Decoding In-Cell Respiratory Enzyme Dynamics by Label-Free In-situ Electrochemistry

Abstract Deciphering metabolic enzyme catalysis in living cells remains a formidable challenge due to the limitations of in vivo assays, which focus on enzymes isolated from respiration. This study introduces an innovative whole-cell electrochemical assay to reveal the Michaelis-Menten landscape of metabolic enzymes amid complex molecular interactions. We controlled the microbial current generation's rate-limiting step, extracting in vivo kinetic parameters (Km, Ki, and kcat) for the periplasmic nitrite and fumarate (FccA) reductases. Despite deleting CymA, a key electron donor, alternative electron transfer pathways sustained the FccA activity. This enabled direct observation of FccA-CymA interaction, uncovering the pivotal role of CymA in altering the post-binding dynamics of FccA, such as catalysis and product release. This finding challenges the long-held belief that the molecular crowding effect primarily drives discrepancies between in vivo and in vitro kinetics. This work offers significant leap in understanding cellular enzymatic processes and opens avenues for future biochemical research.