M?ssbauer, EPR, and optical studies of the corrinoid/iron-sulfur protein involved in the synthesis of acetyl coenzyme A by Clostridium thermoaceticum

We have purified to homogeneity the 88-kDa corrinoid protein from Clostridium thermoaceticum which acts as a methyl carrier in the synthesis of acetyl-CoA. As shown here, this protein contains a [4Fe-4S]1+/2+ cluster in addition to a corrinoid. The corrinoid is 5-methoxybenzimidazolylcobamide, with an OH- group probably present as the upper axial ligand. Co+ is present in the reduced form, Co2+ in the as-isolated form, and Co3+ in the methylated form of the protein. The as-isolated corrinoid/Fe-S protein exhibits a Co2+ EPR signal lacking nitrogen superhyperfine splittings, indicating that the benzimidazole base is uncoordinated ("base-off") in the Co2+ state. Optical studies suggest that the Co3+-CH3 corrinoid is also base-off. In the as-isolated and methylated forms, the iron-sulfur cluster is diamagnetic, with quadrupole splittings and isomer shifts characteristic of [4Fe-4S]2+ clusters. The protein can be reduced by CO and CO dehydrogenase in the absence of ferredoxin. The EPR spectra of the reduced cluster exhibit two components: one with principal g-values at 2.07, 1.93, and 1.82 and the other at 2.02, 1.94, and 1.86. The M?ssbauer data show that these signals result from [4Fe-4S]1+ clusters. Chemical analysis shows that the iron:cobalt atomic ratio is close to 4:1, suggesting that a single [4Fe-4S]1+ cluster occurs in two distinct S = 1/2 spin states in the reduced state. Treatment with 1-2.5 M urea converts the two cluster forms into a single one, with EPR and M?ssbauer spectra of typical [4Fe-4S]1+ clusters. A 27-kDa corrinoid protein (Ljungdahl, L.G., LeGall, J., and Lee, J.P. (1973) Biochemistry 12, 1802-1808) also was purified and found to be inactive in the synthesis of acetyl-CoA, contrary to the suggestion of Ljungdahl et al. (1973).     

Kinetic regulation of a corrinoid‐reducing metallo‐ATPase by its substrates

Corrinoid cofactors play a crucial role as methyl group carriers in the C1 metabolism of anaerobes, e.g. in the cleavage of phenyl methyl ethers by O‐demethylases. For the methylation, the protein‐bound corrinoid has to be in the super‐reduced [Co^I]‐state, which is highly sensitive to autoxidation. The reduction of inadvertently oxidized corrinoids ([Co^II]‐state) is catalysed in an ATP‐dependent reaction by RACE proteins, the r eductive a ctivators of c orrinoid‐dependent e nzymes. In this study, a reductive activator of O‐demethylase corrinoid proteins was characterized with respect to its ATPase and corrinoid reduction activity. The reduction of the corrinoid cofactor was dependent on the presence of potassium or ammonium ions. In the absence of the corrinoid protein, a basal slow ATP hydrolysis was observed which was obviously not coupled to corrinoid reduction. ATP hydrolysis was significantly stimulated by the corrinoid protein in the [Co^II]‐state of the corrinoid cofactor. The stoichiometry of ATP hydrolysed per mol corrinoid reduced was near 1:1. Site‐directed mutagenesis was applied to study the impact of a highly conserved region possibly involved in nucleotide binding of RACE proteins, indicating that an aspartate and a glycine residue may play an essential role for the function of the enzyme.     

