EXAFS Data Indicate a “Normal” Axial Cobalt−Nitrogen Bond of the Organo-B₁₂Cofactor in the Two Coenzyme B₁₂-Dependent Enzymes Glutamate Mutase and 2-Methyleneglutarate Mutase

Abstract

A key step in the catalytic cycle of coenzyme B12-dependent enzymes is the homolysis of the cofactor's organometallic bond, leading to the formation of a 5‘-deoxyadenosyl radical. For the adenosylcobalamin-dependent enzyme methylmalonyl CoA mutase (MCM), it has been suggested that this step is mediated by a protein-induced lengthening of the cofactor's axial cobalt−nitrogen bond, in trans position to the scissile organometallic bond. In fact, such a lengthening was first observed in the crystal structure of MCM (Mancia et al. Structure 1996, 4, 339−350) and was later confirmed by an analysis of EXAFS data on the same protein in frozen solution (Scheuring et al. J. Am. Chem. Soc. 1997, 119, 12192−12200). Here, we report the results of an EXAFS study on the related coenzyme B12-dependent enzymes glutamate mutase from Clostridium cochlearium and 2-methyleneglutarate mutase from Clostridium barkeri. Both apoenzymes were overproduced from E. coli and reconstituted with methylcobalamin (MeCbl) to yield inactive enzymes, whose stability toward (substrate-induced) cobalt−carbon bond homolysis should be higher than for the enzymatically active forms obtained by reconstitution with 5‘-desoxyadenosylcobalamin. X-ray absorption data were collected around the cobalt K-absorption edge at 20 K on freeze-dried and frozen protein preparations. In addition to the two recombinant enzymes, we also collected XAS data on recombinant glutamate mutase, reconstituted with MeCbl in the presence of the inhibitor (2S,4S)-4-fluoroglutamate. As a reference compound for the interpretation of the EXAFS spectra, absorption data were also collected from crystalline MeCbl, whose crystal structure is known (Rossi et al. J. Am. Chem. Soc. 1985, 107, 1729−1738). The XANES parts of the XAS spectra for the four samples look very similar to one another and deviate significantly from the corresponding spectra of aquocob(III)alamin and for cob(II)alamin. Moreover, all four methyl-B12 spectra show a pronounced preedge peak, indicating the presence of a covalently attached sixth carbon ligand to the cobalt center. The EXAFS region of the protein spectra were simulated by deriving the amplitude reduction factor, the energy shift, and the Debye−Waller factors for each scattering path from the spectrum of the model compound. All geometrical parameters were assumed to be equal between model compound and enzyme-bound B12 cofactor, with the exception of the axial cobalt−nitrogen distance, which was varied between 1.7 and 2.9 Å. The resulting optimization profiles show the deepest minimums between 2.1 and 2.2 Å, close to the value observed for methylcobalamin. This is also true for the spectrum of GLM in the presence of inhibitor. In all simulations (including the one for the model compound), a second minimum appeared around 2.8 Å. In conclusion, our EXAFS evidence suggests a “normal” axial Co−N bond in the two coenzyme B12-dependent enzymes glutamate mutase and 2-methyleneglutarate mutase. Although a very long (2.6−2.8 Å) Co−N bond would also be compatible with...

Electronic Properties of the Axial Co−C and Co−S Bonds in B12 Systems − A Density Functional Study

“…[9Ϫ11,19,20] Comparison with those previously determined by EXAFS may be useful in order to establish the reliability of bond lengths determined solely by using the first-shell Fourier filter analysis of EXAFS data. [6,25] In particular, such a comparison is important in view of the recent controversial EX- [26,27] In molecular biology, EXAFS is an ideal complement to biocrystallography in establishing the coordination distances in metalloenzyme and, under favorable circumstances, it can give coordination distances with an accuracy of 0.01 Å . [28] However, the analysis generally furnishes several solutions, [26] often difficult to interpret without comparison with accurate X-ray structural data, which often cannot be obtained for metalloproteins.…”

“…Such a large discrepancy has been ascribed to the presence of the four equatorial nitrogen atoms, which limits the accuracy of the axial distances because their contribution to the EX-AFS spectrum interferes with that of the axial ligands. [27,29] The treatment of EXAFS data with the global mapping technique [26] has solved such a discrepancy in the case of (H 2 O)Cbl ϩ , although it has been pointed out that multiple solutions are generally possible. [27,29] Thus, for (H 2 O)Cbl ϩ , an alternative solution gave CoϪN and CoϪO distances in agreement with the crystallographic results (Table 4).…”

“…[27,29] The treatment of EXAFS data with the global mapping technique [26] has solved such a discrepancy in the case of (H 2 O)Cbl ϩ , although it has been pointed out that multiple solutions are generally possible. [27,29] Thus, for (H 2 O)Cbl ϩ , an alternative solution gave CoϪN and CoϪO distances in agreement with the crystallographic results (Table 4). More recently, an improvement of the global mapping technique, which reduces the parameters/observables ratio, has been proposed for the interpretation of EXAFS spectra of simple cobaloximes, a B 12 model.…”

Coα-(1H-Imidazolyl)-Coβ-methylcob(III)amide: Model for Protein-Bound Corrinoid Cofactors

“…Likewise, the value observed for the length of this bond in the crystal structure of glutamate mutase (2.27 ± 2.35 ) is significantly longer than the value observed for crystalline 5. In view of recent XAS evidence [38] [62], it remains to be shown whether and how such a strained CoÀN ax bond can be linked to activation towards CoÀC bond homolysis in this class of coenzyme-B 12 -dependent mutases.…”

“…In the two mutase structures (MCM, Glm), presumably a mixture of corrinoid cofactors with different oxidation states was present [26]. The length of this bond was also studied by X-ray absorption spectroscopy, which yielded an indication for bond elongation for MCM [37] and a more or less normal bond length for Glm [38]. However, recently we were able to show that under the conditions of a high-brilliance X-ray beam, photoreduction of the cofactor may occur, which would then suggest that at least some of the crystallographically observed bond elongation is in fact an artifact of the experimental technique [39].…”