Noncovalent modulation of pH-dependent reactivity of a Mn-salen cofactor in myoglobin with hydrogen peroxide.

To demonstrate protein modulation of metal-cofactor reactivity through noncovalent interactions, pH-dependent sulfoxidation and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) oxidation reactivity of a designed myoglobin (Mb) containing non-native Mn-salen complex (1) was investigated using H2O2 as the oxidant. Incorporation of 1 inside the Mb resulted in an increase in the turnover numbers through exclusion of water from the metal complex and prevention of Mn-salen dimer formation. Interestingly, the presence of protein in itself is not enough to confer the increase activity as mutation of the distal His64 in Mb to Phe to remove hydrogen-bonding interactions resulted in no increase in the turnover numbers, while mutation His64 to Arg, another residue with ability to hydrogen-bond interactions, resulted in an increase in reactivity. These results strongly suggest that the distal ligand His64, through its hydrogen-bonding interaction, plays important roles in enhancing and fine-tuning reactivity of the Mn-salen complex. Nonlinear least-squares fitting of rate versus pH plots demonstrates that 1.Mb(H64X) (X=H, R and F) and the control Mn-salen 1 exhibit pKa values varying from pH 6.4 to 8.3, and that the lower pKa of the distal ligand in 1.Mb(H64X), the higher the reactivity it achieves. Moreover, in addition to the pKa at high pH, 1.Mb displays another pKa at low pH, with pKa of 5.0+/-0.08. A comparison of the effect of different pH on sulfoxidation and ABTS oxidation indicates that, while the intermediate produced at low pH conditions could only perform sulfoxidation, the intermediate at high pH could oxidize both sulfoxides and ABTS. Such a fine-control of reactivity through hydrogen-bonding interactions by the distal ligand to bind, orient and activate H2O2 is very important for designing artificial enzymes with dramatic different and tunable reactivity from catalysts without protein scaffolds.