Dihydroflavin-driven Adenosylation of 4-Coordinate Co(II) Corrinoids: ARE COBALAMIN REDUCTASES ENZYMES OR ELECTRON TRANSFER PROTEINS?*

The identity of the source of the biological reductant needed to convert cobalamin to its biologically active form adenosylcobalamin has remained elusive. Here we show that free or protein-bound dihydroflavins can serve as the reductant of Co²⁺Cbl bound in the active site of PduO-type ATP-dependent corrinoid adenosyltransferase enzymes. Free dihydroflavins (dihydroriboflavin, FMNH₂, and FADH₂) effectively drove the adenosylation of Co²⁺Cbl by the human and bacterial PduO-type enzymes at very low concentrations (1 μm). These data show that adenosyltransferase enzymes lower the thermodynamic barrier of the Co²⁺ → Co⁺ reduction needed for the formation of the unique organometalic Co–C bond of adenosylcobalamin. Collectively, our in vivo and in vitro data suggest that cobalamin reductases identified thus far are most likely electron transfer proteins, not enzymes.     

Studies of the CobA-type ATP:Co(I)rrinoid adenosyltransferase enzyme of Methanosarcina mazei strain Go1

Although methanogenic archaea use B(12) extensively as a methyl carrier for methanogenesis, little is known about B(12) metabolism in these prokaryotes or any other archaea. To improve our understanding of how B(12) metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain G?1 (open reading frame MM3138, referred to as cobA(Mm) here) was cloned and used to restore coenzyme B(12) synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme. cobA(Mm) protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of Ca(2+) ions, but the activity was not enhanced by Mg(2+) and, unlike the S. enterica CobA enzyme, it was >50% inhibited by Mn(2+). The CobA(Mm) enzyme had a K(m)(ATP) of 3 microM and a K(m)(HOCbl) of 1 microM. Unlike the S. enterica enzyme, CobA(Mm) used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the beta phosphate of ATP was required for binding to the enzyme. A striking difference between CobA(Se) and CobA(Mm) was the use of ADP as a substrate by CobA(Mm), suggesting an important role for the gamma phosphate of ATP in binding. The results from (31)P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PPP(i)) is the reaction by-product; no cleavage of PPP(i) was observed, and the enzyme was only slightly inhibited by pyrophosphate (PP(i)). The data suggested substantial variations in ATP binding and probably corrinoid binding between CobA(Se) and CobA(Mm) enzymes.     

Resonance Raman spectroscopic study of the interaction between Co(II)rrinoids and the ATP:corrinoid adenosyltransferase PduO from <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

The human-type ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri (LrPduO) catalyzes the adenosylation of Co(II)rrinoids to generate adenosylcobalamin (AdoCbl) or adenosylcobinamide (AdoCbi⁺). This process requires the formation of “supernucleophilic” Co(I)rrinoid intermediates in the enzyme active site which are properly positioned to abstract the adeonsyl moiety from co-substrate ATP. Previous magnetic circular dichroism (MCD) spectroscopic and X-ray crystallographic analyses revealed that LrPduO achieves the thermodynamically challenging reduction of Co(II)rrinoids by displacing the axial ligand with a non-coordinating phenylalanine residue to produce a four-coordinate species. However, relatively little is currently known about the interaction between the tetradentate equatorial ligand of Co(II)rrinoids (the corrin ring) and the enzyme active site. To address this issue, we have collected resonance Raman (rR) data of Co(II)rrinoids free in solution and bound to the LrPduO active site. The relevant resonance-enhanced vibrational features of the free Co(II)rrinoids are assigned on the basis of rR intensity calculations using density functional theory to establish a suitable framework for interpreting rR spectral changes that occur upon Co(II)rrinoid binding to the LrPduO/ATP complex in terms of structural perturbations of the corrin ring. To complement our rR data, we have also obtained MCD spectra of Co(II)rrinoids bound to LrPduO complexed with the ATP analogue UTP. Collectively, our results provide compelling evidence that in the LrPduO active site, the corrin ring of Co(II)rrinoids is firmly locked in place by several amino acid side chains so as to facilitate the dissociation of the axial ligand.     

