The role of the invariant glutamate 95 in the catalytic site of Complex I from Escherichia coli.
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 microM 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.
Euro
Liliya
L
Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, Finland. liliya.euro@helsinki.fi
Belevich
Galina
G
Bloch
Dmitry A
DA
Verkhovsky
Michael I
MI
Wikström
Mårten
M
Verkhovskaya
Marina
M
D020134:Catalytic Domain; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000235:genetics*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011808:Quinone Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D005973:Glutamine; D009243:NAD; C502197:NuoF protein, E coli; D011808:Quinone Reductases; D042967:Electron Transport Complex I