Cells Lacking Rieske Iron-Sulfur Protein Have a Reactive Oxygen Species-Associated Decrease in Respiratory Complexes I and IV

Abstract

Mitochondrial respiratory complexes of the electron transport chain (CI, CIII, and CIV) can be assembled into larger structures forming supercomplexes. We analyzed the assembly/stability of respiratory complexes in mouse lung fibroblasts lacking the Rieske iron-sulfur protein (RISP knockout [KO]cells), one of the catalytic subunits of CIII. In the absence of RISP, most of the remaining CIII subunits were able to assemble into a large precomplex that lacked enzymatic activity. CI, CIV, and supercomplexes were decreased in the RISP-deficient cells. Reintroduction of RISP into KO cells restored CIII activity and increased the levels of active CI, CIV, and supercomplexes. We found that hypoxia (1% O₂) resulted in increased levels of CI, CIV, and supercomplex assembly in RISP KO cells. In addition, treatment of control cells with different oxidative phosphorylation (OXPHOS) inhibitors showed that compounds known to generate reactive oxygen species (ROS) (e.g., antimycin A and oligomycin) had a negative impact on CI and supercomplex levels. Accordingly, a superoxide dismutase (SOD) mimetic compound and SOD2 overexpression provided a partial increase in supercomplex levels in the RISP KO cells. Our data suggest that the stability of CI, CIV, and supercomplexes is regulated by ROS in the context of defective oxidative phosphorylation.

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ACKNOWLEDGMENTS

We express our gratitude to Alejandro Ocampo, Xiao Wang, Sofia Garcia, and Ana LaTorre for their help with flow cytometry and technical assistance, to T. Wenz for insightful discussions, and to A. Barrientos and E. Perales-Clemente for critical review of the manuscript. We also thank Mike Murphy and Rob Smith for providing MitoQ and dTPP.

We are indebted to the following funding agencies for supporting our work: the James and Esther King Biomedical Research Program, Florida Department of Health, for grant 08KN-01 (F.D.), PHS for grants NS041777, CA085700, and EY10804 (C.T.M.), and the Spanish Ministry of Education for DGA-B55 grants SAF2009-08007 and CSD2007-00020 (J.A.E.). The CNIC is supported by the Spanish Ministry of Science and Innovation and the Pro-CNIC Foundation.