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Biomedical subjects

M R Chance

Publications and source records attributed to M R Chance.

At least 19 recordsLinked to original sources
PubMed1998

RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting.

Radiolysis of water with a synchrotron x-ray beam permits the hydroxyl radical-accessible surface of an RNA to be mapped with nucleotide resolution in 10 milliseconds. Application of this method to folding of the Tetrahymena ribozyme revealed that the most stable domain of the tertiary structure, P4-P6, formed cooperatively within 3 seconds. Exterior helices became protected from hydroxyl radicals in 10 seconds, whereas the catalytic center required minutes to be completely folded. The results show that rapid collapse to a partially disordered state is followed by a slow search for the active structure.

Animals↗
PubMed1998

A method for examining the chemical basis for bone disease: synchrotron infrared microspectroscopy.

Infrared microspectroscopy combines microscopy and spectroscopy for the purpose of chemical microanalysis. Light microscopy provides a way to generate and record magnified images and visibly resolve microstructural detail. Infrared spectroscopy provides a means for analyzing the chemical makeup of materials. Combining light microscopy and infrared spectroscopy permits the correlation of microstructure with chemical composition. Inherently, the long wavelengths of infrared radiation limit the spatial resolution of the technique. However, synchrotron infrared radiation significantly improves both the spectral and spatial resolution of an infrared microspectrometer, such that data can be obtained with high signal-to-noise at the diffraction limit, which is 3-5 microm in the mid-infrared region. In this study, we use infrared microspectroscopy to study the chemical composition of bone using two mapping methods. In the osteon method, linear maps are collected from the center of an osteon (newer bone) to the periphery (older bone) and their chemical compositions are compared. In the transverse method, applied specifically to subchondral bone, line maps are collected from the edge of the articular cartilage (older bone) to the marrow space (newer bone). A significant advantage of infrared microspectroscopy over other chemical methods is that the bone does not need to be homogenized for testing; we are able to study cross-sectional samples of bone in situ at a resolution better than 5 microm and compare the results with morphological findings on stained serial sections immediately adjacent to those examined by infrared microspectroscopy. The infrared absorption bands of bone proteins and mineral are sensitive to mineral content (i.e. carbonate, phosphate, acid phosphate), mineral crystallinity and the content/nature of the organic matrix. In this study, they are analyzed as a function of (1) age, i.e. distance with respect to the center of an osteon, and (2) morphology, i.e. cortical versus cancellous (notably subchondral) bone. Results show that the protein/mineral ratio is higher in younger bone. As bone matures, mineralization increases, as does carbonate substitution into the hydroxyapatite lattice. Finally, most of the changes in chemical composition of bone occur within 20 microm of the site of new bone growth, e.g. the center of an osteon, demonstrating the need for the high spatial resolution achieved only with the use of a synchrotron infrared source.

Animals↗
PubMed1997

A comparison of functional and structural consequences of the tyrosine B10 and glutamine E7 motifs in two invertebrate hemoglobins (Ascaris suum and Lucina pectinata).

