Abstract

Cytochrome c oxidase (COX) is hypothesized to be an important regulator of oxidative phosphorylation. However, no animal phenotypes have been described due to genetic defects in nuclear-encoded subunits of COX. We knocked down predicted homologues of COX IV and COX Va in the nematode Caenorhabditis elegans. Animals treated with W09C5.8 (COX IV) or Y37D8A.14 (COX Va) RNA interference had shortened lifespans and severe defects in mitochondrial respiratory chain function. Amount and activity of complex IV, as well as supercomplexes that included complex IV, were decreased in COX-deficient worms. The formation of supercomplex I:III was not dependent on COX. We found that COX deficiencies decreased intrinsic complex I enzymatic activity, as well as complex I-III enzymatic activity. However, overall amounts of complex I were not decreased in these animals. Surprisingly, intrinsic complex I enzymatic activity is dependent on the presence of complex IV, despite no overall decrease in the amount of complex I. Presumably the association of complex I with complex IV within the supercomplex I:III:IV enhances electron flow through complex I. Our results indicate that reduction of a single subunit within the electron transport chain can affect multiple enzymatic steps of electron transfer, including movement within a different protein complex. Patients presenting with multiple defects of electron transport may, in fact, harbor a single genetic defect.

Highlights

  • Uid-state model” of the mitochondrial respiratory chain depicts the respiratory complexes embedded in the inner mitochondrial membrane as separate entities, functionally connected to each other by the mobile electron carriers, coenzyme Q and cytochrome c

  • RNA interference (RNAi)—The average percentages of c oxidase (COX) IV and COX Va knockdown determined by qPCR from 4 independent cultures were 53.1 Ϯ 7 and 27.4 Ϯ 4%, respectively

  • Phenotypes—Nematodes treated with RNAi for either subunit COX IV (W09C5.8) or COX Va (Y37D8A.14) required an additional day to reach adulthood compared with N2

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Summary

Mitochondrial Complex IV Modulates Complex I Function

Evidence supports interdependence among MRC complexes, the mechanism regulating this phenomenon remains unclear. We expected that reduced levels of complex IV subunits would in turn reduce amounts of associated components of any supercomplex that includes complex IV. These defects could lead to whole animal phenotypes characteristic of mitochondrial dysfunction. Binding of ATP to the cytosolic domain of COX IV leads to an increase of the Km of cytochrome c [24], which slows mitochondrial respiration. This mechanism can be abolished by the binding of 3,5-diiodothyronine to COX Va [25]. Our results caution that primary complex IV defects could be misinterpreted to be primary complex I-complex IV defects

Nematodes and Bacteria
Worm Culture and Mitochondrial Isolation
Mitochondrial Function
Western Blot Analysis
Mass Spectrometric Analysis of the Polypeptides in BNGs
Statistical Analysis
RESULTS
COX IV
DISCUSSION
Full Text
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