Assembly of Respiratory Complexes I, III, and IV into NADH Oxidase Supercomplex Stabilizes Complex I in Paracoccus denitrificans

Abstract

J. Biol. Chem. 260, 2458 -2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete "respirasome." Inactivation of the gene for tightly bound cytochrome c 552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.

Interaction between Cytochrome caa3 and F1F0-ATP Synthase of Alkaliphilic Bacillus pseudofirmus OF4 Is Demonstrated by Saturation Transfer Electron Paramagnetic Resonance and Differential Scanning Calorimetry Assays

“…Rather, the current results are in line with a growing body of work that now suggests that some mitochondrial electron-transfer complexes specifically interact to form supermolecular structures called supercomplexes in organisms ranging from the yeast Saccharomyces cerevisiae (35,36), beef (19,29,35,37), and plants (38)(39)(40)(41)(42). Similar supermolecular structures have also been described for the respiratory chains of bacteria (43)(44)(45)(46)(47). The roles that have been attributed to respiratory supercomplexes are substrate-channeling, catalytic enhancement, sequestration of reactive intermediates (35), stabilization of protein complexes (48), increasing the capacity of the inner mitochondrial membrane for protein insertion (36), and generating mitochondrial cristae morphology (49).…”

Evolution of Respiratory Complex I

“…Well characterized preparations of intact bacterial complex I have so far been obtained from E. coli (32,33), Aquifex aeolicus (34), and recently T. thermophilus (15). The P. denitrificans enzyme was previously isolated only either as a subcomplex (22) or in low yields as part of a supercomplex with respiratory complexes III and IV (35).…”

Supramolecular organizations in the aerobic respiratory chain of Escherichia coli

“…strain 7 has a terminal oxygen reductase supercomplex resulting from the functional fusion of complexes III and IV, containing cytochromes of the b and a types, and a Rieske-type ironesulfur protein [17,18]. A mitochondrial-like respirasome was identified in Paraccocus denitrificans [19] and supramolecular associations of complexes III and IV were detected in P. denitrificans [19,20], Corynebacterium glutamicum [21], and Bacillus PS3 [22,23].…”

“…Indeed, it has been suggested that since it contains a detergent stable complex I and is devoid of complex III, such supramolecular organization is not necessary [19]. Nevertheless, co-localization of oxidative phosphorylation complexes has been suggested [30].…”