We demonstrate the first application of L-edge X-ray absorption spectroscopy (XAS) to the electronic characterization of biological photolysis products. The experimental L-edge XAS spectra of deoxymyoglobin (deoxy Mb), oxymyoglobin (MbO2), carbonmonoxymyoglobin (MbCO), and the low-temperature photoproducts (Mb*CO and Mb*O2) are presented and compared to simulated spectra using a ligand field multiplet calculation. This analysis indicates that MbCO and MbO2 are both low spin and does not support some previous studies which suggest that MbO2 has an intermediate spin. Both photoproducts, Mb*CO and Mb*O2, are different from deoxy Mb in the FeII electronic structure. In addition, different low-temperature photolysis intermediates are suggested for MbCO and MbO2. The L-edge XAS spectra for FeIII in aquometmyoglobin (met Mb) and azidomet myoglobin (MbN3) provide a comparison of the ferrous versus ferric myoglobin species. Finally, the special advantages of using soft X-ray absorption spectroscopy for understanding the electronic transitions coupled to photolysis-induced structural changes are discussed.

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.

The two available crystallographic structures of cobalamin dependent enzymes, the 27 kDa fragment of the methylcobalamin-dependent enzyme, methionine synthase, from Escherichia coli [Drennan, C. L. et al. Science 1994, 266, 1669] and the 5‘-deoxyadenosylcobalamin-dependent enzyme methylmalonyl-coenzyme A mutase from Propionibacterium shermanii [Mancia, F. et al. Structure 1996, 4, 339], show striking similarities despite the differences in reaction mechanism. In particular, the 5,6-dimethylbenzimidazole group is detached and replaced by a histidine group of the enzyme. Here we present an analysis of Extended X-ray Absorption Fine Structure (EXAFS) spectroscopic data for both 5‘-deoxyadenosylcobalamin and aquocobalamin bound to methylmalonyl-coenzyme A mutase in the absence of substrate. The analysis is conducted with a suite of programs called AUTOFIT 1.0 [Chance, et al. Biochemistry 1996, 35, 9014], which allows an evenhanded comparison of the goodness-of-fit of the EXAFS data to a varied grid of simulations based on the ab initio EXAFS code FEFF 6.01. The X-ray edge data indicate an increase in effective nuclear charge of the metal ion of the enzyme bound 5‘-deoxyadeonsylcobalamin compared to the corresponding free cobalamin, and the EXAFS results show small decreases in equatorial and no significant change in the Co−C bond length (despite the potential elongation of the Co−N(His) bond) upon cofactor binding to the enzyme. Thus, the change in coordination of the nitrogenous axial ligand engineered by the enzyme does not significantly contribute to a trans effect in the ground state. Weakening of the Co−C bond must be initiated by substrate binding. In addition, the global mapping technique resolves discrepancies between previous EXAFS results and crystallographic data on aquocobalamin.

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...

Zinc finger arrays have been established as a critical structural feature of proteins involved in DNA recognition. Retroviral nudeocapsid 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 Cys3Hisj coordination environment for zinc. The average ofthe Zn-S distances is 2.30(1) A and that of the Zn-N distance (to histidine) is 2.01(3) A.Zinc finger domains are known to be of great importance in a number ofproteins that are involved in nucleic acid binding and transcriptional control. All known retroviral nucleocapsid (NC) proteins contain at least one sequence of the form Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys, where Xaa represents various amino acids (1-4). Site-directed mutagenesis experiments involving point mutations of cysteine, histidine, and adjacent residues in the NC protein of murine leukemia virus indicate that the conserved arrays are intimately involved in retroviral RNA recognition, since these mutants give rise to virus particles with little or no detectible viral RNA (5, 6). Subsequent genetic experiments with human immunodeficiency virus type 1 (HIV-1) gag domains, which have two cysteine arrays rather than one, reveal that mutations in either Cys-His array reduce RNA packaging considerably, and infectivity even more drastically (7,8).Prior investigations have conclusively shown that synthetic peptides based on these conserved retroviral sequences bind Zn2+ tightly and stoichiometrically (also Co2+ and Cd2+), and a wealth of spectroscopic evidence points to a three-cysteine one-histidine coordination for metals bound to these peptides (4,(9)(10)(11). The relevance of the peptides to virus function has been questioned, since the zinc content of avian myeloblastosis virus is reportedly insufficient to populate the cysteine arrays (12). However, more recent evidence clearly shows that the zinc content of HIV-1 and human T-cell leukemia virus type I (HTLV-I) is present in near stoichiometric amounts to the NC protein cysteine arrays (13). Thus, the potential importance of retroviral cysteine arrays and zinc to the physiological functions of nucleic acid binding and infectivity is illustrated by a wide range of investigations. We describe experiments that probe the zinc environment of concentrated preparations of equine infectious anemia virus (EIAV), a lentivirus (14, 15), using extended x-ray absorption fine structure (EXAFS) spectroscopy. Data obtained from analysis of virus was compared to that from a well-characterized 18-residue retroviral-type zinc finger peptide, based on sequences from HIV-1. This peptide has the sequence Val-Lys-Cys-Phe-As...