Veratrol-<Emphasis Type="Italic">O</Emphasis>-demethylase of <Emphasis Type="Italic">Acetobacterium dehalogenans</Emphasis>: ATP-dependent reduction of the corrinoid protein

The anaerobic veratrol O-demethylase mediates the transfer of the methyl group of the phenyl methyl ether veratrol to tetrahydrofolate. The primary methyl group acceptor is the cobalt of a corrinoid protein, which has to be in the +1 oxidation state to bind the methyl group. Due to the negative redox potential of the cob(II)/cob(I)alamin couple, autoxidation of the cobalt may accidentally occur. In this study, the reduction of the corrinoid to the superreduced [Co^I] state was investigated. The ATP-dependent reduction of the corrinoid protein of the veratrol O-demethylase was shown to be dependent on titanium(III) citrate as electron donor and on an activating enzyme. In the presence of ATP, activating enzyme, and Ti(III), the redox potential versus the standard hydrogen electrode (E _SHE) of the cob(II)alamin/cob(I)alamin couple in the corrinoid protein was determined to be −290 mV (pH 7.5), whereas E _SHE at pH 7.5 was lower than −450 mV in the absence of either activating enzyme or ATP. ADP, AMP, or GTP could not replace ATP in the activation reaction. The ATP analogue adenosine-5′-(β,γ-imido)triphosphate (AMP-PNP, 2–4 mM) completely inhibited the corrinoid reduction in the presence of ATP (2 mM).     

Monte Carlo studies of phospholipid lamellae. Effects of proteins, cholesterol, bilayer curvature, and lateral mobility on order parameters

In this paper we present the results of a Monte Carlo study of the effects of protein, cholesterol, bilayer curvature, and mobility on the chain order parameters of a lipid layer. The Monte Carlo method used is identical to the version developed earlier (Scott, Jr., H.L. (1977) Biochim. Biophys. Acta 469, 264–271). Simulations of protein and cholesterol effects are accomplished by insertion of a rigid stationary cylinder into the lipid matrix. The protein studies show the presence of boundary lipid (Jost, P., Griffith, O.H., Capaldi, R.H. and Vanderkooi, G. (1973) Biochim. Biophys. Acta 311, 141–152). The effect of cholesterol is dependent upon the length of the lipid hydrocarbon chains relative to the cholesterol depth of penetration. Our computer studies of bilayer curvature show the manner in which this curvature disrupts chain packing and are consistent with experimental results (Chrzeszczyk, A., Wishnia, A. and Springer, C.S. (1977) Biochim. Biophys. Acta, 470, 161–171). We also find that restricting lateral motion in chains, the simplest manner in which head group interactions can affect hydrocarbon chain order, does not measurably alter the order parameters. We argue that this provides some support for an earlier hypothesis by Scott (Scott, Jr., H.L. (1975) Biochim. Biophys. Acta 406, 329–346) regarding head group-chain interaction in monolayer experiments.     

Characterization of an O-demethylase of Desulfitobacterium hafniense DCB-2

Besides acetogenic bacteria, only Desulfitobacterium has been described to utilize and cleave phenyl methyl ethers under anoxic conditions; however, no ether-cleaving O-demethylases from the latter organisms have been identified and investigated so far. In this study, genes of an operon encoding O-demethylase components of Desulfitobacterium hafniense strain DCB-2 were cloned and heterologously expressed in Escherichia coli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer of the methyl group to the superreduced corrinoid of a corrinoid protein. Desulfitobacterium methyltransferase I had 66% identity (80% similarity) to that of the vanillate-demethylating methyltransferase I (OdmB) of Acetobacterium dehalogenans. The substrate spectrum was also similar to that of the latter enzyme; however, Desulfitobacterium methyltransferase I showed a higher level of activity for guaiacol and used methyl chloride as a substrate. Methyltransferase II catalyzed the transfer of the methyl group from the methylated corrinoid protein to tetrahydrofolate. It also showed a high identity (～70%) to methyltransferases II of A. dehalogenans. The corrinoid protein was produced in E. coli as cofactor-free apoprotein that could be reconstituted with hydroxocobalamin or methylcobalamin to function in the methyltransferase I and II assays. Six COG3894 proteins, which were assumed to function as activating enzymes mediating the reduction of the corrinoid protein after an inadvertent oxidation of the corrinoid cofactor, were studied with respect to their abilities to reduce the recombinant reconstituted corrinoid protein. Of these six proteins, only one was found to catalyze the reduction of the corrinoid protein.     

