PMID- 34274318 OWN - NLM STAT- MEDLINE DCOM- 20211122 LR - 20211122 IS - 1083-351X (Electronic) IS - 0021-9258 (Print) IS - 0021-9258 (Linking) VI - 297 IP - 3 DP - 2021 Sep TI - Critical roles of the Cu(B) site in efficient proton pumping as revealed by crystal structures of mammalian cytochrome c oxidase catalytic intermediates. PG - 100967 LID - S0021-9258(21)00768-7 [pii] LID - 10.1016/j.jbc.2021.100967 [doi] LID - 100967 AB - Mammalian cytochrome c oxidase (CcO) reduces O(2) to water in a bimetallic site including Fe(a3) and Cu(B) giving intermediate molecules, termed A-, P-, F-, O-, E-, and R-forms. From the P-form on, each reaction step is driven by single-electron donations from cytochrome c coupled with the pumping of a single proton through the H-pathway, a proton-conducting pathway composed of a hydrogen-bond network and a water channel. The proton-gradient formed is utilized for ATP production by F-ATPase. For elucidation of the proton pumping mechanism, crystal structural determination of these intermediate forms is necessary. Here we report X-ray crystallographic analysis at approximately 1.8 A resolution of fully reduced CcO crystals treated with O(2) for three different time periods. Our disentanglement of intermediate forms from crystals that were composed of multiple forms determined that these three crystallographic data sets contained approximately 45% of the O-form structure, approximately 45% of the E-form structure, and approximately 20% of an oxymyoglobin-type structure consistent with the A-form, respectively. The O- and E-forms exhibit an unusually long Cu(B)(2+)-OH(-) distance and Cu(B)(1+)-H(2)O structure keeping Fe(a3)(3+)-OH(-) state, respectively, suggesting that the O- and E-forms have high electron affinities that cause the O-->E and E-->R transitions to be essentially irreversible and thus enable tightly coupled proton pumping. The water channel of the H-pathway is closed in the O- and E-forms and partially open in the R-form. These structures, together with those of the recently reported P- and F-forms, indicate that closure of the H-pathway water channel avoids back-leaking of protons for facilitating the effective proton pumping. CI - Copyright (c) 2021 The Authors. Published by Elsevier Inc. All rights reserved. FAU - Shimada, Atsuhiro AU - Shimada A AD - Picobiology Institute, Graduate School of Life Science, University of Hyogo, kamigori, Akoh, Hyogo, Japan. FAU - Hara, Fumiyoshi AU - Hara F AD - Picobiology Institute, Graduate School of Life Science, University of Hyogo, kamigori, Akoh, Hyogo, Japan. FAU - Shinzawa-Itoh, Kyoko AU - Shinzawa-Itoh K AD - Picobiology Institute, Graduate School of Life Science, University of Hyogo, kamigori, Akoh, Hyogo, Japan; Department of Life Science, Graduate School of Life Science, University of Hyogo, kamigori, Akoh, Hyogo, Japan. FAU - Kanehisa, Nobuko AU - Kanehisa N AD - Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan. FAU - Yamashita, Eiki AU - Yamashita E AD - Institute for Protein Research, Osaka University, Suita, Osaka, Japan. FAU - Muramoto, Kazumasa AU - Muramoto K AD - Department of Life Science, Graduate School of Life Science, University of Hyogo, kamigori, Akoh, Hyogo, Japan. Electronic address: muramoto@sci.u-hyogo.ac.jp. FAU - Tsukihara, Tomitake AU - Tsukihara T AD - Picobiology Institute, Graduate School of Life Science, University of Hyogo, kamigori, Akoh, Hyogo, Japan; Institute for Protein Research, Osaka University, Suita, Osaka, Japan. Electronic address: tsuki@protein.osaka-u.ac.jp. FAU - Yoshikawa, Shinya AU - Yoshikawa S AD - Picobiology Institute, Graduate School of Life Science, University of Hyogo, kamigori, Akoh, Hyogo, Japan; Department of Life Science, Graduate School of Life Science, University of Hyogo, kamigori, Akoh, Hyogo, Japan. Electronic address: yoshi@sci.u-hyogo.ac.jp. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20210715 PL - United States TA - J Biol Chem JT - The Journal of biological chemistry JID - 2985121R RN - 0 (Proton Pumps) RN - 789U1901C5 (Copper) RN - EC 1.9.3.1 (Electron Transport Complex IV) SB - IM MH - Animals MH - Catalysis MH - Cattle MH - Copper/*metabolism MH - Crystallography, X-Ray MH - Electron Transport Complex IV/chemistry/*metabolism MH - Mitochondria, Heart/*enzymology MH - Protein Conformation MH - Proton Pumps/*metabolism PMC - PMC8390519 OTO - NOTNLM OT - X-ray crystallography OT - bioenergetics OT - catalytic intermediate OT - copper OT - cytochrome c oxidase (Complex IV) OT - enzyme mechanism OT - heme OT - metalloenzyme OT - mitochondrial membrane potential OT - proton pump COIS- Conflict of interest Each of the authors of this manuscript declares that they have no conflicts of interest with regard to the contents of this article. The authors state that none of the new findings in this manuscript have been published or are under consideration for publication elsewhere. EDAT- 2021/07/19 06:00 MHDA- 2021/11/23 06:00 PMCR- 2021/07/15 CRDT- 2021/07/18 20:33 PHST- 2020/10/03 00:00 [received] PHST- 2021/07/07 00:00 [revised] PHST- 2021/07/12 00:00 [accepted] PHST- 2021/07/19 06:00 [pubmed] PHST- 2021/11/23 06:00 [medline] PHST- 2021/07/18 20:33 [entrez] PHST- 2021/07/15 00:00 [pmc-release] AID - S0021-9258(21)00768-7 [pii] AID - 100967 [pii] AID - 10.1016/j.jbc.2021.100967 [doi] PST - ppublish SO - J Biol Chem. 2021 Sep;297(3):100967. doi: 10.1016/j.jbc.2021.100967. Epub 2021 Jul 15.