Recent progress in experimental studies on the catalytic mechanism of cytochrome c oxidase

Cytochrome c oxidase (CcO) reduces molecular oxygen (O2) to water, coupled with a proton pump from the N-side to the P-side, by receiving four electrons sequentially from the P-side to the O2-reduction site—including Fea3 and CuB—via the two low potential metal sites; CuA and Fea. The catalytic cycle includes six intermediates as follows, R (Fea32+, CuB1+, Tyr244OH), A (Fea32+-O2, CuB1+, Tyr244OH), Pm (Fea34+ = O2−, CuB2+-OH−, Tyr244O•), F (Fea34+ = O2−, CuB2+-OH-, Tyr244OH), O (Fea33+-OH-, CuB2+-OH−, Tyr244OH), and E (Fea33+-OH-, CuB1+-H2O, Tyr244OH). CcO has three proton conducting pathways, D, K, and H. The D and K pathways connect the N-side surface with the O2-reduction site, while the H-pathway is located across the protein from the N-side to the P-side. The proton pump is driven by electrostatic interactions between the protons to be pumped and the net positive charges created during the O2 reduction. Two different proton pump proposals, each including either the D-pathway or H-pathway as the proton pumping site, were proposed approximately 30 years ago and continue to be under serious debate. In our view, the progress in understanding the reaction mechanism of CcO has been critically rate-limited by the resolution of its X-ray crystallographic structure. The improvement of the resolutions of the oxidized/reduced bovine CcO up to 1.5/1.6 Å resolution in 2016 provided a breakthrough in the understanding of the reaction mechanism of CcO. In this review, experimental studies on the reaction mechanism of CcO before the appearance of the 1.5/1.6 Å resolution X-ray structures are summarized as a background description. Following the summary, we will review the recent (since 2016) experimental findings which have significantly improved our understanding of the reaction mechanism of CcO including: 1) redox coupled structural changes of bovine CcO; 2) X-ray structures of all six intermediates; 3) spectroscopic findings on the intermediate species including the Tyr244 radical in the Pm form, a peroxide-bound form between the A and Pm forms, and Fr, a one-electron reduced F-form; 4) time resolved X-ray structural changes during the photolysis of CO-bound fully reduced CcO using XFEL; 5) a simulation analysis for the Pm→Pr→F transition

Substrate binding induces structural changes in cytochrome P450cam

X-ray structures of ferric cytochrome P450cam partially complexed with the substrate (+)-camphor to two different extents were determined at 1.30–1.35 Å resolution, revealing the protein structures of the substrate-free and substrate-bound forms

A description of the structural determination procedures of a gap junction channel at 3.5 Å resolution

The structural determination procedures of a gap junction channel at 3.5 Å resolution are described, including the preparation of crystals, intensity data collection, data processing, phasing and structural refinement

The Structure of the Mammalian 20S Proteasome at 2.75 Å Resolution

AbstractThe 20S proteasome is the catalytic portion of the 26S proteasome. Constitutively expressed mammalian 20S proteasomes have three active subunits, β1, β2, and β5, which are replaced in the immunoproteasome by interferon-γ-inducible subunits β1i, β2i, and β5i, respectively. Here we determined the crystal structure of the bovine 20S proteasome at 2.75 Å resolution. The structures of α2, β1, β5, β6, and β7 subunits of the bovine enzyme were different from the yeast enzyme but enabled the bovine proteasome to accommodate either the constitutive or the inducible subunits. A novel N-terminal nucleophile hydrolase activity was proposed for the β7 subunit. We also determined the site of the nuclear localization signals in the molecule. A model of the immunoproteasome was predicted from this constitutive structure

Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa

This research was originally published in Journal of Biological Chemistry. Hiroyuki Akama, Takanori Matsuura, Sachiko Kashiwagi, Hiroshi Yoneyama, Shin-ichiro Narita, Tomitake Tsukihara, Atsushi Nakagawa and Taiji Nakae. Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa. Journal of Biological Chemistry. 2004; 279, 25939-25942. © the American Society for Biochemistry and Molecular Biology

Crystal structure of the drug discharge outer membrane protein, OprM, of Pseudomonas aeruginosa : Dual modes of membrane anchoring and occluded cavity end

This research was originally published in Journal of Biological Chemistry. Hiroyuki Akama, Misa Kanemaki, Masato Yoshimura, Tomitake Tsukihara, Tomoe Kashiwagi, Hiroshi Yoneyama, Shin-ichiro Narita, Atsushi Nakagawa and Taiji Nakae. Crystal structure of the drug discharge outer membrane protein, OprM, of Pseudomonas aeruginosa : Dual modes of membrane anchoring and occluded cavity end. Journal of Biological Chemistry. 2004; 279, 52816-52819. © the American Society for Biochemistry and Molecular Biology