Crystal Structure of Cytochrome b/c1 Complex, A Large Membrane Protein Complex from Bovine Heart Mitochondria. Di Xia¹, Hoeon Kim¹, Johann Deisenhofer¹ Chang-An Yu², Anatoly Kachurin², Jia-Zhi Xia², Linda Yu², ¹Howard Hughes Medical Institute and Department of Biochemistry, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX 75235-9050, USA, ²Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078-3035, USA

Cytochrome b/c1 complex (bc1) is an integral part of the respiratory chain in most aerobic organisms and of photosynthetic apparatus in purple bacteria. The b/c1 from bovine heart mitochondria, a large heme containing membrane protein complex of eleven different subunits, was purified and crystallized. Cryo-frozen crystals of b/c1 diffracted X-ray up to 2.6 Å resolution using synchrotron radiation, allowing successful diffraction data collection for a native and several heavy atom derivative data sets. Electron density calculated with initial MIR phases reveals a closely interacting dimeric b/c1 in the crystal. Iron positions of the four redox prosthetic groups, hemes b_H, b_L, c₁, and Rieske FeS cluster were located by anomalous difference Fourier at 1.5 Å wavelength, and confirmed using data collected at iron absorption edge. The crystal structure of b/c1 consists of three regions, a large matrix region, a trans-membrane helix region of 13 membrane spanning helices and a less ordered inter-membrane space region. Amino acid sequence assignments of electron density have been completed for subunits core1, core2, cytochrome b, subunit 6 and 7, as well as for the C-terminal part of the cytochrome c₁ and N-terminal part of the Rieske protein, accounting for over 70% of the total molecular mass of the b/c1. Specific respiratory inhibitors such as antimycin A and myxothiazol were co-crystallized with the b/c1 complex. Difference Fourier unveiled the inhibitor binding pockets inside the cytochrome b. The position of the Rieske FeS cluster in the crystal changes upon binding of some inhibitors.