A nanosecond time-resolved XFEL analysis of structural changes associated with CO release from cytochrome c oxidase

Shimada, Atsuhiro; Kubo, Minoru; Baba, Seiki; Yamashita, Keitaro; Hirata, Kunio; Ueno, Go; Nomura, Takashi; Kimura, Tetsunari; Shinzawa‐Itoh, Kyoko; Baba, Jumpei; Hatano, K.; Eto, Y.; Miyamoto, Akari; Murakami, Hideo; Kumasaka, Takashi; Owada, Shigeki; Tono, Kensuke; Yabashi, Makina; Yamaguchi, Yoshihiro; Yanagisawa, Sachiko; Sakaguchi, Miyuki; Ogura, Takashi; Komiya, Ryo; Yan, Jiwang; Yamashita, Eiki; Yamamoto, Masaki; Ago, Hideo; Yoshikawa, Shinya; Tsukihara, Tomitake

doi:10.1126/sciadv.1603042

Cited by 76 publications

(79 citation statements)

References 49 publications

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“…In order to characterize the room-temperature structure of photoswitching intermediate states that only exist transiently, another method is needed that allows collecting structural data on a timescale from a few ps to ms. Such a method is time-resolved serial femtosecond (fs) crystallography (TR-SFX) using X-ray free-electron lasers (XFELs) 22,23 , which has already provided intermediate-state structures of several photosensitive proteins [24][25][26][27][28][29][30][31][32][33][34][35][36][37] . Recently, we combined ultrafast optical spectroscopy and TR-SFX to study excited-state intermediates of rsEGFP2 on the ps timescale during off-to-on switching 27 .…”

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confidence: 99%

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy

et al. 2020

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Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an Xray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final onstate. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.

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Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy

et al. 2020

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“…Subsequent binding of oxygen closes this channel, and one proton is proposed to be released into the P phase with each reduction/oxidation of heme a via an amide bond gate between Tyr440 and Ser441 (24) and an H-bonded network to Asp51 at the P-phase surface (10). Support has come from structural perturbations induced by redox/ligand state changes in the water channel (25)(26)(27)(28), the Asp51 residue (29), and the proposed proton-collecting site around the bound Mg 2+ (23). These observations, together with effects of H-channel mutations on coupling efficiencies in a chimeric bovine/human CcO construct (30,31), have led to the proposal that these structures provide the route for translocated protons in mammalian mitochondrial CcOs.…”

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Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations

Sharma

Jambrina

Kaukonen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Proton pumping A-type cytochrome oxidase (CO) terminates the respiratory chains of mitochondria and many bacteria. Three possible proton transfer pathways (D, K, and H channels) have been identified based on structural, functional, and mutational data. Whereas the D channel provides the route for all pumped protons in bacterial A-type COs, studies of bovine mitochondrial CO have led to suggestions that its H channel instead provides this route. Here, we have studied H-channel function by performing atomistic molecular dynamics simulations on the entire, as well as core, structure of bovine CO in a lipid-solvent environment. The majority of residues in the H channel do not undergo large conformational fluctuations. Its upper and middle regions have adequate hydration and H-bonding residues to form potential proton-conducting channels, and Asp51 exhibits conformational fluctuations that have been observed crystallographically. In contrast, throughout the simulations, we do not observe transient water networks that could support proton transfer from the N phase toward heme via neutral His413, regardless of a labile H bond between Ser382 and the hydroxyethylfarnesyl group of heme In fact, the region around His413 only became sufficiently hydrated when His413 was fixed in its protonated imidazolium state, but its calculated pK is too low for this to provide the means to create a proton transfer pathway. Our simulations show that the electric dipole moment of residues around heme changes with the redox state, hence suggesting that the H channel could play a more general role as a dielectric well.

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“…Normal electron transfer rates have been reported for all three variants, as would be expected for ideally uncoupled oxidases. These data, together with redox-and ligand-induced structural changes in two domains of the H-channel (56)(57)(58)(59), have led to the proposal that it is the H-channel that provides the route for pumped protons both into and out of the proton trap. However, mutagenesis of H-channel residues of bacterial HCOs failed to support any crucial H-channel function.…”

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A common coupling mechanism for A-type heme-copper oxidases from bacteria to mitochondria

Maréchal

Genko

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Mitochondria metabolize almost all the oxygen that we consume, reducing it to water by cytochrome c oxidase (CcO). CcO maximizes energy capture into the protonmotive force by pumping protons across the mitochondrial inner membrane. Forty years after the H+/e− stoichiometry was established, a consensus has yet to be reached on the route taken by pumped protons to traverse CcO’s hydrophobic core and on whether bacterial and mitochondrial CcOs operate via the same coupling mechanism. To resolve this, we exploited the unique amenability to mitochondrial DNA mutagenesis of the yeast Saccharomyces cerevisiae to introduce single point mutations in the hydrophilic pathways of CcO to test function. From adenosine diphosphate to oxygen ratio measurements on preparations of intact mitochondria, we definitely established that the D-channel, and not the H-channel, is the proton pump of the yeast mitochondrial enzyme, supporting an identical coupling mechanism in all forms of the enzyme.

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A nanosecond time-resolved XFEL analysis of structural changes associated with CO release from cytochrome c oxidase

Abstract: XFEL and IR analyses suggest that O2 bound at CuB blocks proton backflow for unidirectional H+ transport by water channel closure.

Cited by 76 publications

References 49 publications

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy

Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations

A common coupling mechanism for A-type heme-copper oxidases from bacteria to mitochondria

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