Three-dimensional structure of respiratory complex I from Escherichia coli in ice in the presence of nucleotides

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Abstract

Complex I (NADH:ubiquinone oxidoreductase) is the largest protein complex of bacterial and mitochondrial respiratory chains. The first three-dimensional structure of bacterial complex I in vitrified ice was determined by electron cryo-microscopy and single particle analysis. The structure of the Escherichia coli enzyme incubated with either NAD+ (as a reference) or NADH was calculated to 35 and 39 Å resolution, respectively. The X-ray structure of the peripheral arm of Thermus thermophilus complex I was docked into the reference EM structure. The model obtained indicates that Fe–S cluster N2 is close to the membrane domain interface, allowing for effective electron transfer to membrane-embedded quinone. At the current resolution, the structures in the presence of NAD+ or NADH are similar. Additionally, side-view class averages were calculated for the negatively stained bovine enzyme. The structures of bovine complex I in the presence of either NAD+ or NADH also appeared to be similar. These observations indicate that conformational changes upon reduction with NADH, suggested to occur by a range of studies, are smaller than had been thought previously. The model of the entire bacterial complex I could be built from the crystal structures of subcomplexes using the EM envelope described here.

Abbreviations

CTF
contrast transfer function
DDM
dodecyl-maltoside
EEDQ
N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
EM
electron microscopy
FEG
field emission gun
FMN
flavin mononucleotide
HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
MES
2-(N-morpholino)-ethanesulfonic acid
ROS
reactive oxygen species

Keywords

Complex I
NADH:ubiquinone oxidoreductase
Membrane protein structure
Single particle analysis
Electron microscopy

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