The modification occurs when reducing the well-folded cyanomet state. A priori, steric (i.e., correct seating of the heme), electrostatic, or heme electronic factors can all contribute to the reactivity. Attempts to crosslink in the presence of azide as an alternative anionic ligand were not successful (Supporting Information Fig. S21) and imidazole coordination led only to partial crosslinking (Supporting Information Fig. S22). The possibility that cyanide promotes the covalent modification
by stabilizing the protonated vinyl seems unlikely given that binding of this anionic ligand is not expected to perturb significantly the electron distribution of the ferrous heme [54]. Thus, the data point to steric effects as the major determinants of reactivity in the absence of inhibiting ligands.