A single amino acid residue controls ROS production in the respiratory C omplex I from E scherichia coli

Summary Reactive oxygen species ( ROS ) production by respiratory C omplex I from E scherichia coli was studied in bacterial membrane fragments and in the isolated and purified enzyme, either solubilized or incorporated in proteoliposomes. We found that the replacement of a single amino acid residue in close proximity to the nicotinamide adenine dinucleotide ( NADH )‐binding catalytic site ( E95 in the NuoF subunit) dramatically increases the reactivity of C omplex I towards dioxygen ( O 2 ). In the E95Q variant short‐chain ubiquinones exhibit strong artificial one‐electron reduction at the catalytic site, also leading to a stronger increase in ROS production. Two mechanisms can contribute to the observed kinetic effects: (a) a change in the reactivity of flavin mononucleotide ( FMN ) towards dioxygen at the catalytic site, and (b) a change in the population of the ROS ‐generating state. We propose the existence of two ( closed and open ) states of the NAD + ‐bound enzyme as one feature of the substrate‐binding site of C omplex I . The analysis of the kinetic model of ROS production allowed us to propose that the population of C omplex I with reduced FMN is always low in the wild‐type enzyme even at low ambient redox potentials, minimizing the rate of reaction with O 2 in contrast to E95Q variant.