Identification of specific corrinoids reveals corrinoid modification in dechlorinating microbial communities

Cobalamin and other corrinoids are essential cofactors for many organisms. The majority of microbes with corrinoid‐dependent enzymes do not produce corrinoids de novo, and instead must acquire corrinoids produced by other organisms in their environment. However, the profile of corrinoids produced in corrinoid‐dependent microbial communities, as well as the exchange and modification of corrinoids among community members have not been well studied. In this study, we applied a newly developed liquid chromatography tandem mass spectrometry‐based corrinoid detection method to examine relationships among corrinoids, their lower ligand bases and specific microbial groups in microbial communities containing Dehalococcoides mccartyi that has an obligate requirement for benzimidazole‐containing corrinoids for trichloroethene respiration. We found that p‐cresolylcobamide ([p‐Cre]Cba) and cobalamin were the most abundant corrinoids in the communities. It suggests that members of the family Veillonellaceae are associated with the production of [p‐Cre]Cba. The decrease of supernatant‐associated [p‐Cre]Cba and the increase of biomass‐associated cobalamin were correlated with the growth of D. mccartyi by dechlorination. This supports the hypothesis that D. mccartyi is capable of fulfilling its corrinoid requirements in a community through corrinoid remodelling, in this case, by importing extracellular [p‐Cre]Cba and 5,6‐dimethylbenzimidazole (DMB) (the lower ligand of cobalamin), to produce cobalamin as a cofactor for dechlorination. This study also highlights the role of DMB, the lower ligand produced in all of the studied communities, in corrinoid remodelling. These findings provide novel insights on roles played by different phylogenetic groups in corrinoid production and corrinoid exchange within microbial communities. This study may also have implications for optimizing chlorinated solvent bioremediation.     

Growth characteristics and corrinoid production of Methanosarcina barkeri on methanol-acetate medium

Methanosarcina barkeri strain Fusaro was grown on a mixed substrate medium of methanol and acetate. When 50 mM of acetate was added to the methanol basal medium (250 mM), the rates of methane production, methanol consumption, cell growth and corrinoid production were stimulated 3.2, 2.7, 3.5, and 2.4 times, respectively compared with those in methanol alone. Addition of acetate also has significant influence on corrinoid distribution decreasing the intracellular corrinoid content from 6.8 to 3.0 mg/g dry cell and increasing the extracellular corrinoid concentration from 4.0 to 5.4 mg/l. The carbon balance analysis for methanogenesis and cellular growth with or without acetate addition revealed that about 50% of the utilized acetate carbon might be incorporated in the cellular materials and the remaining might be oxidized to generate the electrons which stimulate the methanol reduction to methane, accelerating the metabolic activities of the methanogenesis from methanol consequently enhancing the rates of methane and corrinoid production, and cell growth.     

Redox potential changes during ATP‐dependent corrinoid reduction determined by redox titrations with europium(II)–DTPA

Corrinoids are essential cofactors of enzymes involved in the C₁ metabolism of anaerobes. The active, super‐reduced [Co^I] state of the corrinoid cofactor is highly sensitive to autoxidation. In O‐demethylases, the oxidation to inactive [Co^II] is reversed by an ATP‐dependent electron transfer catalyzed by the activating enzyme (AE). The redox potential changes of the corrinoid cofactor, which occur during this reaction, were studied by potentiometric titration coupled to UV/visible spectroscopy. By applying europium(II)–diethylenetriaminepentaacetic acid (DTPA) as a reductant, we were able to determine the midpoint potential of the [Co^II]/[Co^I] couple of the protein‐bound corrinoid cofactor in the absence and presence of AE and/or ATP. The data revealed that the transfer of electrons from a physiological donor to the corrinoid as the electron‐accepting site is achieved by increasing the potential of the corrinoid cofactor from ?530 ± 15 mV to ?250 ± 10 mV (E_SHE, pH 7.5). The first 50 to 100 mV of the shift of the redox potential seem to be caused by the interaction of nucleotide‐bound AE with the corrinoid protein or its cofactor. The remaining 150–200 mV had to be overcome by the chemical energy of ATP hydrolysis. The experiments revealed that Eu(II)–DTPA, which was already known as a powerful reducing agent, is a suitable electron donor for titration experiments of low‐potential redox centers. Furthermore, the results of this study will contribute to the understanding of thermodynamically unfavorable electron transfer processes driven by the power of ATP hydrolysis.     

