Critical Roles of Subunit NuoH (ND1) in the Assembly of Peripheral Subunits with the Membrane Domain of Escherichia coli NDH-1

Prem Kumar Sinha,Jesus Torres-Bacete,Eiko Nakamaru-Ogiso,Norma Castro-Guerrero,Akemi Matsuno-Yagi,Takao Yagi

doi:10.1074/jbc.m809468200

Prem Kumar Sinha, Jesus Torres-Bacete + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m809468200

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Abstract

The bacterial proton-translocating NADH:quinone oxidoreductase (NDH-1) consists of two domains, a peripheral arm and a membrane arm. NuoH is a counterpart of ND1, which is one of seven mitochondrially encoded hydrophobic subunits, and is considered to be involved in quinone/inhibitor binding. Sequence comparison in a wide range of species showed that NuoH is comprehensively conserved, particularly with charged residues in the cytoplasmic side loops. We have constructed 40 mutants of 27 conserved residues predicted to be in the cytoplasmic side loops of Escherichia coli NuoH by utilizing the chromosomal DNA manipulation technique and investigated roles of these residues. Mutants of Arg(37), Arg(46), Asp(63), Gly(134), Gly(145), Arg(148), Glu(220), and Glu(228) showed low deamino-NADH-K(3)Fe(CN)(6) reductase activity, undetectable NDH-1 in Blue Native gels, low contents of peripheral subunits (especially NuoB and NuoCD) bound to the membranes, and a significant loss of the membrane potential and proton-pumping function coupled to deamino-NADH oxidation. The results indicated that these conserved residues located in the cytoplasmic side loops are essential for the assembly of the peripheral subunits with the membrane arm. Implications for the involvement of NuoH (ND1) in maintaining the structure and function of NDH-1 are discussed.

Highlights

Electron microscopic analyses have established that NDH-1/complex I has a characteristic L-shaped structure consisting of two domains, a peripheral arm protruding into the cytoplasm and a membrane domain embedded within the cytoplasmic membrane [11,12,13]
Based on the projection structure of the membrane domain and detergent-based fractionation study that led to the disruption of the membrane arm into fragments containing NuoL/M, NuoA/ K/N, and NuoH/J subunits, a speculative arrangement of the membrane segment of E. coli NDH-1 has been proposed, wherein subunits NuoA, NuoK, NuoN, NuoJ, and NuoH are present in the vicinity of the peripheral arm, whereas the NuoL and NuoM subunits are distantly located from the peripheral segment [17,18,19,20]
The results indicate that NuoH, through its conserved residues in the cytoplasmic side loops, is involved in connecting the membrane arm with the peripheral arm, presumably with the NuoB and/or NuoCD subunit of NDH-1

Summary

Introduction

The results indicate that NuoH, through its conserved residues in the cytoplasmic side loops, is involved in connecting the membrane arm with the peripheral arm, presumably with the NuoB and/or NuoCD subunit of NDH-1. To clarify the structural and functional roles of the conserved residues as well as residues corresponding to those linked to human diseases, we have generated a set of 40 point mutations covering 27 different residues of the E. coli NuoH subunit using homologous recombination targeted directly to the chromosome [9] (see Table 1 for the complete list of mutants).

Results

Conclusion