Structural and Electronic Factors in Heterolytic Cleavage: Formation of the Co(I) Intermediate in the Corrinoid/Iron-Sulfur Protein from Clostridium thermoaceticum

Abstract

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)

Spectroscopic Studies of the Corrinoid/Iron−Sulfur Protein from Moorella thermoacetica

“…Further insight into the axial ligation of Factor III m in CFeSP is provided by the EPR spectrum of Co 2+ CFeSP obtained in isotopically labeled water (Figure 3). The dramatic loss of intensity in the g ∥ region of this spectrum relative to the spectrum in natural abundance water is readily attributed to 17 O-induced hyperfine broadening and conclusively proves that H 2 O coordinates to the metal center in one of the open axial positions.…”

“…16,17 Yet, on the basis of our recent computational studies on a series of Co 2+ Cbi + models with varying Co-OH 2 bond lengths, lower axial ligand dissociation should drastically alter the energies of the Co 3d-based ligand field (LF) transitions and the EPR parameters (e.g., g-values and 59 Co hyperfine coupling constants). 27 However, no such dramatic alterations are actually observed in the case of asisolated Co 2+ CFeSP, thus arguing against the presence of a four-coordinate Co 2+ corrinoid species in this enzyme.…”

Visualizing molecular juggling within a B12-dependent methyltransferase complex

“…As-isolated, reduced, and methylated CFeSP samples in solution were prepared in a room temperature anaerobic chamber (MBraun) following similar procedures to those previously described 18,19,22,23,26,27 . Briefly, purified CFeSP (20 µM) was used for the as-isolated sample, CFeSP mixed with Ti(III) citrate (1 mM) was used for the reduced sample, and CFeSP mixed with equimolar MeTr, Ti(III) citrate (1 mM), and CH 3 -H 4 folate (1 mM) was used for the methylated sample.…”

“…Following established protocols 18,19,22,23,26,27 , B 12 reduction was achieved with Ti(III) citrate, yielding a sharp 390 nm peak indicative of active Co(I) in both solution and in crystallo spectra (black lines). Further treatment with CH 3 -H 4 folate yields decreased absorbance at 390 nm and a new peak at 450 nm (red lines), characteristic of the product complex (protein-bound CH 3 -Co(III) 18,19,22,23 ).…”

Catalysis of Methyl Group Transfers Involving Tetrahydrofolate and B12

“…In the M. thermoaceticum MTHF:CFeSP methyltransferase involved in the Wood-Ljungdahl pathway, the inactive Co(II) state of the CFeSP is already "base-off " in the resting enzyme, which represents a "ready" state for electron transfer to form a four-coordinate Co(I) state (Harder et al, 1989;Ragsdale et al, 1987;Stich et al, 2006;Wirt et al, 1993Wirt et al, , 1995. The direct electron donor is the [4Fe-4S] cluster of the AcsC subunit of the CFeSP (Menon and Ragsdale, 1999).…”