Radical S-adenosylmethionine (SAM) enzymes represent one of the largest and most versatile superfamilies of iron–sulfur proteins, catalyzing an extraordinary range of chemically challenging transformations. Found in all domains of life, these enzymes participate in diverse biological processes including cofactor biosynthesis, primary and secondary metabolism, DNA repair, and post-translational modifications. Within this superfamily, B12-dependent radical SAM enzymes constitute a major functional subgroup, uniquely integrating radical chemistry initiated by a [4Fe–4S] cluster with the organometallic reactivity of vitamin B12. Despite their biological importance, the molecular mechanisms governing catalysis, active-site regulation, and cofactor recognition remain incompletely understood.

This thesis combines structural biology, biochemistry, and spectroscopy to investigate the catalytic mechanism of a representative B12-dependent radical SAM enzyme. A robust crystallographic approach was developed to capture distinct catalytic states and trap reaction intermediates. Together with biochemical characterization of enzyme variants and functional analysis of alternative cobamide cofactors, this work provides a unique mechanistic view of this enzyme.

The molecular basis of cobamides selectivity was investigated through structural and biochemical characterization. Although the enzyme can accommodate different cobamides, important differences in catalytic efficiency were measured, demonstrating that cofactor structure modulates enzyme activity. The high-resolution crystal structures obtained provide the first structural characterization of a B12-dependent enzyme in interaction with cobamides, offering unique insights into corrinoid recognition.

To further investigate the enzyme mechanism, we develop a novel strategy to access a series of structurally distinct reaction intermediates that collectively define a continuous catalytic trajectory, linking radical SAM-mediated chemistry with cobalamin-dependent SN2 methyl transfer. In addition, mutational studies revealed that two highly conserved tyrosine residues play distinct yet complementary roles in catalysis. One residue is required to maintain the productive positioning of SAM cofactor, whereas the second acts as a conformational switch that couples substrate binding to productive coordination of SAM. Together, these findings establish an active site regulatory mechanism that links substrate recognition with radical initiation.

Overall, this work provides a deep view of the mechanism of B12-dependent radical SAM enzymes, defining new principles that govern cofactor interaction, active site regulation, and catalytic control.