The architecture of the distal heme pocket in hemoglobins and myoglobins can play an important role in controlling ligand binding dynamics. The size and polarity of the residues occupying the distal pocket may contribute steric and dielectric effects. In vertebrate systems, the distal pocket typically contains a "distal" histidine at position E7 and a leucine at position B10. There are several invertebrate organisms that have hemoglobins or myoglobins that display a pattern in which residues E7 and B10 are a glutamine and tyrosine, respectively. These proteins often have very high oxygen affinities stemming from very slow ligand off rates. In this study, two such hemoglobins, one from the nematode Ascaris suum and the other from the sulfide-fixing clam Lucina pectinata, are compared with respect to conformational and functional properties. Ultraviolet resonance Raman spectroscopy and visible resonance Raman spectroscopy are used to probe, respectively, the ligand-dependent hydrogen bonding pattern of the tyrosine residues and the proximal heme pocket interactions. Fourier transform infrared absorption spectroscopy is used to probe the dielectric properties of the distal heme pocket through the stretching frequency of carbon monoxide bound to the heme. Functionality is probed through the geminate rebinding of both CO and O2. The findings reveal two very different patterns indicative of two different mechanisms for achieving low oxygen off rates. In Hb Ascaris, a hydrogen bonding network that includes the E7 Gln, B10 Tyr, and oxygen bound to the heme results in a tight cage for the oxygen. Dissociation of the O2 requires a large amplitude conformational fluctuation that results both in a spontaneous dissociation of the oxygen through the loss of hydrogen bond stabilization and in an enhanced probability for ligand escape though the transient disruption and opening of the tight distal cage. In the case of the Hb from Lucina, there is no evidence for a tight cage. Instead the data support a model in which the hydrogen bonding network is far more tenuous and the equilibrium state of distal pocket is far more open and accessible than is the case in Ascaris. The results explain why Hb Ascaris has one of the highest oxygen affinities known (P50 approximately 10(-)3 Torr) while Hb Lucina II has an oxygen affinity comparable to that of Mb (P50 = 0.13 Torr) even though both of these Hbs contain the B10 Tyr and E7 Gln motif and display very low oxygen off rates. The roles of water and proximal strain are discussed.

Amino Acid Substitution↗
PubMed1997

Identification of conformational substates involved in nitric oxide binding to ferric and ferrous myoglobin through difference Fourier transform infrared spectroscopy (FTIR).

Hemeproteins play an important role in the signaling processes mediated by nitric oxide (NO). For example, the production of NO by nitric oxide synthase, the activation of guanylate cyclase by binding NO, and the scavenging of NO by hemoglobin, myoglobin, and cytochrome c oxidase all occur through unique mechanisms of interaction between NO and hemeproteins. Unlike carbon monoxide (CO) and oxygen (O2), which have been studied extensively, the reactions of NO with ferric and ferrous hemeproteins are not as well characterized. In this work, NO binding to myoglobin is studied using cryogenic optical spectroscopy and Fourier transform infrared spectroscopy (FTIR) in order to characterize the ligand-bound and photoproduct states involved in the interaction of NO with the heme iron and the distal pocket of the protein. For ferrous nitrosyl myoglobin (MbIINO), optical spectroscopy is used to show that the ligand-bound state can be converted to >95% stable photoproduct below 10 K. The Soret peak of the photoproduct is red-shifted by 4 nm relative to deoxy-myoglobin (Mb), similar to previous results for carbonmonoxy- (MbCO) and oxy-myoglobin (MbO2) (Miller et al., 1996). MbIINO completely rebinds by 35 K, indicating that the rebinding barrier for NO is lower than MbCO, consistent with room temperature picosecond kinetic measurements. For ferric nitrosyl myoglobin (MbIIINO), we find that the photoproduct yield at cryogenic temperatures is less than unity and dependent on the distal pocket residue. Native MbIIINO has a lower photoproduct yield than the mutant, MbIII(H64L)NO, where the distal histidine is replaced by leucine. The rebinding rates for the native and mutant species are similar to each other and to MbIINO. By using FTIR difference spectroscopy (photolyzed/unphotolyzed) of isotopically labeled ferrous nitrosyl myoglobin (MbIINO), the NO stretching frequencies in both the ligand-bound states and photoproduct states are determined. Two ligand-bound conformational states (1607 and 1613 cm-1) and two photoproduct conformational states (1852 and 1857 cm-1) are observed for MbIINO. This is the first direct observation of photolyzed NO in the distal pocket of myoglobin. The ligand-bound frequencies are consistent with a bent MbIINO moiety, where the unpaired pi*(NO) electron remains localized on NO, causing nu(N-O) to be approximately 300 cm-1 lower than MbIIINO. Similar to MbO2, we suggest that Nepsilon of the distal histidine is protonated, forming a hydrogen bond to the NO ligand. For native MbIIINO, a single ligand-bound conformational state with respect to nu(N-O) is observed at 1927 cm-1. This frequency decreases to 1904 cm-1 for the mutant, MbIII(H64L)NO, contrary to the increase of the carbon monoxide (CO) stretching frequency in the isoelectronic MbII(H64L)CO mutant versus native MbCO. For linear MbIIINO, we suggest that backbonding from the unpaired pi*(NO) electron to iron results in an increased positive charge on the NO ligand, Fe(delta-)-NO(delta+). This can be facilitated by tautomerism of the distal histidine, leaving Nepsilon of the imidazole ring unprotonated and able to accept positive charge from the Fe(delta-)-NO(delta+) moiety, resulting in a higher bond order (and a 23 cm-1 shift to higher frequency) for native MbIIINO versus MbIII(H64L)NO, where this interaction is absent. These different interactions between the distal histidine and the ferrous versus ferric species illustrate potential ways the protein can stabilize the bound ligand and demonstrate the versatile nature by which NO can bind to hemeproteins.