Structural and functional analyses of the human-type corrinoid adenosyltransferase (PduO) from Lactobacillus reuteri

ATP:Co(I)rrinoid adenosyltransferase (ACA) catalyzes the conversion of cobalamin to coenzyme B12, an essential cofactor in animal metabolism. Several mutations of conserved residues in the active site of human ACA have been identified in humans with methylmalonic aciduria. However, the catalytic role of these residues remains unclear. To better understand the function of these residues and to determine how the enzyme promotes catalysis, several variants of a human-type ACA from the lactic acid bacterium Lactobacillus reuteri (LrPduO) were kinetically and structurally characterized. Kinetic analyses of a series of alternate nucleotides were also performed. Substrate inhibition was observed at subsaturating concentrations of ATP, consistent with an ordered binding scheme where ATP is bound first by the enzyme. Modification or elimination of an active site, inter-subunit salt bridge resulted in a reduced "on" rate for ATP binding, with a less significant disruption in the rate of subsequent catalytic steps. Kinetic and structural data demonstrate that residue Arg132 is not involved in orienting ATP in the active site but, rather, it stabilizes the altered substrate in the transition state. Two functional groups of ATP explain the reduced ability of the enzyme to use alternate nucleotides: the amino group at the C-6 position of ATP contributes approximately 6 kcal/mol of free energy to ground state binding, and the nitrogen at the N-7 position assists in coordinating the magnesium ion in the active site. This study provides new insight into the role of substrate binding determinants and active site residues in the reaction catalyzed by a human-type ACA.     

Co+–H interaction inspired alternate coordination geometries of biologically important cob(I)alamin: possible structural and mechanistic consequences for methyltransferases

A detailed computational analysis employing density functional theory (DFT), atoms in molecules, and quantum mechanics/molecular mechanics (QM/MM) tools has been performed to investigate the primary coordination environment of cob(I)alamin (Co(+)Cbx), which is a ubiquitous B(12) intermediate in methyltransferases and ATP:corrinoid adenosyltransferases. The DFT calculations suggest that the simplified (Co(+)Cbl) as well as the complete (Co(+)Cbi) complexes can adapt to the square pyramidal or octahedral coordination geometry owing to the unconventional H-bonding between the Co(+) ion and its axial ligands. These Co(+)-H bonds contain appreciable amounts of electrostatic, charge transfer, long-range correlation, and dispersion components. The computed reduction potentials of the Co(2+)/Co(+) couple imply that the Co(+)-H(H(2)O) interaction causes a greater anodic shift [5-98?mV vs. the normal hydrogen electrode (NHE) in chloroform solvent] than the analogous Co(+)-H(imidazole) interaction (1?mV vs. NHE) in the reduction potential of the Co(2+)/Co(+) couple. This may explain why a β-axial H(2)O ligand has specifically been found in the active sites of certain methyltransferases. The QM/MM analysis of methionine synthase bound Co(+)Cbx (Protein Data Bank ID 1BMT, resolution 3.0??) indicates that the enzyme-bound Co(+)Cbx can also form a Co(+)-H bond, but can only exist in square pyramidal form because of the steric constraints imposed by the cellular environment. The present calculations thus support a recently proposed alternate mechanism for the enzyme-bound Co(2+)/Co(+) reduction that involves the conversion of square pyramidal Co(2+)Cbx into square pyramidal Co(+)Cbx (Kumar and Kozlowski in Angew. Chem. Int. Ed. 50:8702-8705, 2011).     

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.     

Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

The MgATP complex analogue cobalt-tetrammine-ATP [Co(NH3)4ATP] inactivates (Na+ + K+)-ATPase at 37 degrees C slowly in the absence of univalent cations. This inactivation occurs concomitantly with incorporation of radioactivity from [alpha-32P]Co(NH3)4ATP and from [gamma-32P]Co(NH3)4ATP into the alpha subunit. The kinetics of inactivation are consistent with the formation of a dissociable complex of Co(NH3)4ATP with the enzyme (E) followed by the phosphorylation of the enzyme: (Formula: see text). The dissociation constant of the enzyme-MgATP analogue complex at 37 degrees C is Kd = 500 microM, the inactivation rate constant k2 = 0.05 min-1. ATP protects the enzyme against the inactivation by Co(NH3)4ATP due to binding at a site from which it dissociates with a Kd of 360 microM. It is concluded, therefore, that Co(NH3)4ATP binds to the low-affinity ATP binding site of the E2 conformational state. K+, Na+ and Mg2+ protect the enzyme against the inactivation by Co(NH3)4ATP. Whilst Na+ or Mg2+ decrease the inactivation rate constant k2, K+ exerts its protective effect by increasing the dissociation constant of the enzyme.Co(NH3)4ATP complex. The Co(NH3)4ATP-inactivated (Na+ + K+)-ATPase, in contrast to the non-inactivated enzyme, incorporates [3H]ouabain. This indicates that the Co(NH3)4ATP-inactivated enzyme is stabilized in the E2 conformational state. Despite the inactivation of (Na+ + K+)-ATPase by Co(NH3)4ATP from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 microM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na+ + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the E1 and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na+ + K+)-transport.     

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)     

Effects of single nucleotide changes on the binding and activity of RNA aptamers to human papillomavirus 16 E7 oncoprotein

ATP:Cobalamin adenosyltransferases catalyze the transfer a 5′-deoxyadenosyl moiety from ATP to cob(I)alamin in the synthesis of the Co–C bond of coenzyme B₁₂. There are three types of adenosyltransferases, CobA, PduO and EutT. Among these adenosyltransferases, the PduO-type adenosyltransferases is the most widely distributed enzyme. Structural comparisons between apo BcPduO and BcPduO in complex with MgATP revealed that the N-terminal strands of both structures were ordered, which is in contrast with the most previously available PduO-type adenosyltransferase structures. Furthermore, unlike other reported structures, apo BcPduO was bound to additional dioxane molecules causing a side chain conformational change at the Tyr30 residue, which is an important residue that mediates hydrogen bonding with ATP molecules upon binding of cobalamin to the active site. This study provides more structural information into the role of active site residues on substrate binding.     

Electronic structure studies of the adenosylcobalamin cofactor in glutamate mutase

Glutamate mutase (GM) is a cobalamin-dependent enzyme that catalyzes the reversible interconversion of L-glutamate and L-threo-3-methylaspartate via a radical-based mechanism. To initiate catalysis, the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor's Co-C bond is cleaved homolytically to generate an adenosyl radical and Co2+ Cbl. In this work, we employed a combination of spectroscopic and computational tools to evaluate possible mechanisms by which the Co-C bond is activated for homolysis. Minimal perturbations to the electronic absorption (Abs), circular dichroism (CD), and magnetic CD (MCD) spectra of AdoCbl are observed upon formation of holoenzyme, even in the presence of substrate (or a substrate analogue), indicating that destabilization of the Co3+ Cbl "ground state" is an unlikely mechanism for Co-C bond activation. In contrast, striking alterations are observed in the spectroscopic data of the post-homolysis product Co2+ Cbl when bound to glutamate mutase in the presence of substrate (or a substrate analogue) as compared to unbound Co2+ Cbl. These enzymatic perturbations appear to most strongly affect the metal-to-ligand charge-transfer transitions of Co2+ Cbl, suggesting that the cofactor/active-site interactions give rise to a fairly uniform stabilization of the Co 3d orbitals. Remarkable similarities between the results obtained in this study and those reported previously for the related Cbl-dependent isomerase methylmalonyl-CoA mutase indicate that a common mechanism by which the cofactor's Co-C bond is activated for homolytic cleavage may be operative for all base-off/His-on Cbl-dependent isomerases.     

