Design and synthesis of sugar-responsive semiartificial myoglobin triggered by modulation of apoprotein-cofactor interactions

Phenylboronic acid groups as sugar recognition sites were successfully introduced into native myoglobin by a cofactor-reconstitution method. Spectrophotometric pH titration demonstrated the sugar-induced pK(a) shift of the H₂O coordinated to the heme center of the semisynthetic myoglobin bearing phenylboronic acids (met-Mb(PhBOH)₂). By means of circular dichroism (CD) and paramagnetic ¹H NMR spectroscopies, it was proven that sugars that were bound to phenylboronic acid sites induced the rearrangement of the heme crevice to reinforce the heme cofactor-apoprotein interactions. The structural changes that were induced by the binding of sugars subsequently enhanced the dioxygen storage activity of Mb(PhBOH)₂. Such sugar-induced structural and functional changes did not occur for other modified Mbs that had no sugar-recognition units. Interestingly, a randomly modified Mb with phenylboronic acid units did not show any sugar response. In Mb(PhBOH)₂, the information from the sugar-binding event was efficiently transmitted to the active center, so that the activity was efficiently altered upon sugar binding. In conclusion, the active site specific incorporation of molecular recognition units as nonnatural functional molecules can provide a novel strategy for the design of stimuli-responsive semisynthetic proteins.