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)­FeIII(H2O)­(HisH)] (1), [(AcMP-11)­FeIII(OH)­(HisH)] (2), and [(AcMP-11)­FeIII(OH)­(His–)] (3). From the pH dependence
of the second-order rate constant for NO binding (kon), we determined individual rate constants characterizing
forms 1–3, revealing only a ca. 10-fold
decrease in the NO binding rate on going from 1 (kon(1) = 3.8 × 106 M–1 s–1) to 2 (kon(2) = 4.0 × 105 M–1 s–1) and the inertness of 3. 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 2. The reactivity of 2 is accounted for
through the existence of a tautomeric equilibrium between the major
[(AcMP-11)­FeIII(OH)­(HisH)] (2a) and minor
[(AcMP-11)­FeIII(H2O)­(His–)]
(2b) species, of which the second one is assigned as
the NO binding target due to its labile Fe–OH2 bond.
The proposed mechanism is further substantiated by quantum-chemical
calculations, which confirmed both the significant labilization of
the Fe–OH2 bond in the [(AcMP-11)­FeIII(H2O)­(His–)] tautomer and the feasibility
of the tautomer formation, especially after introducing empirical
corrections to the computed relative acidities of the H2O and HisH ligands based on the experimental pKa values. It is shown that the “effective lability”
of the axial ligand (OH–/H2O) in 2 may be comparable to the lability of the H2O
ligand in 1.