Sulfur-Containing Cobalamins: X-ray Absorption Spectroscopic Characterization

Scheuring, Eva M.; Sagi, Irit; Chance, Mark R.

doi:10.1021/bi00186a034

Cited by 39 publications

(60 citation statements)

References 13 publications

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“…The four in-plane Co-N bonds are similar to those reported for other Co-S containing Cbls, and for most other Cbl complexes in general. These equatorial bonds appear to be rather insensitive to the nature of the axial ligand and the Co-ligand bond distance, as previously suggested 9. The GS − ligand is bound to the cobalt through the sulfur atom as expected, with a Co-S bond distance of 2.295(1) Å.…”

Section: Resultssupporting

confidence: 78%

The X-Ray Crystal Structure of Glutathionylcobalamin Revealed

2010

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The first evidence of a complex between glutathione and cobalamin, glutathionylcobalamin (GSCbl), was presented by Wagner and Bernhauer more than 40 years ago (Ann N Y Acad Sci, 1964, 112, 580). More recently, NMR and EXAFS solution studies by Brown et al (Biochemistry, 1993, 32, 8421) and Scheuring et al. (Biochemistry, 1994, 33, 6310), respectively, provided evidence that the glutathionyl moiety in GSCbl is bound to the cobalt center via a Co-S bond. Despite continued efforts, the structural analysis of glutathionylcobalamin in the solid state has remained elusive. Here we report the first atomic resolution crystal structure of GSCbl, refined to a crystallographic R-factor of 0.0683. The glutathione moiety is bound to the cobalt center through the sulfur atom as expected, with a Co-S bond distance of 2.295(1) A. This distance agrees with the distance obtained from the EXAFS analysis of GSCbl (2.280(5) Å). However, the bond to the axial α-5,6-dimethylbenzimidazole base (DMB), 2.074(3) Å, is significantly shorter than that determined from the EXAFS measurements (Co-N3B = 2.15(3) Å). The corrin fold angle is 24.7°, the highest ever reported for a cobalamin structure, and points in the direction of the β-face of the corrin, towards the glutathione (GS − ). The GS − ligand has been modeled in two conformations, each featuring distinct hydrogen bonding interactions. In both conformations, the α-carboxylate group of the GS − ligand interacts with the generally rigid side chain a of the cobalamin molecule, resulting in two distinct conformations. A comparison with the structure of other thiolatocobalamins revealed high similarity in the positions of the atoms in the cysteinyl moiety, the fold of the corrin rings, and the Co-S bond distances.

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Section: Resultssupporting

confidence: 78%

The X-Ray Crystal Structure of Glutathionylcobalamin Revealed

2010

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“…Further details of the simulations are discussed in Results and Discussion. Data processing, where specified, was carried out using a PC-based Bell Labs EXAFS package compiled with Microsoft FORTRAN 77 (Rowlett et al, 1994;Scheuring et al, 1994). For Figures 4 and 6-8 the experimental data and the simulations were k 3 weighted, Fourier transformed, filtered from 0.8 to 4.8 Å, backtransformed, and processed by using the Bell Labs EXAFS package.…”

Section: Methodsmentioning

confidence: 99%

Global Mapping of Structural Solutions Provided by the Extended X-ray Absorption Fine Structure ab Initio Code FEFF 6.01: Structure of the Cryogenic Photoproduct of the Myoglobin−Carbon Monoxide Complex