Quantification of corrinoids in methanogenic bacteria

Corrinoids in several diverse species of methanogens were quantified by a bioassay utilizingEscherichia coli 113–3, a corrinoid auxotroph. All five species examined contained >0.65 nmol corrinoid/mg dry cells when grown on H₂/CO₂ as carbon and energy source. The highest corrinoid levels (4.1 nmol/mg cells) were found inMethanosarcina barkeri grown on methanol. The amount of corrinoids found in this species was dependent on growth conditions, but, regardless of energy source, metabolized levels inMethanosarcina barkeri were higher than those found in theMethanobacterium species examined (M. arbophilicum, M. formicium, M. ruminantium, andM. thermoautotrophicum).     

Coenzyme M methylase activity of the 480-kilodalton corrinoid protein from Methanosarcina barkeri.

Activity staining of extracts of Methanosarcina barkeri electrophoresed in polyacrylamide gels revealed an additional methylcobalamin:coenzyme M (methylcobalamin:CoM) methyltransferase present in cells grown on acetate but not in those grown on trimethylamine. This methyltransferase is the 480-kDa corrinoid protein previously identified by its methylation following inhibition of methyl-CoM reductase in otherwise methanogenic cell extracts. The methylcobalamin:CoM methyltransferase activity of the purified 480-kDa protein increased from 0.4 to 3.8 micromol/min/mg after incubation with sodium dodecyl sulfate (SDS). Following SDS-polyacrylamide gel electrophoresis analysis of unheated protein samples, a polypeptide with an apparent molecular mass of 48 kDa which possessed methylcobalamin:CoM methyltransferase activity was detected. This polypeptide migrated with an apparent mass of 41 kDa when the 480-kDa protein was heated before electrophoresis, indicating that the alpha subunit is responsible for the activity. The N-terminal sequence of this subunit was 47% similar to the N termini of the A and M isozymes of methylcobalamin:CoM methyltransferase (methyltransferase II). The endogenous methylated corrinoid bound to the beta subunit of the 480-kDa protein could be demethylated by CoM, but not by homocysteine or dithiothreitol, resulting in a Co(I) corrinoid. The Co(I) corrinoid could be remethylated by methyl iodide, and the protein catalyzed a methyl iodide:CoM transmethylation reaction at a rate of 2.3 micromol/min/mg. Methyl-CoM was stoichiometrically produced from CoM, as demonstrated by high-pressure liquid chromatography with indirect photometric detection. Two thiols, 2-mercaptoethanol and mercapto-2-propanol, were poorer substrates than CoM, while several others tested (including 3-mercaptopropanesulfonate) did not serve as methyl acceptors. These data indicate that the 480-kDa corrinoid protein is composed of a novel isozyme of methyltransferase II which remains firmly bound to a corrinoid cofactor binding subunit during isolation.     

Reductive activation of the corrinoid-containing enzyme involved in methyl group transfer between methyl-tetrahydromethanopterin and coenzyme M inMethanosarcina barkeri

The conversion of methyl-tetrahydromethanopterin to methylcoenzyme M inMethanosarcina barkeri is catalyzed by two enzymes: an enzyme with a bound corrinoid, which becomes methylated during the reaction and an enzyme which tranfers the methyl group from this corrinoid to coenzyme M. As in the similar methyltransfer reaction inMethanobacterium thermoautotrophicum the corrinoid enzyme inM. barkeri needs to be activated by H₂ and ATP. ATP can be replaced by Ti(III)citrate or CO.     

