Complex II (CII) activity controls phenomena that require crosstalk between metabolism and signaling, including neurodegeneration, cancer metabolism, immune activation, and ischemia-reperfusion injury. CII activity can be regulated at the level of assembly, a process that leverages metastable assembly intermediates. The nature of these intermediates and how CII subunits transfer between metastable complexes remains unclear. In this work, we identify metastable species containing the SDHA subunit and its assembly factors, and we assign a preferred temporal sequence of appearance of these species during CII assembly. Structures of two species show that the assembly factors undergo disordered-to-ordered transitions without the appearance of significant secondary structure. The findings identify that intrinsically disordered regions are critical in regulating CII assembly, an observation that has implications for the control of assembly in other biomolecular complexes.

1st Faculty of Medicine Charles University 128 00 Prague 2 Czech Republic

Center for Structural Biology Vanderbilt University Nashville TN 37232 USA

Department of Biochemistry and Biophysics University of California San Francisco CA 94158 USA

Department of Biochemistry Vanderbilt University Nashville TN 37232 USA

Department of Chemistry Vanderbilt University Nashville TN 37232 USA

Department of Medicine Division of Endocrinology and Metabolism University of Florida College of Medicine and Malcom Randall VA Medical Center Gainesville FL 32608 USA

Department of Pharmacology Vanderbilt University Nashville TN 37232 USA

Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health Bethesda MD 20814 USA

Faculty of Science Charles University 128 00 Prague 2 Czech Republic

Institute of Biotechnology Czech Academy of Sciences 252 50 Prague West Czech Republic

Institute of Physiology Czech Academy of Sciences Prague 4 142 20 Prague Czech Republic

Molecular Biology Division San Francisco VA Health Care System San Francisco CA 94121 USA

School of Pharmacy and Medical Science Griffith University Southport QLD 4222 Australia

Vanderbilt Institute of Chemical Biology Vanderbilt University Nashville TN 37232 USA

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