Assembly of respiratory complex I is regulated by FMN insertion into the N-module.

Respiratory complex I plays a crucial role in the mitochondrial electron transport chain and shows promise as a therapeutic target for various human diseases. While most studies focus on inhibiting complex I at the Q-site, little is known about inhibitors targeting other sites within the complex. In this study, we demonstrate that diphenyleneiodonium (DPI), a known N-site inhibitor, uniquely affects the stability of complex I by inhibiting its flavin cofactor FMN. Treatment with DPI blocks the final stage of complex I assembly, leading to the accumulation of assembly intermediates that lack the N-module. Remarkably, high-dose DPI treatment can completely and reversibly degrade complex I in different cellular models. The mechanism involves the accumulation of DPI in mitochondria, where it reacts with FMN, depleting the effective pool of FMN and halting complex I maturation. Consistently, growing cells in medium lacking the FMN precursor riboflavin or knocking out the mitochondrial FAD carrier gene SLC25A32 results in complex I degradation and impairs complex I-dependent respiration. Overall, our findings establish a direct connection between mitochondrial flavin homeostasis and complex I stability and assembly, paving the way for novel pharmacological strategies to regulate respiratory complex I.