Animals↗
PubMed1997

Time-resolved synchrotron X-ray "footprinting", a new approach to the study of nucleic acid structure and function: application to protein-DNA interactions and RNA folding.

Hydroxyl radicals (.OH) can cleave the phosphodiester backbone of nucleic acids and are valuable reagents in the study of nucleic acid structure and protein-nucleic acid interactions. Irradiation of solutions by high flux "white light" X-ray beams based on bending magnet beamlines at the National Synchrotron Light Source (NSLS) yields sufficient concentrations of .OH so that quantitative nuclease protection ("footprinting") studies of DNA and RNA can be conducted with a duration of exposure in the range of 50 to 100 ms. The sensitivity of DNA and RNA to X-ray mediated .OH cleavage is equivalent. Both nucleic acids are completely protected from synchrotron X-ray induced cleavage by the presence of thiourea in the sample solution, demonstrating that cleavage is suppressed by a free radical scavenger. The utility of this time-dependent approach to footprinting is demonstrated with a synchrotron X-ray footprint of a protein-DNA complex and by a time-resolved footprinting analysis of the Mg(2+)-dependent folding of the Tetrahymena thermophilia L-21 ScaI ribozyme RNA. Equilibrium titrations reveal differences among the ribozyme domains in the cooperativity of Mg(2+)-dependent .OH protection. RNA .OH protection progress curves were obtained for several regions of the ribozyme over timescales of 30 seconds to several minutes. Progress curves ranging from > or = 3.5 to 0.4 min-1 were obtained for the P4-P6 and P5 sub-domains and the P3-P7 domain, respectively. The .OH protection progress curves have been correlated with the available biochemical, structural and modeling data to generate a model of the ribozyme folding pathway. Rate differences observed for specific regions within domains provide evidence for steps in the folding pathway not previously observed. Synchrotron X-ray footprinting is a new approach of general applicability for the study of time-resolved structural changes of nucleic acid conformation and protein-nucleic acid complexes.

Animals↗
PubMed1996

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.

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 spectroscopic data, including EXAFS, shows that the crystallographic results of Schlichting et al. (1994) are highly relevant to the physiological solution state and must be taken into account in any attempt to understand the incomplete relaxation process of the heme iron for the Mb*CO photoproduct at low temperature.

Animals↗
PubMed1995

Structural and electronic factors that influence oxygen affinities: a spectroscopic comparison of ferrous and cobaltous oxymyoglobin.