Reactions of nitric oxide with vitamin B12 and its precursor,cobinamide

Despite early claims that nitric oxide does not react with cobalamin under any circumstances, it is now accepted that NO has a high affinity for cobalamin in the 2+ oxidation state [Cbl(II)]. However, it is still the consensus that NO does not react with Cbl(III). We confirmed that NO coordinates to Cbl(II) at all pH values and that Cbl(III) does not react with NO at neutral pH. At low pH, however, Cbl(III) does react with NO by way of a two-step process that also reduces Cbl(III) to Cbl(II). To account for the pH dependence, and because of its intrinsic interest, we also studied reactions of NO with cobinamide [Cbi] in the 2+ and 3+ oxidation states. Both Cbi(II) and Cbi(III) react readily with NO at all pH values. Again, Cbi(III) is reduced during the process of coordinating NO. Compared to cobalamin, cobinamide lacks the tethered 5,6-dimethylbenzamidazolyl moiety bound to the cobalt ion. It may, therefore, be considered a "base-off" form of Cbl. To explain the reaction of Cbl(III) at low pH, we infer that the base-off form of Cbl(III) exists in trace amounts that are rapidly reduced to Cbl(II), which then binds NO efficiently. Base dissociation, we postulate, is the rate-limiting step. Interestingly, Cbi(II) has 100 times greater affinity for NO than does Cbl(II), proving that there is a strong trans effect due to the tethered base in nitrosyl derivatives of both Cbl(II) and Cbl(III). The affinity of Cbi(II) for NO is so high that it is a very efficient NO trap and, consequently, may have important biomedical uses.     

Altering of the metal specificity of Escherichia coli alkaline phosphatase

Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands. The Zn(2+)-requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328. Co(2+)-requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively. The mutations D153H, K328W, and D153H/K328W were induced in E. coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme. The wild-type and D153H enzymes showed very little activity in the presence of Co(2+), but the K328W and especially the D153H/K328W enzymes effectively use Co(2+) for catalysis. Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co(2+). These data together indicate that the active site of the D153H/K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co(2+) for catalysis. These studies suggest that the active site residues His and Trp at the E. coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.     

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).     

Involvement of an activation protein in the methanol:2-mercaptoethanesulfonic acid methyltransferase reaction in Methanosarcina barkeri.

Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) is the first of two enzymes required for transfer of the methyl group of methanol to 2-mercaptoethanesulfonic acid in Methanosarcina barkeri. MT1 binds the methyl group of methanol to its corrinoid prosthetic group only when the central cobalt atom of the corrinoid is present in the highly reduced Co(I) state. However, upon manipulation of MT1 and even during catalysis, the enzyme becomes inactivated as the result of Co(I) oxidation. Reactivation requires H2, hydrogenase, and ATP. Ferredoxin stimulated the apparent reaction rate of methyl group transfer. Here we report that one more protein fraction was found essential for the overall reaction and, more specifically, for formation of the methylated MT1 intermediate. The more of the protein that was present, the shorter the delay of the start of methyl group transfer. The maximum velocity of methyl transfer was not substantially affected by these varying amounts of protein. This demonstrated that the protein was involved in the activation of MT1. Therefore, it was called methyltransferase activation protein.     

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.     

Comparative analysis of cobalamin binding kinetics and ligand protection for intrinsic factor, transcobalamin, and haptocorrin.