Identification and quantitation of corrinoid precursors of cobalamin from Pseudomonas denitrificans by high-performance liquid chromatography

After initial pretreatment for removal of interfering substances, corrinoid precursors of cobalamin from cultures of Pseudomonas denitrificans were separated by HPLC with a gradient elution system. In this system, all the following compounds are separated in their dicyano form, and retention times are given: cobyrinic acid; cobyrinic acid a-amide; cobyrinic acid c-amide; cobyrinic acid g-amide; cobyrinic acid a,g-diamide; cobyrinic acid c,g-diamide; cobyrinic acid a,c-diamide; cobyrinic acid a,c,g-triamide; cobyrinic acid triamide, tetraamide, and pentaamide isolated from P. denitrificans; cobyric acid; cobinamide; cobinamide phosphate; GDP-cobinamide; cyanocobalamin 5'-phosphate; and cyanocobalamin. Application of this HPLC method to culture samples of P. denitrificans revealed that in this microorganism the level of cobyrinic acid and cobyrinic acid monoamide is far lower than that of all other corrinoid precursors of cobalamin and suggested that (i) the (R)-1-amino-2-propanol group is incorporated only after completion of all the other amidations and (ii) the amidations follow only one sequence. The usefulness of this HPLC method was further demonstrated by identifying the 57Co-labeled corrinoid precursors of cobalamin accumulated by cobalamin-deficient mutants of Agrobacterium tumefaciens. A TLC system that separates the different corrinoid intermediates (in their dicyano form) and cyanocobalamin is also described.     

Vitamin B-12 biosynthesis a model system for isopropanolamine formation by reaction between reduced corrinoid and threonine

When a solution of ¹⁴C-labeled L-threonine is incubated with aquocobalamin in the presence of excess of a reductant such as 2-mercaptoethanol or sodium borohydride, a labeled corrinoid adduct is formed. The corrinoid participates stoichiometrically, and not catalytically. Since ³⁵S-labeled 2-mercaptoethanol is not part of the adduct, the reduction of the corrinoid from the Co(III) to the Co(II) form is a prerequisite for the reaction. Various independent tests show the formation of isopropanolamine. The reaction system is an attractive model for the so far unexplained decarboxylation of threonine to isopropanolamine in the course of corrinoid biosynthesis.     

Isolation and characterization of a veratrol:corrinoid protein methyl transferase from Acetobacterium dehalogenans

From 3-methoxyphenol-grown cells of Acetobacterium dehalogenans, an inducible enzyme was purified that mediated the transfer of the methyl groups of veratrol (1,2-dimethoxybenzene) to a corrinoid protein enriched from the same cells. In this reaction, veratrol was converted via 2-methoxyphenol to 1,2-dihydroxybenzene. The veratrol:corrinoid protein methyl transferase, designated MTIver, had an apparent molecular mass of about 32 kDa. With respect to the N-terminal amino acid sequence and other characteristics, MTIver is different from the vanillate:corrinoid protein methyl transferase (MTIvan) isolated earlier from the same bacterium. For the methyl transfer from veratrol to tetrahydrofolate, two additional protein fractions were required, one of which contained a corrinoid protein. This protein was not identical with the corrinoid protein of the vanillate O-demethylase system. However, the latter corrinoid protein could also serve as methyl acceptor for the veratrol:corrinoid protein methyl transferase. MTIver catalyzed the demethylation of veratrol, 3,4-dimethoxybenzoate, 2-methoxyphenol, and 3-methoxyphenol. Vanillate (3-methoxy-4-hydroxybenzoate), 2-methoxybenzoate, or 4-methoxybenzoate could not serve as substrates.     

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO(2)/H(2), CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl-CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol-induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537-540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co-transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X-ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the alpha-axial ligand replacing benziimidazole, suggesting base-off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica.     

