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Kyoko Shinzawa-Itoh

Kyoko Shinzawa-Itoh

Hyogo University, Japan

Title: Complex Structure of Mammalian Cytochrome c–Cytochrome c Oxidase Reveals a Novel Protein-Protein Interaction Mode

Biography

Biography: Kyoko Shinzawa-Itoh

Abstract

Mitochondrial cytochrome c oxidase (CcO) transfers electrons from cytochrome c (Cyt.c) to O2 to generate H2O, a process coupled to proton pumping. To elucidate the mechanism of electron transfer, a crystal structure of the complex of CcO and Cyt.c would be invaluable for mechanistic studies. Two-dimensional (2D) crystals of the mammalian Cyt.c–CcO complex were prepared at higher pH (7.4–9.0) with both proteins in the oxidized state (Osuda et al, 2016), but these 2D crystals could not provide us with a structure of sufficient resolution. We optimized 3D crystallization conditions for ferri-Cyt.c and oxidized CcO at high pH and solved the X-ray structure of the complex at 2.0 Å resolution. The specific interaction between Cyt.c and CcO is stabilized by only six electrostatic interactions between side chains within a small contact surface. From a theoretical calculation based on the complex structure, we identified an electron transfer pathway from the heme c of Cyt.c to CuA of CcO via Lys-13 of Cyt.c. Between the two proteins are three water layers, one of which lies between the other two layers without significant direct interaction with either protein. The inter-molecular span between Cyt.c and CcO is longer than those of other complexes by more than 3.0 Å, and the contact surface area of Cyt.c and CcO is smaller than one-third the size of those of other complexes. Cyt.c undergoes large structural fluctuations, using the interacting regions with CcO as a fulcrum. These features of the protein–protein interaction at the docking interface represent the first known example of a new class of inter-protein interaction, which we term “soft and specific”.  This interaction is predicted to contribute to the rapid association/dissociation of the Cyt.c–CcO complex, which facilitates the sequential supply of four electrons for the O2 reduction reaction.

References:

  1. Osuda Y, Shinzawa-Itoh K, Tani K, Maeda S, Yoshikawa S, Tsukihara T, Gerle C (2016) Two-dimensional crystallization of monomeric bovine cytochrome c oxidase with bound cytochrome c in reconstituted lipid membranes. Microscopy. 65(3):263-267.
  2. Shimada S, Shinzawa-Itoh K, Baba J, Aoe S, Shimada A, Yamashita E, Kang J, Tateno M, Yoshikawa S, Tsukihara T (2017) Complex structure of cytochrome c-cytochrome c oxidase reveals a novel protein-protein interaction mode. EMBO J. 36(3):291-300.
  3. Shinzawa-Itoh K, Shimomura H, Yanagisawa S, Shimada S, Takahashi R, Oosaki M, Ogura T, TsukiharaT (2016) Purification of active respiratory supercomplex from bovine heart mitochondria enables functional studies. J. Biol. Chem. 291(8):4178-84.
  4. Hirata K, Shinzawa-Itoh K, Yano N, Takemura S, Kato K, Hatanaka M, Muramoto K, Kawahara T, Tsukihara T, Yamashita E, Tono K, Ueno G, Hikima T, Murakami H, Inubushi Y, Yabashi M, Ishikawa T, Yamamoto M, Ogura T, Sugimoto H, Shen JR, Yoshikawa S, Ago H (2014) Determination of damage-free crystal structure of an X-ray-sensitive protein using an XFEL. Nat Methods. 11(7):734-736.