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Fig.3 FMN of EQ mutant is reduced fast by an anionic reductant
Table 1 ROS production is increased 15 times by single E95Q mutation
Table 2 ROS production is stimulated by the addition of water soluble
quinones. There is not as dramatic increase in ROS production by EQ
mutation in native membrane vesicles
A single amino acid residue controls ROS production
in the respiratory Complex I from Escherichia coli
Juho Knuuti, Galina Belevich, Mårten Wikström, Dmitry A. Bloch*, Marina Verkhovskaya*
Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, Finland.
Fig.4 The
rate of ROS
production
plotted as a
function of
theoretical
Nernst
potential
differs in the
case of EQ
mutant
Fig.5 FMN is covered from the
other side by backbone nitrogens
Fig.2
Fig.1
Fig.6 Only half of DQ reductase and almost none of Q1
reductase are inhibited by native quinone site inhibitor
ABSTRACT
Reactive oxygen species formation by wild type and
mutated E.coli Complex I was studied in different enzyme
preparations. Replacing conserved glutamate in catalytic
cavity of NuoF subunit with glutamine results in strong
increase of the rate of NADH supported hydrogen peroxide
formation (Table 1). FMN of the mutant is reduced fast by
dithionite (Fig.3). Titration of the rate of ROS production
varying potential set by NADH:NAD+ redox pair shows
positive shift and can be fitted with Nernst plot with n=2.
Measurements with two variants, of which quinone
reductase activity is strongly compromised by single amino
acid mutations in NuoCD or NuoM, give similar rates of
hydrogen peroxide formation and shape as well as midpoint
redox potential of titration as wild type (Fig.4).
RESULTS
Elevated mid-point redox potential of
ROS production agree with previously
determined two electron potential of
FMN [Euro et al., 2009]. Fifteen times
increase in ROS production velocity
however could be explained only if
chemistry between oxygen and FMN is
enhanced either thermodynamically or
spatially. Results argue against
significant role of N2 and quinone in
ROS production in used forward
electron transfer conditions. ROS
production by Complex I is also
stimulated by addition of water soluble
quinones. A model to explain previous
and current experimental results was
Variant Complex I activity, mol mg-1 min-
1
Rate of NADH-dependent
ROS production, nmol mg-1
min-1NADH:HAR
oxidoreductase
NADH:DQ
oxidoreductase
Wt 107.4 ±7.0 26.9 ±1.8 51.7 ±3.5
NuoM E144A 109.7 ±8.5 3.6±0.5 55.6 ±7.5
NuoCD R274A 72.4 ±2.3 5.7±0.3 44.1 ±0.4
NuoF E95Q 24.4 ±1.5 11.5 ±1.5 749.4 ±1.7
Sample ROS production*
- DQ Q1 Menadione
Purified wt 1.00±0.02 1.16±0.01 9.28±0.28 12.10±0.31
Purified E95Q 15.98±0.46 22.78±0.04 95.46±2.25 144.87±1.17
Membranes wt 1.00±0.14 1.07±0.09 3.93±0.02 5.55±0.02
Membranes E95Q** 6.04±0.90 5.98±0.26 40.17±1.47 59.07±2.25