Boosting the catalytic performance of an enzyme through controlled conformational regulation in vitro remains challenging due to its intrinsic structural complexity and conformational instability. Herein, we report a straightforward thermal activation strategy that induces permanent conformational remodeling of cytochrome c (Cyt c) toward a catalytically favorable state. The activated Cyt c exhibits up to 4.4-fold biocatalytic activity enhancement compared to the native enzyme and maintains its full activity over at least 7 days of storage. Mechanistic investigations combining experimental characterization and molecular simulations reveal that activity enhancement is governed by a well-defined structural evolution at the molecular level. Thermal activation drives a local unfolding-refolding transition in the vicinity of the catalytic center, leading to the opening of the catalytic gate and a pronounced increase in substrate accessibility. Simultaneously, the coordination environment between heme Fe and Met80 S changes to a relaxed conformation, whereas the Fe-N (His18) coordination remains relatively rigid during the thermal activation. This asymmetric coordination modulation establishes a balanced heme microenvironment that ensures both high substrate accessibility and structural integrity of the heme center, thus enabling efficient catalysis without compromising robustness. These findings elucidate a clear structure-activity relationship linking thermally induced conformational evolution to enhanced enzyme-substrate interactions and catalytic performance and demonstrate thermal activation as an effective strategy for modulating metalloenzyme catalysis.
Liu et al. (Fri,) studied this question.