Various structural and electronic factors that result in similar rates of oxygen association (kon) and differing rates of oxygen dissociation (koff) for ferrous (FeMb) and cobaltous (CoMb) myoglobin have been investigated. Similar values for kon indicate similar barriers to oxygen binding for CoMb and FeMb. Through optical spectroscopy, we have found that the stable quantum yields of photolysis for CoMbO2 (0.55 +/- 0.05) and FeMbO2 (0.50 +/- 0.05) at 10 K are the same. The X-ray absorption near edge spectra (XANES) of CoMb and FeMb reveal similar metal-heme displacements for the deoxy, oxy, and low temperature photoproduct states of CoMb and FeMb. Thus, similar barriers to ligand binding, indicated by similar kon's and photoproduct yields for CoMb and FeMb, correlate with the metal-heme displacements for the oxy, deoxy, and low temperature photoproduct states of CoMb and FeMb. Lower values of koff for FeMbO2 versus CoMbO2 imply different barriers to oxygen release for the two species. X-ray edge positions of CoMb and FeMb indicate a substantial transfer of electron density from the metal to the ligand upon oxygenation. The distribution of electron density throughout the M-O-O moiety differs for CoMbO2 and FeMbO2. Resonance Raman spectroscopy has demonstrated that the Co-O bond is weaker when compared to Fe-O [Tsubaki, M., & Yu, N. T. (1981) Proc. Natl. Acad. Sci., U.S.A. 78, 3581]. We have used photolyzed/unphotolyzed Fourier Transform Infrared (FTIR) difference spectra of CoMb16O2, CoMb18O2, FeMb16O2, and FeMb18O2 to show that the dioxygen stretching frequency, v(O-O), in CoMbO2 (approximately 1138 cm-1) is higher than FeMbO2 (approximately 1131 cm-1). The dioxygen stretching frequency in CoMbO2 is closer to that of heme protein models lacking a hydrogen bond to the distal histidine, suggesting that formation of the hydrogen bond in FeMbO2 provides a greater effect on the distribution of electron density throughout the Fe-O-O... HN moiety, potentially stabilizing a more ionic Fe-O-O bond. These findings demonstrate important electrostatic differences in the distal environments of CoMbO2 and FeMbO2, resulting in different barriers to oxygen release.

Animals↗
PubMed1995

Purification of a variant-specific surface protein of Giardia lamblia and characterization of its metal-binding properties.

Giardia lamblia, an intestinal parasite of humans and other vertebrates, undergoes surface antigenic variation by modulating the expression of different variant-specific surface proteins (VSP). VSPs are cysteine-rich surface proteins that bind zinc and other heavy metals in vitro. We developed an immunoaffinity chromatographic method to purify a VSP in order to determine its biochemical properties. The sequences of two different proteolytic fragments agreed with the sequence deduced from the cloned gene, and amino-terminal sequence indicated the removal of a 14-residue signal peptide, consistent with the transport of VSP to the cell surface. The protein is not glycosylated and has an isoelectric point of 5.3. X-ray microanalyses indicated that the major metals in Giardia trophozoites, as well as purified VSP, are zinc and iron. The zinc concentration in Giardia cells was found to be 0.43 mM and the iron concentration 0.80 mM when compared with standard samples (zinc) or calculated from a known physical constants (iron). We propose that metal coordination stabilizes VSPs, rendering them resistant to proteolytic attack in the upper small intestine. Moreover, the ability to bind ions by Giardia may play a role in nutritional deficiency and/or malabsorption in heavily infected persons.

Amino Acid Sequence↗
PubMed1995

Structural and electronic factors in heterolytic cleavage: formation of the Co(I) intermediate in the corrinoid/iron-sulfur protein from Clostridium thermoaceticum.

