Abstract
Corrinoid iron‑sulfur protein (CFeSP) is the methyltransferase in the Wood-Ljungdahl pathway, yet the geometric and electronic structure that enables its higher catalytic efficiency relative to small-molecule analogs remains a subject of debate. While axial ligation is known to tune the reactivity of B(12)-dependent enzymes, weak interactions in "base-off" species like methylated CFeSP (Me-CFeSP) have been difficult to define using standard structural probes, despite that solution-phase spectroscopy has suggested their presence. Here we utilize Co K-edge X-ray absorption spectroscopy (XAS) and theoretical analysis (DFT, TD-DFT, and QM/MM) to investigate a series of methylated corrinoids: methylcobalamin, methylcobinamide, and Me-CFeSP. Our results show that despite similar Co-CH(3) bond distances across these species, the Co K-pre-edge feature undergoes a diagnostic increase in intensity and a shift to lower energy. Our analysis reveals that these spectral signatures are driven by modulations in the trans-axial Co-ligand distance, and because of the lower symmetry of the corrin ring, the spectral signature of the trans-axial ligand is decoupled from the methyl group. These results provide additional quantitative evidence that Me-CFeSP in solution possesses a weakly bound trans-axial water ligand rather than a 5-coordinate structure estimated by recent structural and XAS studies. This subtle ligation modulates frontier orbital energies to lower the CoC bond dissociation energy, explaining, in part, the enhanced methyl transfer rates of CFeSP. Collectively, this work establishes a spectroscopic and computational basis for diagnosing unresolved axial ligation in corrinoids, suggesting that such interactions may be a widespread, yet underappreciated, feature in "base-off" B(12) enzymology.