The role of the invariant glutamate 95 in the catalytic site of Complex I from Escherichia coli

Abstract

Replacement of glutamate 95 for glutamine in the NADH- and FMN-binding NuoF subunit of E. coli Complex I decreased NADH oxidation activity 2.5–4.8 times depending on the used electron acceptor. The apparent K m for NADH was 5.2 and 10.4 μM for the mutant and wild type, respectively. Analysis of the inhibitory effect of NAD + on activity showed that the E95Q mutation caused a 2.4-fold decrease of K i NAD+ in comparison to the wild type enzyme. ADP-ribose, which differs from NAD + by the absence of the positively charged nicotinamide moiety, is also a competitive inhibitor of NADH binding. The mutation caused a 7.5-fold decrease of K i ADP-ribose relative to wild type enzyme. Based on these findings we propose that the negative charge of Glu95 accelerates turnover of Complex I by electrostatic interaction with the negatively charged phosphate groups of the substrate nucleotide during operation, which facilitates release of the product NAD +. The E95Q mutation was also found to cause a positive shift of the midpoint redox potential of the FMN, from − 350 mV to − 310 mV, which suggests that the negative charge of Glu95 is also involved in decreasing the midpoint potential of the primary electron acceptor of Complex I.