Substrate-induced Conformational Change in Bacterial Complex I

Aygun A Mamedova,Peter J Holt,Joe Carroll,Leonid A Sazanov

doi:10.1074/jbc.m401539200

Aygun A Mamedova, Peter J Holt + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m401539200

Copy DOI

Abstract

The mechanism coupling electron transfer and proton pumping in respiratory complex I (NADH-ubiquinone oxidoreductase) has not been established, but it has been suggested that it involves conformational changes. Here, the influence of substrates on the conformation of purified complex I from Escherichia coli was studied by cross-linking and electron microscopy. When a zero-length cross-linking reagent was used, the presence of NAD(P)H, in contrast to that of NAD+, prevented the formation of cross-links between the hydrophilic subunits of the complex, including NuoB, NuoI, and NuoCD. Comparisons using different cross-linkers suggested that NuoB, which is likely to coordinate the key iron-sulfur cluster N2, is the most mobile subunit. The presence of NAD(P)H led also to enhanced proteolysis of subunit NuoG. These data indicate that upon NAD(P)H binding, the peripheral arm of the complex adopts a more open conformation, with increased distances between subunits. Single particle analysis showed the nature of this conformational change. The enzyme retains its L-shape in the presence of NADH, but exhibits a significantly more open or expanded structure both in the peripheral arm and, unexpectedly, in the membrane domain also.

Highlights

NADH-ubiquinone oxidoreductase is the first enzyme of the mitochondrial and bacterial respiratory chains
NAD(P)H-induced Proteolysis of Subunit NuoG—Before proceeding with the study of near-neighbor relationships between subunits of E. coli complex I using cross-linking reagents, control experiments were performed in which the enzyme was incubated at 4 °C with different substrates and analyzed by SDS-PAGE
Addition of complex I inhibitors rotenone and piericidin A, quinone analogue decyl-ubiquinone, or the reducing agent sodium dithionite at 10 mM did not lead to proteolysis either

Summary

Introduction

NADH-ubiquinone oxidoreductase (complex I, EC 1.6.5.3) is the first enzyme of the mitochondrial and bacterial respiratory chains. 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]. The first model employs modifications of the Q cycle and assumes that some electron carriers are located in the membrane and are directly involved in proton translocation [19, 20] In this case, quinone-binding sites and proton-translocation machinery need to be located close to the peripheral arm with its redox centers to allow direct interaction. These data indicate that NAD(P)H binding induced significant conformational change in bovine complex I Both NADH and NADPH bind to the FMN-containing 51-kDa. This paper is available on line at http://www.jbc.org subunit (NuoF homologue) [27]. The extent and nature of any conformational changes in the context of the L-shaped structure of the enzyme are not known

Methods

Results

Conclusion