The Location of NuoL and NuoM Subunits in the Membrane Domain of the Escherichia coli Complex I

Abstract

The molecular organization of bacterial NADH: ubiquinone oxidoreductase (complex I or NDH-1) is not established, apart from a rough separation into dehydrogenase, connecting and membrane domains. In this work, complex I was purified from Escherichia coli and fragmented by replacing dodecylmaltoside with other detergents. Exchange into decyl maltoside led to the removal of the hydrophobic subunit NuoL from the otherwise intact complex. Diheptanoyl phosphocholine led to the loss of NuoL and NuoM subunits, whereas other subunits remained in the complex. The presence of N,Ndimethyldodecylamine N-oxide or Triton X-100 led to further disruption of the membrane domain into fragments containing NuoL/M/N, NuoA/K/N, and NuoH/J subunits. Among the hydrophilic subunits, NuoCD was most readily dissociated from the complex, whereas NuoB was partially dissociated from the peripheral arm assembly in N,N-dimethyldodecylamine N-oxide. A model of subunit arrangement in bacterial complex I based on these data is proposed. Subunits NuoL and NuoM, which are homologous to antiporters and are implicated in proton pumping, are located at the distal end of the membrane arm, spatially separated from the redox centers of the peripheral arm. This is consistent with proposals that the mechanism of proton pumping by complex I is likely to involve long range conformational changes.NADH:ubiquinone oxidoreductase (complex I, EC 1.6.5.3) is the first enzyme of the respiratory chains of most mitochondria and many bacteria. It catalyzes the transfer of two electrons from NADH to quinone, coupled to the translocation of about 4 protons across the membrane (for reviews, see Refs. 1-4). Complex I is one of the largest known membrane protein complexes. The bovine enzyme has a mass of ϳ980 kDa and is composed of about 46 subunits, including the seven hydrophobic ND subunits encoded in the mitochondrial genome (5). The simplest version of the complex in terms of protein content is the prokaryotic enzyme that has 13-14 subunits and a combined molecular mass of about 550 kDa (6). All of the subunits of bacterial complex I (also referred to as NDH-1) have analogues in the mitochondrial enzyme (4). Because of its relatively simple subunit composition, NDH-1 represents a useful "minimal" model to study the structure and function of complex I. It is currently the least understood component of the respiratory chain. In contrast to other enzymes of the respiratory chain, the atomic structure of complex I is not known and the mechanisms of proton pumping and electron transfer are not established.Electron microscopy has shown that both the mitochondrial and the bacterial enzyme have a characteristic L-shaped structure. One arm is embedded in the membrane and the other, the peripheral arm, protrudes into the mitochondrial matrix or bacterial cytoplasm. This was demonstrated at about 20 to 30 Å resolution for the enzyme from fungus Neurospora crassa (7, 8), yeast Yarrowia lipolytica (9), beef heart mitochondria (10), Escherichia coli (11,12), and the thermophile Aqu...

Substrate-induced Conformational Change in Bacterial Complex I

“…Recently we have proposed a detailed model of this arrangement (18), which is shown in Fig. 5 alongside the two different conformations of complex I obtained in this study.…”

“…Substrate-induced conformation change in complex I from E. coli. Two representative class-sum images of complex I in the presence of either NADH ("open" conformation) or NAD ϩ ("closed" conformation), with the membrane domain being horizontal, are shown alongside a model of subunit arrangement in bacterial complex I (18). The dehydrogenase domain consists of subunits NuoE, -F, and -G; the connecting domain consists of subunits NuoCD, -B, and -I; the remaining subunits make up the membrane domain.…”

“…Hydrophobic Nuo subunits are not readily identified by peptide mass mapping (13). The additional subunit may be NuoH (36 kDa), judging from its molecular mass and likely proximity to the peripheral arm (18). The apparent small shift in molecular mass of NuoI in the presence of cross-linkers might indicate a modification of this subunit, such as an internal cross-link.…”

“…On the basis of further fragmentation studies, we have proposed recently a more detailed model of subunit arrangement in bacterial complex I, in particular in the membrane domain. In this model, the large hydrophobic subunits NuoL and NuoM are located in a distal part of the membrane arm, spatially separated from the redox centers of the peripheral arm (18).…”

Molecular Biology of Microbial Hydrogenases

“…Subunit NuoL, (or NuoM, or NuoN, which apparently evolved by triplication of an ancestral gene related to bacterial H + /K + antiporters (Fearnley and Walker, 1992;Friedrich and Weiss, 1997) (Appel and Schulz, 1996;Schmitz et al 2002), E. coli H 2 ase-3 (HycCDEFG) (Sauter et al, 1992) and Methanosarcina barkeri (EchABCDEF) (Künkel et al, 1998) (Adapted from Dupuis et al, 2001;Holt et al, 2003;Hedderich 2004 164 Vignais and Colbeau would have provided the transmembrane channel required to complete the proton (or Na + ) pump (Dupuis et al, 2001;Mathiesen and Hagerhall, 2002;Hedderich, 2004).…”