Experimental and Computational Insight into the Mechanism of NO Binding to Ferric Microperoxidase. The Likely Role of Tautomerization to Account for the pH Dependence.

According to the current paradigm, the metal-hydroxo bond in a six-coordinate porphyrin complex is believed to be significantly less reactive in ligand substitution than the analogous metal-aqua bond, due to a much higher strength of the former bond. Here, we report kinetic studies for nitric oxide (NO) binding to a heme-protein model, acetylated microperoxidase-11 (AcMP-11), that challenge this paradigm. In the studied pH range 7.4-12.6, ferric AcMP-11 exists in three acid-base forms, assigned in the literature as [(AcMP-11)Fe(HO)(HisH)] (), [(AcMP-11)Fe(OH)(HisH)] (), and [(AcMP-11)Fe(OH)(His)] (). From the pH dependence of the second-order rate constant for NO binding (), we determined individual rate constants characterizing forms -, revealing only a ca. 10-fold decrease in the NO binding rate on going from ( = 3.8 × 10 M s) to ( = 4.0 × 10 M s) and the inertness of . These findings lead to the abandonment of the dissociatively activated mechanism, in which the reaction rate can be directly correlated with the Fe-OH bond energy, as the mechanistic explanation for the process with regard to . The reactivity of is accounted for through the existence of a tautomeric equilibrium between the major [(AcMP-11)Fe(OH)(HisH)] () and minor [(AcMP-11)Fe(HO)(His)] () species, of which the second one is assigned as the NO binding target due to its labile Fe-OH bond. The proposed mechanism is further substantiated by quantum-chemical calculations, which confirmed both the significant labilization of the Fe-OH bond in the [(AcMP-11)Fe(HO)(His)] tautomer and the feasibility of the tautomer formation, especially after introducing empirical corrections to the computed relative acidities of the HO and HisH ligands based on the experimental p values. It is shown that the "effective lability" of the axial ligand (OH/HO) in may be comparable to the lability of the HO ligand in .