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At least 145 records · Page 8Linked to original sources
PubMed1998

Heteronuclear NMR studies of cobalt corrinoids. 20. 31P chemical shift anisotropy of aquacobalamin and its complex with a haptocorrin from chicken serum.

Static light scattering measurements have been used to determine the molecular mass (65.3 kDa) and second virial coefficient (3.66 x 10(-4) mol mL g-2) for the complex between chicken serum haptocorrin (HC) and aquacobalamin (H2OCbl). Dynamic light scattering measurements have also been used to determine the translational diffusion coefficient of the H2OCbl-HC complex as a function of concentration. From the diffusion coefficient at infinite dilution (4.71 x 10(-7) cm2 s-1), the hydrodynamic radius (45.5 A) and rotational correlation time (85.4 ns) have been calculated. Using the latter, and measured values of the 31P NMR linewidths of the H2OCbl-HC complex at several field strengths, a detailed analysis of the 31P nuclear relaxation is possible. The chemical shift anisotropy term from the transverse relaxation equation is determined to be 95.7 ppm, and the average phosphorus-proton internuclear distance is 2.05 A. For comparison to protein-free H2OCbl, the chemical shift anisotropy of the phosphorus atom was studied by solid state NMR spectroscopy and the 31P relaxation by solution T1 measurements. These studies give values of 110.3 ppm for the chemical shift anisotropy term and 2.45 A for the average phosphorus-proton internuclear distance. The results are consistent with a significant change in the conformation of the H2OCbl phosphodiester upon binding to haptocorrin which could be due to a shortening of the axial Co-N bond.

Animals↗
PubMed1993

Heteronuclear nuclear magnetic resonance studies of cobalt corrinoids. 15. The structure of glutathionylcobalamin: a 1H and 13C two-dimensional nuclear magnetic resonance study at 600 MHz.

Glutathionylcobalamin (GSCbl), the complex formed between glutathione (GSH, gamma-glutamylcysteinylglycine) and aquacobalamin (H2OCbl), has been implicated as an intermediate in the pathway for the formation of the cobalamin coenzymes. In chemical model studies, GSCbl has been shown to be a substrate for methylcobalamin formation in the presence of S-adenosylmethionine and a thiol reductant. Although GSCbl was first described in 1964, the structure of this compound, particularly the site of GSH coordination, has been unknown. GSCbl was prepared by reacting GSH (5-fold molar excess) with H2OCbl in 0.1 M sodium phosphate (pH 6.5) and was purified by gel-permeation chromatography on a Bio-Gel P2 polyacrylamide column. By use of a combination of homonuclear [homonuclear J-correlated spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), and absorption-mode nuclear Overhauser effect spectroscopy (NOESY)] and inverse detected heteronuclear [1H-detected heteronuclear multiple-quantum coherence (HMQC) and 1H-detected multiple-bond heteronuclear multiple-quantum coherence (HMBC) spectroscopies] two-dimensional NMR methods at 600 MHz, the complete 1H and 13C NMR spectra of GSCbl have now been assigned. Comparison of the 1H and 13C NMR chemical shifts of the GS moiety of GSCbl to those of free GSH and GS- shows that by far the largest differences occur at the cysteine alpha and beta positions. This result strongly suggests that GSH is coordinated to the cobalt atom in GSCbl via the cysteine sulfur atom.

Carbon Isotopes↗
PubMed2001

Three-dimensional structure of ATP:corrinoid adenosyltransferase from Salmonella typhimurium in its free state, complexed with MgATP, or complexed with hydroxycobalamin and MgATP.

In Salmonella typhimurium, formation of the cobalt-carbon bond in the biosynthetic pathway for adenosylcobalamin is catalyzed by the product of the cobA gene which encodes a protein of 196 amino acid residues. This enzyme is an ATP:co(I)rrinoid adenosyltransferase which transfers an adenosyl moiety from MgATP to a broad range of co(I)rrinoid substrates that are believed to include cobinamide, its precursor cobyric acid and probably others as yet unidentified, and hydroxocobalamin. Three X-ray structures of CobA are reported here: its substrate-free form, a complex of CobA with MgATP, and a ternary complex of CobA with MgATP and hydroxycobalamin to 2.1, 1.8, and 2.1 A resolution, respectively. These structures show that the enzyme is a homodimer. In the apo structure, the polypeptide chain extends from Arg(28) to Lys(181) and consists of an alpha/beta structure built from a six-stranded parallel beta-sheet with strand order 324516. The topology of this fold is very similar to that seen in RecA protein, helicase domain, F(1)ATPase, and adenosylcobinamide kinase/adenosylcobinamide guanylyltransferase where a P-loop is located at the end of the first strand. Strikingly, the nucleotide in the MgATP.CobA complex binds to the P-loop of CobA in the opposite orientation compared to all the other nucleotide hydrolases. That is, the gamma-phosphate binds at the location normally occupied by the alpha-phosphate. The unusual orientation of the nucleotide arises because this enzyme transfers an adenosyl group rather than the gamma-phosphate. In the ternary complex, the binding site for hydroxycobalamin is located in a shallow bowl-shaped depression at the C-terminal end of the beta-sheet of one subunit; however, the active site is capped by the N-terminal helix from the symmetry-related subunit that now extends from Gln(7) to Ala(24). The lower ligand of cobalamin is well-ordered and interacts mostly with the N-terminal helix of the symmetry-related subunit. Interestingly, there are few interactions between the protein and the polar side chains of the corrin ring which accounts for the broad specificity of this enzyme. The corrin ring is oriented such that the cobalt atom is located approximately 6.1 A from C5' of the ribose and is beyond the range of nucleophilic attack. This suggests that a conformational change occurs in the ternary complex when Co(III) is reduced to Co(I).