Spectroelectrochemical studies of the corrinoid/iron-sulfur protein involved in acetyl coenzyme A synthesis by Clostridium thermoaceticum

An 88-kDa corrinoid/iron-sulfur protein (C/Fe-SP) is the methyl carrier protein in the acetyl-CoA pathway of Clostridium thermoaceticum. In previous studies, it was found that this C/Fe-SP contains (5-methoxybenzimidazolyl)cobamide and a [4Fe-4S]2+/1+ center, both of which undergo redox cycling during catalysis, and that the benzimidazole base is uncoordinated to the cobalt (base off) in all three redox states, 3+, 2+, and 1+ [Ragsdale, S.W., Lindahl, P.A., & Münck, E. (1987) J. Biol. Chem. 262, 14289-14297]. In this paper, we have determined the midpoint reduction potentials for the metal centers in this C/Fe-SP by electron paramagnetic resonance and UV-visible spectroelectrochemical methods. The midpoint reduction potentials for the Co3+/2+ and the Co2+/1 couples of the corrinoid were found to be 300-350 and -504 mV (+/- 3 mV) in Tris-HCl at pH 7.6, respectively. We also removed the (5-methoxybenzimidazolyl)cobamide cofactor from the C/Fe-SP and determined that its Co3+/2+ reduction potential is 207 mV at pH 7.6. The midpoint potential for the [4Fe-4S]2+/1+ couple in the C/Fe-SP was determined to be -523 mV (+/- 5 mV). Removal of this cluster totally inactivates the protein; however, there is little effect of cluster removal on the midpoint potential of the Co2+/1+ couple. In addition, removal of the cobamide has an insignificant effect on the midpoint reduction potential of the [4Fe-4S] cluster. A 27-kDa corrinoid protein (CP) also was studied since it contains (5-methoxybenzimidazolyl)cobamide in the base-on form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a Corrinoid Compound in the Edible (Blue-Green) Alga,Suizenji-nori

The edible blue-green alga (cyanobacterium), Suizenji-nori, contained 143.8±22.4 μg of vitamin B₁₂ per 100 g dry weight of the alga (mean±SE, n=4). A corrinoid compound was purified from the dried Suizenji-nori, and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified corrinoid compound were not identical to those of true vitamin B₁₂, but to those of pseudovitamin B₁₂ which is inactive for humans.     

Kinetic and spectroscopic studies of the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri: substrate specificity and insights into the mechanism of Co(II)corrinoid reduction

The PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus reuteri ( LrPduO) catalyzes the formation of the essential Co-C bond of adenosylcobalamin (coenzyme B 12) by transferring the adenosyl group from cosubstrate ATP to a transient Co (1+)corrinoid species generated in the enzyme active site. While PduO-type enzymes have previously been believed to be capable of adenosylating only Co (1+)cobalamin (Co (1+)Cbl (-)), our kinetic data obtained in this study provide in vitro evidence that LrPduO can in fact also utilize the incomplete corrinoid Co (1+)cobinamide (Co (1+)Cbi) as an alternative substrate. To explore the mechanism by which LrPduO overcomes the thermodynamically challenging reduction of its Co (2+)corrinoid substrates, we have examined how the enzyme active site alters the geometric and electronic properties of Co (2+)Cbl and Co (2+)Cbi (+) by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopic techniques. Our data reveal that upon binding to LrPduO that was preincubated with ATP, both Co (2+)corrinoids undergo a partial ( approximately 40-50%) conversion to distinct paramagnetic Co (2+) species. The spectroscopic signatures of these species are consistent with essentially four-coordinate, square-planar Co (2+) complexes, based on a comparison with the results obtained in our previous studies of related enzymes. Consequently, it appears that the general strategy employed by adenosyltransferases for effecting Co (2+) --> Co (1+) reduction involves the formation of an "activated" Co (2+)corrinoid intermediate that lacks any significant axial bonding interactions, to stabilize the redox-active, Co 3d z (2) -based molecular orbital.