We have completed the first direct structural characterization of an enzyme-bound four-coordinate Co(I) intermediate, in this case for the corrinoid/iron-sulfur protein (C/Fe-SP) from Clostridium thermoaceticum. Extended X-ray absorption fine structure and X-ray edge spectroscopy of the active Co(I) state of the C/Fe-SP indicates a four-coordinate (distorted) square-planar structure where the best fit gives average Co-N(equatorial) distances of 1.87 +/- 0.01 A, corresponding to 4.2 +/- 0.3 ligands. The X-ray edge spectrum of Co(I) C/Fe-SP contains a moderate intensity 1s-4p + "shake-down" (SD) transition and no 1s-3d peak (where SD transitions are indicative of square-planar geometries). X-ray edge results for the methyl-Co(III) form, reported earlier [Wirt, M. D., Kumar, M., Ragsdale, S. W., & Chance, M. R. (1993) J. Am. Chem. Soc. 115, 2146-2150], are consistent with a base-off methylcobamide structure. The absence of a ligated 5-methoxybenzimidazole base in the methyl-Co(III) state is important since the base-off form is predicted to predispose the Co-C bond toward heterolytic cleavage to form the four-coordinate Co(I) species concurrent with methyl transfer. Additionally, we have examined first-derivative X-ray edge spectra of Co(I) C/Fe-SP, relative to edge spectra of a cobalt foil, as an indicator of effective nuclear charge on cobalt. The Co(I) C/Fe-SP edge position at 7720.5 +/- 0.3 eV is less than, but very close to, the value seen for the corresponding free Co(I) cobalamin.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1994

Kinetic and structural characterization of spinach carbonic anhydrase.

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.

Bicarbonates↗
PubMed1994

Sulfur-containing cobalamins: X-ray absorption spectroscopic characterization.

Sulfur-containing cobalamins are thought to have a special role in the intracellular conversion of cyanocobalamin to its coenzyme forms through a Co(I) intermediate. Glutathionylcobalamin is especially interesting as a possible precursor of cobalamin coenzymes [Wagner et al. (1969) Ann. N.Y. Acad. Sci. 112, 580; Pezacka et al. (1990) Biochem. Biophys. Res. Commun. 169, 443]. Recent NMR data [Brown et al. (1993) Biochemistry 32, 8421] strongly support the hypothesis that glutathione coordinates ito the cobalt through the sulfur atom in glutathionylcobalamin. In this study three-sulfur containing cobalamin derivatives (glutathionylcobalamin, sulfitocobalamin, and cysteinylcobalamin) have been characterized by X-ray absorption spectroscopy. We give evidence for the sulfur coordination in these compounds and present the corresponding structural information. The Co-Neq distances are also distances in the sulfur-containing cobalamins are very close to one another (1.90 +/- 0.01 A). The Co-S and Co-Nax distances are also similar (Co-S: 2.28-2.35 A and Co-Nax: 2.13-2.16 A) and in the expected range. The X-ray edge positions for the sulfur derivatives shift to lower energies with respect to cyanocobalamin. This indicates strong electron donation from the sulfur to the cobalt and suggests that the effective charge on the cobalt ion in sulfur cobalamins is largely reduced from +3.

Chemical Phenomena↗
PubMed1993

Human cobalophilin: the structure of bound methylcobalamin and a functional role in protecting methylcobalamin from photolysis.

The interactions of methylcobalamin with cobalophilin from human serum were analyzed using extended X-ray absorption fine structure (EXAFS) spectroscopy, photolysis of the cobalt-carbon bond of methylcobalamin, and a pKa determination of the protonation of the coordinated nitrogen of 5,6-dimethylbenzimidazole (DMB). These results are consistent with the idea that the DMB nitrogen is still coordinated when protein is bound; however, the ability of a methyl radical (generated by photolysis) to escape the geminate cage of the protein is considerably reduced. For methylcobalamin in solution, the DMB nitrogen ligand is at a distance of 2.20 +/- 0.03 A from cobalt [Sagi, I., & Chance, M. R. (1992) J. Am. Chem. Soc. 114, 8061-8066]. This distance to the lower axial ligand does not change when protein binds (2.20 +/- 0.04 A), nor do the optical spectra exhibit any base-off character. The average of the distance from cobalt to the four equatorial nitrogens of the corrin plane is also unchanged. The pKa for the conversion of the "base-on" to the "base-off" form of methylcobalamin, where the above DMB nitrogen becomes protonated and the Co-N axial bond is cleaved, does not deviate from the free cobalamin value of 2.7 when methylcobalamin is bound to cobalophilin. These results indicate that replacement of the DMB ligand with a ligand from the protein is unlikely. Although the background-subtracted EXAFS data sets for free methylcobalamin and for the protein complex are extremely similar, more accurate data with explicit higher shell analysis would be required to entirely rule out ligand replacement. The chemical and electronic nature of the ligand changes little.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

Continuous-wave quantum yields of various cobalamins are influenced by competition between geminate recombination and cage escape.