Changes in the absorbance spectrum of aquo-cobalamin (Cbl x OH(2)) revealed that its binding to transcobalamin (TC) is followed by slow conformational reorganization of the protein-ligand complex (Fedosov, S. N., Fedosova, N. U., Nex?, E., and Petersen, T. E. (2000) J. Biol. Chem. 275, 11791-11798). Two phases were also observed for TC when interacting with a Cbl-analogue cobinamide (Cbi), but not with other cobalamins. The slow phase had no relation to the ligand recognition, since both Cbl and Cbi bound rapidly and in one step to intrinsic factor (IF) and haptocorrin (HC), namely the proteins with different Cbl specificity. Spectral transformations observed for TC in the slow phase were similar to those upon histidine complexation with Cbl x OH(2) and Cbi. In contrast to a closed structure of TC x Cbl x OH(2), the analogous IF and HC complexes revealed accessibility of Cbl's upper face to the external reagents. The binders decreased sensitivity of adenosyl-Cbl (Cbl x Ado) to light in the range: free ligand, IF x, HC x, TC x Cbl x Ado. The spectrum of TC x Cbl small middle dotAdo differed from those of IF and HC and mimicked Cbl x Ado participating in catalysis. The above data suggest presence of a histidine-containing cap shielding the Cbl-binding site in TC. The cap coordinates to certain corrinoids and, possibly, produces an incapsulated Ado-radical when Cbl small middle dotAdo is bound.     

A blue corrinoid from partial degradation of vitamin B12 in aqueous bicarbonate: spectra, structure, and interaction with proteins of B12 transport

Cobalamin (Cbl) is a complex cofactor produced only by bacteria but used by all animals and humans. Cyanocobalamin (vitamin B(12), CNCbl) is one commonly isolated form of cobalamin. B(12) belongs to a large group of corrinoids, which are characterized by a distinct red color conferred by the system of conjugated double bonds of the corrin ring retaining a Co(III) ion. A unique blue Cbl derivative was produced by hydrolysis of CNCbl in a weakly alkaline aqueous solution of bicarbonate. This corrinoid was purified and isolated as dark blue crystals. Its spectroscopic analysis and X-ray crystallography revealed B-ring opening with formation of 7,8-seco-cyanocobalamin (7,8-sCNCbl). The unprecedented structural change was caused by cleavage of the peripheral C-C bond between saturated carbons 7 and 8 of the corrin macrocycle accompanied by formation of a C═C bond at C7 and a carbonyl group at C8. Additionally, the C-amide was hydrolyzed to a carboxylic acid. The extended conjugation of the π-system caused a considerable red shift of the absorbance spectrum. Formation and degradation of 7,8-sCNCbl were analyzed qualitatively. Its interaction with the proteins of mammalian Cbl transport revealed both a slow binding kinetics and a low overall affinity. The binding data were compared to those of other monocarboxylic derivatives and agreed with the earlier proposed scheme for two-step ligand recognition. The obtained results are consistent with the structural models of 7,8-sCNCbl and the transport proteins intrinsic factor and transcobalamin. Potential applications of the novel derivative for drug conjugation are discussed.     

Preparation and characterization of cobalt-substituted anthrax lethal factor

Anthrax lethal factor (LF) is a zinc-dependent endopeptidase involved in the cleavage of mitogen-activated protein kinase kinases near their N-termini. The current report concerns the preparation of cobalt-substituted LF (CoLF) and its characterization by electronic spectroscopy. Two strategies to produce CoLF were explored, including (i) a bio-assimilation approach involving the cultivation of LF-expressing Bacillus megaterium cells in the presence of CoCl(2), and (ii) direct exchange by treatment of zinc-LF with CoCl(2). Independent of the method employed, the protein was found to contain one Co(2+) per LF molecule, and was shown to be twice as active as its native zinc counterpart. The electronic spectrum of CoLF suggests the Co(2+) ion to be five-coordinate, an observation similar to that reported for other Co(2+)-substituted gluzincins, but distinct from that documented for the crystal structure of native LF. Furthermore, spectroscopic studies following the exposure of CoLF to thioglycolic acid (TGA) revealed a sequential mechanism of metal removal from LF, which likely involves the formation of an enzyme: Co(2+):TGA ternary complex prior to demetallation of the active site. CoLF reported herein constitutes the first spectroscopic probe of LF's active site, which may be utilized in future studies to gain further insight into the enzyme's mechanism and inhibitor interactions.     

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.