Abstract
Respiratory complex I is a multi-subunit membrane protein complex that reversibly couples NADH oxidation and ubiquinone reduction with proton translocation against transmembrane potential. Complex I from Escherichia coli is among the best functionally characterized complexes, but its structure remains unknown, hindering further studies to understand the enzyme coupling mechanism. Here, we describe the single particle cryo-electron microscopy (cryo-EM) structure of the entire catalytically active E. coli complex I reconstituted into lipid nanodiscs. The structure of this mesophilic bacterial complex I displays highly dynamic connection between the peripheral and membrane domains. The peripheral domain assembly is stabilized by unique terminal extensions and an insertion loop. The membrane domain structure reveals novel dynamic features. Unusual conformation of the conserved interface between the peripheral and membrane domains suggests an uncoupled conformation of the complex. Considering constraints imposed by the structural data, we suggest a new simple hypothetical coupling mechanism for the molecular machine.
Highlights
Complex I, NADH:ubiquinone oxidoreductase, is a multi-subunit enzyme found in many bacteria and most eukaryotes
The peripheral arm, exposed to the cytoplasm in bacteria or the mitochondrial matrix in eukaryotes, contains binding sites for NADH, ubiquinone, and flavin mononucleotide (FMN) as well as eight or nine iron-sulfur clusters, seven of which connect the NADH and ubiquinone-binding sites (Sazanov, 2015) enabling rapid electron transfer (Verkhovskaya et al., 2008). The membrane-embedded arm includes a chain of three antiporter-like subunits, NuoL, NuoM, and NuoN (E. coli nomenclature is used for the subunits hereafter) (Efremov and Sazanov, 2011), which are found in the Mrp family of multisubunit H+/Na antiporters (Steiner and Sazanov, 2020)
Here we present a single particle cryo17 electron microscopy (cryo-EM) structure of the entire E. coli complex I reconstituted into lipid nanodiscs, with the peripheral arm structure solved at 2.1 Å resolution and that of the membrane domain at 3.7 Å
Summary
Complex I, NADH:ubiquinone oxidoreductase, is a multi-subunit enzyme found in many bacteria and most eukaryotes It facilitates transfer of two electrons from NADH to ubiquinone, or its analogues, coupled reversibly with translocation of four protons across the membrane against trans-membrane potential (Galkin et al, 2006; Sazanov, 2015). The membrane arm features a continuous chain of conserved and functionally important ionizable residues positioned in the middle of the membrane These are suggested to be involved in proton translocation and its coupling to electron transfer (Baradaran et al, 2013; Efremov and Sazanov, 2011). We present a single particle cryo-EM structure of the entire E. coli complex I reconstituted into lipid nanodiscs, with the peripheral arm structure solved at 2.1 Å resolution and that of the membrane domain at 3.7 Å
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