Adenosine Triphosphate↗
PubMed1996

Heteronuclear NMR Studies of Cobalt Corrinoids. 18. Correlation of Structure and Magnetic Resonance Parameters in Base-On Cobalamins(1).

Recent X-ray crystal structure determinations (including a new X-ray determination of the structure of cyano-13-epicobalamin reported herein) create a series of seven base-on cobalamins structurally characterized by modern crystallographic techniques in which the intramolecular equilibrium constant for coordination of the axial benzimidazole ligand (Bzm) varies from 76.6 to 4.90 x 10(7). For the five normal, unepimerized cobalamins, the free energy change for this equilibrium correlates linearly with the axial Co-N bond length (r(2) = 0.99). Absolute assignment of the (1)H and (13)C NMR spectra of two of these structurally characterized cobalamins (CH(3)Cbl and CN-13-epiCbl) together with literature assignments for the other complexes now provides reliable (13)C NMR assignments and chemical shifts for all seven complexes. The magnetic anisotropies of the central cobalt atom of all seven complexes, estimated by a method described earlier, are well correlated with the axial Co-N bond distance (r(2) = 0.97) and the free energy of coordination of the Bzm ligand (r(2) = 0.95). The (31)P NMR chemical shift of the phosphodiester moiety of the nucleotide loop is excellently correlated to the axial Co-N bond length (r(2) = 0.996) of the unepimerized cobalamins and provides a reliable method of estimating this bond length. The (15)N chemical shifts of the axially coordinated Bzm nitrogen vary strongly with the axial Co-N bond distance and correlate linearly with this structural parameter (r(2) = 0.991) except for the case of H(2)OCbl(+), which deviates substantially. However, there is a good linear correlation (r(2) = 0.98) of this (15)N chemical shift with the free energy of Bzm coordination for the five unepimerized cobalamins. Attempts to correlate (13)C NMR chemical shifts with structural, thermodynamic, and corrin ring conformational parameters are discussed.

Journal Article↗
PubMed1996

Solvent Cage Effects in Organocobalt Corrinoid Chemistry: Thermal Homolysis of alpha- and beta-(Cyanomethyl)cobinamides(1).

The equilibrium constant for the thermal isomerization of the diastereomeric alpha- and beta-(cyanomethyl)cobinamides (NCCH(2)Cbi(+)'s) has been measured over the temperature range 70-95 degrees C. Although the beta diastereomer is the thermodynamically more stable isomer, it is favored by the entropy change, but disfavored by the enthalpy change. In the presence of >/=5 x 10(-)(3) M concentration of the radical trap 4-hydroxy-2,2,6,6,-tetramethylpiperidinyloxy (4-HTEMPO), thermolysis of either isomer leads to cob(II)inamide and the trapped NCCH(2)(*) radical (NCCH(2)-4-HTEMPO) in high yield and no isomerization can be detected. The kinetics of the 4-HTEMPO-trapped thermal homolysis of alpha- and beta-NCCH(2)Cbi(+) have been studied in anaerobic glycerol/water mixtures of varying viscosity. The observed first-order rate constants for thermolysis show the expected inverse dependence on viscosity indicating that the process is at least partially diffusion controlled. From these data, the primary rate constant, k(1), for carbon-cobalt bond homolysis and the ratio of the rate constants for in-cage recombination and diffusional separation (k(c)/k(d)) can be extracted. The enthalpies of activation for Co-C bond homolysis are identical (29.0 +/- 0.3 kcal mol(-)(1)) while the entropy of activation is 2-fold higher for the alpha diastereomer. In water, the fractional cage efficiencies, F(c), are quite small (0.12 +/- 0.01, alpha; 0.049 +/- 0.008, beta) and invariant for each complex in the temperature range 75-95 degrees C. Assuming that the rate constant for diffusional separation of the caged radical pairs is the same for both isomers, the ratio of the in-cage recombination rate constants, k(c)(alpha)/k(c)(beta), can be calculated to be 2.6 +/- 0.6. This surprising kinetic preference for the alpha diastereomer results from enthalpic stabilization of the recombination transition state for the alpha diastereomer, since the beta diastereomer is entropically favored.

Journal Article↗
PubMed1981

Ring contraction of hydroporphinoid to corrinoid complexes.

Crystalline nickel(II) and dicyanocobalt(III) complexes of a racemic 1,2,2,7,7,12,12,17,17,20-decamethyl-20-hydroxy-1,2,3,7,8,12,13,20-octahydro- 17H-porphyrin rearrange to the corresponding complexes of racemic 19-acetyl-1,2,2,7,7,12,12,17,17-nonamethyl-trans-corrin on melting (approximately 290 degrees C and 260 degrees C, respectively). The nickel(II) 19-acetylcorrinate formed in this way is shown to deacetylate to racemic nickel(II) 1,2,2,7,7,12,12,17,17-nonamethyl-trans-corrinate on treatment with 2 M KOH. These reactions are being studied as potentially biomimetic chemical models for the elusive ring contraction step in vitamin B(12) biosynthesis.

Journal Article↗
PubMed2003

Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus.

The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 +/- 1 kDa, whereas the native molecular mass was 71 +/- 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-beta-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 +/- 0.1 mol of cobalamin, 0.6 +/- 0.02 mol of cobalt, 7.1 +/- 0.6 mol of iron, and 5.8 +/- 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 +/- 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The K(m) values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 +/- 3.2, 23.7 +/- 5.2, and 47 +/- 10 micro M, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.

Bacteria, Anaerobic↗