Quantum yields of photolysis of the cobalt-carbon bond for three cobalamin compounds were measured with a continuous-wave laser at 442 nm under both aerobic and anaerobic conditions. Aerobically, the initial homolysis product, Co(II) cobalamin, is trapped by oxygen to form aquocobalamin. Use of an excess of the radical trapping reagent 2,2,6,6-tetramethyl-1-piperidinyloxyl, under anaerobic conditions, scavenges the carbon radical and allows detection of the cobalt(II) photoproduct. Quantum yields measured under anaerobic conditions for 5'-deoxyadenosylcobalamin (phi (Co-C alpha),442 = 0.20 +/- 0.03) and methylcobalamin (phi (Co-C alpha),442 = 0.35 +/- 0.03) are in agreement with the values obtained under aerobic conditions (phi (Co-C alpha),442 = 0.19 +/- 0.04 and phi (Co-C alpha),442 = 0.36 +/- 0.04, respectively). Additionally, the quantum yield values for 5'-deoxyadenosylcobalamin and its base-off derivative (phi (Co-C alpha),442 = 0.045 +/- 0.015) match those obtained on a nanosecond time scale [Chen, E., & Chance, M. R. (1990) J. Biol. Chem. 256, 12987-12994]. A comparison of quantum yields obtained anaerobically for 5'-deoxyadenosylcobalamin and methylcobalamin in H2O versus ethylene glycol shows a 4-fold decrease for the former cobalamin and no change for the latter. These quantum yields are evaluated in terms of time-independent radical separation distances.

Aerobiosis↗
PubMed1993

Temperature dependent coordination effects in base-off adenosyl and methylcobalamin by X-ray edge spectroscopy.

Examination of the role of base-off cobalamin species (where the 5,6-dimethylbenzimidazole ligand coordinated to cobalt is detached by protonation of the imidazole nitrogen) in differentiation between homolytic and heterolytic cobalt-carbon bond cleavage mechanisms is a primary step in better understanding B12-dependent enzyme catalysis. X-ray absorption edge spectroscopy provides the first direct structural evidence of five-coordination in base-off adenosyl- and base-off methylcobalamin complexes at room temperature. Integration of 1s-3d pre-edge transitions of the base-off species reveals the dependence of coordination number on temperature. Gradual increases in 1s-3d transition intensities, as the temperature is increased from 180 K to 298 K, reflect a change in the coordination number from six (where a water molecule is presumed to occupy the coordination site vacated by the 5,6-dimethylbenzimidazole ligand) to primarily five-coordinate. Base-off configurations that strengthen the Co-C bond may be both decreasing the tendency for homolytic cleavage while increasing the tendency for hetrolytic Co(I) B12 formation.

Cobamides↗
PubMed1992

Extended x-ray absorption fine structure studies of a retrovirus: equine infectious anemia virus cysteine arrays are coordinated to zinc.

Zinc finger arrays have been established as a critical structural feature of proteins involved in DNA recognition. Retroviral nucleocapsid proteins, which are involved in the binding of viral RNA, contain conserved cysteine-rich arrays that have been suggested to coordinate zinc. We provide metalloprotein structural data from an intact virus preparation that validate this hypothesis. Extended x-ray absorption fine structure (EXAFS) spectroscopy of well-characterized and active preparations of equine infectious anemia virus, compared with a peptide with known coordination and in combination with available biochemical and genetic data, defines a Cys3His1 coordination environment for zinc. The average of the Zn-S distances is 2.30(1) A and that of the Zn-N distance (to histidine) is 2.01(3) A.

Absorptiometry, Photon↗