A single amino acid mutation (E95Q) in the NuoF subunit of respiratory Complex I from Escherichia coli results in a 15-fold increase in the rate of reactive oxygen species (ROS) production. The mutant's flavin mononucleotide (FMN) is reduced faster by an anionic reductant. Titration of the ROS production rate as a function of redox potential shows a positive shift that fits a Nernst plot with n=2, indicating a two-electron process. Measurements using variants with impaired quinone reductase activity show similar ROS production rates and redox potentials as the wild type, arguing against a significant role for quinones or the N2 channel