et al. 1996

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X-ray methods based on synchrotron technology have the promise of providing time-resolved structural data based on the high flux and brightness of the X-ray beams. One of the most closely examined problems in this area of time-resolved structure determination has been the examination of intermediates in ligand binding to myoglobin. Recent crystallographic experiments using synchrotron radiation have identified the protein tertiary and heme structural changes that occur upon photolysis of the myoglobin--carbon monoxide complex at cryogenic temperatures [Schlichting, I., Berendzen, J., Phillips, G., & Sweet, R. (1994) Nature 371, 808--812]. However, the precision of protein crystallographic data (approximately 0.2 A) is insufficient to provide precise metrical details of the iron--ligand bond lengths. Since bond length changes on this scale can trigger reactivity changes of several orders of magnitude, such detail is critical to a full understanding of metalloprotein structure--function relationships. Extended X-ray absorption fine structure (EXAFS) spectroscopy has the potential for analyzing bond distances to a precision of 0.02 A but is hampered by its relative insensitivity to the geometry of the backscattering atoms. Thus, it is often unable to provide a unique solution to the structure without ancillary structural information. We have developed a suite of computer programs that incorporate this ancillary structural information and compute the expected experimental spectra for a wide ranging series of Cartesian coordinate sets (global mapping). The programs systematically increment the distance of the metal to various coordinating ligands (along with their associated higher shells). Then, utilizing the ab initio EXAFS code FEFF 6.01, simulated spectra are generated and compared to the actual experimental spectra, and the differences are computed. Finally, the results for hundreds of simulations can be displayed (and compared) in a single plot. The power of this approach is demonstrated in the examination of high signal to noise EXAFS data from a photolyzed solution sample of the myoglobin--carbon monoxide complex at 10 K. Evaluation of these data using our global mapping procedures placed the iron to pyrrole nitrogen average distances close to the value for deoxymyoglobin (2.05 +/- 0.01 A), while the distance from iron to the proximal histidine nitrogen is seen to be 2.20 +/- 0.04 A. It is also shown that one cannot uniquely position the CO ligand on the basis of the EXAFS data alone, as a number of reasonable minima (from the perspective of the EXAFS) are observed. This provides a reasonable explanation for the multiplicity of solutions that have been previously reported. The results presented here are seen to be in complete agreement with the crystallographic results of Schlichting et al. (1994) within the respective errors of the two techniques; however, the extended X-ray absorption fine structure data allow the iron--ligand bond lengths to be precisely defined. An examination of the available spectroscopi...

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“…This analysis was checked against fits analyzed using the Bell labs EXAFS package run on a personal computer (Scheuring & Chance, 1994). We compared several fits with different nitrogen:sulfur (N:S) coordination numbers held fixed in the fitting procedure.…”

Section: Methodsmentioning

confidence: 99%

Kinetic and Structural Characterization of Spinach Carbonic Anhydrase

Rowlett¹,

Chance²,

Wirt³

et al. 1994

Biochemistry

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We have carried out kinetics studies of spinach carbonic anhydrase (CA) using stopped-flow spectrophotometry at steady state and 13C-NMR exchange at chemical equilibrium. We found that the rate of CO2<-->HCO3- exchange catalyzed by spinach CA at pH 7.0 to be 3-5 times faster than the maximal kcat for either CO2 hydration or HCO3- dehydration at steady state, suggesting a rate-determining H+ transfer step in the catalytic mechanism. Correspondingly, we measured a pH-independent solvent deuterium isotope effect on kcat of approximately 2.0, and found that the rate of catalysis was significantly decreased at external buffer concentrations below 5 mM. Our results are consistent with a zinc-hydroxide mechanism of action with for spinach CA, similar to that of animal carbonic anhydrases. We have also collected X-ray absorption spectra of spinach CA. Analysis of the extended fine structure (EXAFS) suggests that the coordination sphere of Zn in spinach CA must have one or more sulfur ligands, in contrast to animal CAs which have only nitrogen and oxygen ligands. The models which best fit the data have average Zn-N(O) distances of 1.99-2.06 A, average Zn-S distances of 2.31--2.32 A, and a total coordination number of 4-6. We conclude that animal and spinach CAs are convergently evolved enzymes which are structurally quite different, but functionally equivalent.

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Sulfur-Containing Cobalamins: X-ray Absorption Spectroscopic Characterization

Cited by 39 publications

References 13 publications

The X-Ray Crystal Structure of Glutathionylcobalamin Revealed

The X-Ray Crystal Structure of Glutathionylcobalamin Revealed

Global Mapping of Structural Solutions Provided by the Extended X-ray Absorption Fine Structure ab Initio Code FEFF 6.01: Structure of the Cryogenic Photoproduct of the Myoglobin−Carbon Monoxide Complex

Kinetic and Structural Characterization of Spinach Carbonic Anhydrase

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