10-dimethyl-2-pinene- 10-carbonitrile ( 9 ) is first transformed to the menthene derivative 11 (see Schemes 4 and 5). The ring opening is then followed by a fast saturation of the disubstituted olefinic system in 11 , and ultimately the remaining double bond is reduced in a slow reaction. The cis-configurated saturated menthane derivative 16 is the main final product ( 16 / 17 ≈ 10:1).     

Interaction between super-reduced cobalamin and selenite

The kinetics of the reaction between the two-electron reduced form of cobalamin (super-reduced cobalamin, cob(I)alamin, or Cbl(I)) and sodium selenite in an alkaline medium is studied spectrophotometrically. It is shown that the selenite rapidly oxidizes Cbl(I) to cob(II)alamin (Cbl(II)). It is established that the active form of the oxidant is the protonated selenite anion (HSeO₃^-), which receives six electrons during the reaction and transforms into HSe^–. The reactions of cob(I)alamin oxidation by selenite and sulfite are compared.     

Evidence for the formation of a cis-dichlorovinyl anion upon reduction of cis-1,2-dichlorovinyl(pyridine)cobaloxime

The reduction of cis-1,2-dichlorovinyl(pyridine)cobaloxime, a model complex for the organometallic intermediate proposed in the dechlorination of trichloroethylene by cobalamin, was studied. Two mechanisms were considered for the Co-C bond cleavage following reduction. In the first, the Co-C bond cleaves to produce Co(I) and a chlorovinyl radical, while the second pathway results in the formation of Co(II) and a chlorovinyl anion. Four reducing agents, cobaltocene, decamethylcobaltocene, cob(I)alamin, and chromium(II), were used in the presence of H atom and proton donor species to identify the presence of chlorovinyl radical or chlorovinyl anion intermediates. Mechanistic conclusions were based on comparisons of the final product ratios of cis-dichloroethylene (cDCE) and chloroacetylene, which were found to have a direct relationship to the amount of proton donor available, with increased proton donor leading to increased cDCE production. The results support the intermediacy of a cis-1,2-dichlorovinyl anion.     

Electron transfer: 150. The reaction of corrin-bound cobalt(III) with indium(I) — an additional mechanistic variation

(Corrin)cobalt(III) is reduced by indium(I) to its Co(II) counterpart (cob(II)alamin, B_12r) at pH 1-2 in aqueous chloride. In these solutions the aqua oxidant (B_12a) is in mobile anation equilibrium with its chloro derivative. At [Cl^–] 0.03 M, consumption of Co(III) is exponential and proceeds by parallel paths involving the aqua- and chloro-substituted oxidants. At [Cl^–] 0.10 M, rates are governed mainly by an preliminary act requiring In(I) and 2 Cl^–, but no Co(III). This initiation step generates a more reactive In(I) species which is taken to result from a slow heterolysis and loss of ligating water from InCl₂ ^–(aq).     

Characterization of protein contributions to cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase by using photolysis in the ternary complex

Protein contributions to the substrate-triggered cleavage of the cobalt-carbon (Co-C) bond and formation of the cob(II)alamin-5'-deoxyadenosyl radical pair in the adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied by using pulsed-laser photolysis of AdoCbl in the EAL-AdoCbl-substrate ternary complex, and time-resolved probing of the photoproduct dynamics by using ultraviolet-visible absorption spectroscopy on the 10(-7)-10(-1) s time scale. Experiments were performed in a fluid dimethylsulfoxide/water cryosolvent system at 240 K, under conditions of kinetic competence for thermal cleavage of the Co-C bond in the ternary complex. The static ultraviolet-visible absorption spectra of holo-EAL and ternary complex are comparable, indicating that the binding of substrate does not labilize the cofactor cobalt-carbon (Co-C) bond by significantly distorting the equilibrium AdoCbl structure. Photolysis of AdoCbl in EAL at 240 K leads to cob(II)alamin-5'-deoxyadenosyl radical pair quantum yields of <0.01 at 10(-6) s in both holo-EAL and ternary complex. Three photoproduct states are populated following a saturating laser pulse, and labeled, P(f), P(s), and P(c). The relative amplitudes and first-order recombination rate constants of P(f) (0.4-0.6; 40-50 s(-1)), P(s) (0.3-0.4; 4 s(-1)), and P(c) (0.1-0.2; 0) are comparable in holo-EAL and in the ternary complex. Time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy shows that visible irradiation alters neither the kinetics of thermal cob(II)alamin-substrate radical pair formation, nor the equilibrium between ternary complex and cob(II)alamin-substrate radical pair, at 246 K. The results indicate that substrate binding to holo-EAL does not "switch" the protein to a new structural state, which promptly stabilizes the cob(II)alamin-5'-deoxyadenosyl radical pair photoproduct, either through an increased barrier to recombination, a decreased barrier to further radical pair separation, or lowering of the radical pair state free energy, or a combination of these effects. Therefore, we conclude that such a change in protein structure, which is independent of changes in the AdoCbl structure, and specifically the Co-C bond length, is not a basis of Co-C bond cleavage catalysis. The results suggest that, following the substrate trigger, the protein interacts with the cofactor to contiguously guide the cleavage of the Co-C bond, at every step along the cleavage coordinate, starting from the equilibrium configuration of the ternary complex. The cleavage is thus represented by a diagonal trajectory across a free energy surface, that is defined by chemical (Co-C separation) and protein configuration coordinates.     

Cob(I)alamin als Katalysator. Retention der Konfiguration bei der reduktiv-protonenübertragenden Spaltung der Co,C-Bindung eines Alkylcobalamins. 7. Mitteilung [1]

Cob(I)alamin as Catalyst. 7. Communication [1]. Retention of Configuration during the Reductive Cleavage of the Co, C-Bond of an Alkylcobalamin Using catalytic amounts of cob(I)alamin (see Scheme 1) in aqueous acetic acid (?)-α-pinen ( 1 ) and (?)-β-pinen ( 2 ; s. Scheme 3) have been reduced. A large excess of metallic zinc served as electron source. The saturated products 5–8 (see Scheme 3) and the mechanistic aspects of their generation are discussed. The relative amounts of cis- ( 5 ) and trans-pinane ( 6 ) lead to the conclusion that the reductive cleavage of the Co, C-bond accompanied by H⁺ transfer in an alkylcobalamin occurs with retention of configuration. This result is in agreement with the corresponding cleavage of the Co,C-bond of an alkyl[hydroxy-diazaoctahydroporphinato]cobalt complex [9].