In-Solution Conformation Dynamics of Hemoprotein Catalytic Adaptability Revealed by Ultraviolet Photodissociation Mass Spectrometry

Despite structure-based mutagenesis being widely used for the rational evolution of engineering enzymes, the in-solution conformation dynamics of enzyme catalytic adaptability is still hard to profile and modulate. Herein, we utilize native mass spectrometry to probe the integrity of hemoprotein overall structure and 193 nm ultraviolet photodissociation to provide residue-level conformation dynamics of catalytic hotspots in peroxidation reaction. We demonstrate that the structure of hemoprotein is generally stable in 25% acetonitrile and methanol aqueous solutions, yet the hotspot conformation dynamics and peroxidase activity are significantly different. The hydrophobic heme-binding pocket becomes more flexible within 25% acetonitrile solution, releasing more space between heme and His64 to adapt hydrogen peroxide to form a peroxidation intermediate. In contrast, a His93-heme-His64 double coordination is formed in 25% methanol solution, preventing the formation of a peroxidation intermediate. These findings represent a paradigm shift in biocatalytic design, enabling the rational modulation of enzyme conformation in-solution to optimize the biocatalysis efficiency.