Hydrogen-deuterium exchange reveals catalytically linked protein flexibility in myoglobin-mediated intramolecular...

A comprehensive understanding of the biophysical parameters that dictate high catalytic efficiency in enzymes is essential for advancing both fundamental enzymology and its applications. Experimental evidence suggests that protein dynamics play a pivotal role in transiently shaping active site configurations, facilitating the efficient traversal of reaction barriers. In a previous study, protein engineering led to the development of a triple mutant of myoglobin, which enabled the successful synthesis of an array of chiral flavanone compounds through myoglobin-mediated carbene transfer. To gain deeper insights into the molecular mechanisms underlying the evolution of structural dynamics that contribute to the accelerated catalytic properties, we first performed hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses on both the wild-type myoglobin and the engineered triple mutant in the presence and absence of a substrate analog to elucidate conformational changes and impacts of mutations on protein flexibility and functional dynamics. HDX-MS analysis identified distinct regions of the protein, both proximal and distal to the mutation sites, which exhibited differential HDX behaviors in response to either ligand binding or mutation, thereby providing insights into the structural and dynamic evolution of the mutant. We postulate that the mutation reconfigures the conformational ensemble of the protein, thereby promoting favorable conformational sampling and enhancing the efficiency of the catalyzed reaction. Computational studies further support this conclusion, providing additional insights into the structural and dynamic factors influencing enzymatic efficiency. This study highlights the critical role of protein structural dynamics in evolved enzymes, underscoring the potential of probing and harnessing these dynamics for advancements in protein engineering and redesign.