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)

We have made significant improvements in pump-probe time-resolved X-ray absorption spectroscopy that enable us to structurally describe chemical intermediates with short lifetimes. We demonstrate that X-ray preedge data for a 1 mM compound can be acquired with a high signal-to-noise ratio by time-resolved discrimination of fluorescent signals from a 13-element germanium detector. With the utilization of this novel time-multiplexed laser photolysis system coupled to a flow cell, we characterized the structure of the initial photoproduct of five-coordinate base-off Co(III) methylcobalamin. The structure of the primary photoproduct could have included five-or six-coordinate species with water ligation, or a four-coordinate square-planar species. A four-coordinate Co(II) species is expected to be unstable but its biological relevance is highlighted by our recent discovery of a four-coordinate Co(II) species, (existing as the inactive, as isolated, form) in the corrinoid protein of Clostridium thermoaceticum. The X-ray preedge spectra of five-and sixcoordinate species have a strong 1s-3d transition at about 10 eV below the edge. In four-coordinate, squareplanar species the 1s-3d intensity is significantly reduced, but they show a 1s-4p z peak at about 6 eV below the edge. We used this "fingerprint" to monitor the structural change upon photolysis. Since the quantum yield of the base-off species is 0.48, the observed spectrum upon photolysis is a mixture of photoproduct and initial states. The photoproduct of the base-off methylcobalamin shows a substantial decrease in the 1s-3d peak and significant increase in the 1s-4p z peak. This indicates the formation of a four-coordinate species. The four-coordinate species in the free cobalamin is very unstable and can only be detected by time-resolved methods. This indicates a special role for the protein in maintaining an unusual four-coordinate Co(II) corrinoid.

Electron spin echo envelope modulation (ESEEM) and extended X-ray absorption fine structure (EXAFS) spectroscopic studies of oxygenated cobalt (oxyCo) [tetraphenylporphyrin(TPP)][1-methylimidazole (1-MeIm)], an active site model of oxyCo-substituted globins (functional and EPR-active [S = 1/2] analogues of oxygen carrying hemoproteins), are carried out in order to examine the correlation of oxygen affinity with electron-nuclear coupling parameters and metal−ligand bond lengths. ESEEM demonstrates that the magnitude of the electron-nuclear hyperfine and nuclear quadrupole couplings to the directly-coordinated 14N of 1-MeIm decrease (A iso, from 3.54 to 3.04 MHz; e 2 qQ, from 2.39 to 2.08 MHz) as the solvent compositon is varied from 0 to 50% (v/v) dichloromethane in toluene. For oxyCo[(o-R)1TPP][1-MeIm] (where R = −H, −NHCOC(CH3)3, −NHCOCH3, or −NHCONHC6H5, an ortho substitutent on one of the four meso phenyls of TPP), couplings to the axial nitrogen decrease (A iso, from 3.54 to 3.07 MHz; e 2 qQ, from 2.39 to 2.09 MHz) with increased electron-withdrawing strength of R, i.e., with increased acidity of the amide proton of R that may interact with the bound dioxygen. EXAFS measurements, and analysis using ab initio EXAFS codes and global mapping, find that the cobalt−axial nitrogen (Nax) bond of oxyCoTPP-1-MeIm shortens by 0.18 ± 0.06 Å when the solvent is changed from 100% toluene (Co−Nax = 2.12 Å) to 50% toluene/50% dichloromethane (Co−Nax = 1.94 Å). The average cobalt−equatorial nitrogens (1.94−1.96 Å) and cobalt−oxygen (1.95−1.98 Å) distances are unchanged within the error. Similar results were obtained when oxyCoTPP-1-MeIm was compared (in 100% toluene) with its (o-NHCONHC6H5)1TPP counterpart, where the cobalt−ligand bond lengths are indistinguishable from those of oxyCoTPP-1-MeIm in 50% toluene/50% dichloromethane. Increasing the polarity of the solvent and of the vicinity of the bound dioxygen increases oxygen affinity of the metal due to an increase in the ionicity of the cobalt−dioxygen bond that is manifested in reduction in electron-nuclear couplings to the axial nitrogen [Lee et al. Biochemistry 1994, 33, 7609] and shortening of the cobalt−axial nitrogen bond. These ESEEM and EXAFS characterizations of metal−ligand interactions demonstrate the correlation of electron-nuclear coupling and metal−ligand bond lengths with oxygen affinity of hemoprotein model complexes.