Atsuhiro Shimada

The Mg2+-containing water cluster of mammalian cytochrome c oxidase collects four pumping proton equivalents in each catalytic cycle

Bovine heart cytochrome c oxidase (CcO) pumps four proton equivalents per catalytic cycle through the H-pathway, a proton-conducting pathway, which includes a hydrogen bond network and a water channel operating in tandem. Protons are transferred by H3O+ through the water channel from the N-side into the hydrogen bond network, where they are pumped to the P-side by electrostatic repulsion between protons and net positive charges created at heme a as a result of electron donation to O2 bound to heme a3. To block backward proton movement, the water channel remains closed after O2 binding until the sequential four-proton pumping process is complete. Thus, the hydrogen bond network must collect four proton equivalents before O2 binding. However, a region with the capacity to accept four proton equivalents was not discernable in the x-ray structures of the hydrogen bond network. The present x-ray structures of oxidized/reduced bovine CcO are improved from 1.8/1.9 to 1.5/1.6 Å resolution, increasing the structural information by 1.7/1.6 times and revealing that a large water cluster, which includes a Mg2+ ion, is linked to the H-pathway. The cluster contains enough proton acceptor groups to retain four proton equivalents. The redox-coupled x-ray structural changes in Glu198, which bridges the Mg2+ and CuA (the initial electron acceptor from cytochrome c) sites, suggest that the CuA-Glu198-Mg2+ system drives redox-coupled transfer of protons pooled in the water cluster to the H-pathway. Thus, these x-ray structures indicate that the Mg2+-containing water cluster is the crucial structural element providing the effective proton pumping in bovine CcO.

Complex structure of cytochrome c-cytochrome c oxidase reveals a novel protein-protein interaction mode

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, we determined the structure of the mammalian Cyt.c–CcO complex at 2.0‐Å resolution and identified an electron transfer pathway from Cyt.c to CcO. The specific interaction between Cyt.c and CcO is stabilized by a few electrostatic interactions between side chains within a small contact surface area. Between the two proteins are three water layers with a long inter‐molecular span, one of which lies between the other two layers without significant direct interaction with either protein. 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 protein–protein interaction, which we term “soft and specific”. This interaction is likely 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.

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

XFEL and IR analyses suggest that O2 bound at CuB blocks proton backflow for unidirectional H+ transport by water channel closure. Bovine cytochrome c oxidase (CcO), a 420-kDa membrane protein, pumps protons using electrostatic repulsion between protons transferred through a water channel and net positive charges created by oxidation of heme a (Fea) for reduction of O2 at heme a3 (Fea3). For this process to function properly, timing is essential: The channel must be closed after collection of the protons to be pumped and before Fea oxidation. If the channel were to remain open, spontaneous backflow of the collected protons would occur. For elucidation of the channel closure mechanism, the opening of the channel, which occurs upon release of CO from CcO, is investigated by newly developed time-resolved x-ray free-electron laser and infrared techniques with nanosecond time resolution. The opening process indicates that CuB senses completion of proton collection and binds O2 before binding to Fea3 to close the water channel using a conformational relay system, which includes CuB, heme a3, and a transmembrane helix, to block backflow of the collected protons.

Respiratory Conservation of Energy with Dioxygen: Cytochrome c Oxidase

Cytochrome c oxidase (CcO) is the terminal oxidase of cell respiration which reduces molecular oxygen (O₂) to H2O coupled with the proton pump. For elucidation of the mechanism of CcO, the three-dimensional location and chemical reactivity of each atom composing the functional sites have been extensively studied by various techniques, such as crystallography, vibrational and time-resolved electronic spectroscopy, since the X-ray structures (2.8 Å resolution) of bovine and bacterial CcO have been published in 1995.X-ray structures of bovine CcO in different oxidation and ligand binding states showed that the O₂reduction site, which is composed of Fe (heme a 3) and Cu (CuB), drives a non-sequential four-electron transfer for reduction of O₂to water without releasing any reactive oxygen species. These data provide the crucial structural basis to solve a long-standing problem, the mechanism of the O₂reduction.Time-resolved resonance Raman and charge translocation analyses revealed the mechanism for coupling between O₂reduction and the proton pump: O₂is received by the O₂reduction site where both metals are in the reduced state (R-intermediate), giving the O₂-bound form (A-intermediate). This is spontaneously converted to the P-intermediate, with the bound O₂fully reduced to 2 O²⁻. Hereafter the P-intermediate receives four electron equivalents from the second Fe site (heme a), one at a time, to form the three intermediates, F, O, and E to regenerate the R-intermediate. Each electron transfer step from heme a to the O₂reduction site is coupled with the proton pump.X-ray structural and mutational analyses of bovine CcO show three possible proton transfer pathways which can transfer pump protons (H) and chemical (water-forming) protons (K and D). The structure of the H-pathway of bovine CcO indicates that the driving force of the proton pump is the electrostatic repulsion between the protons on the H-pathway and positive charges of heme a, created upon oxidation to donate electrons to the O₂reduction site. On the other hand, mutational and time-resolved electrometric findings for the bacterial CcO strongly suggest that the D-pathway transfers both pump and chemical protons. However, the structure for the proton-gating system in the D-pathway has not been experimentally identified. The structural and functional diversities in CcO from various species suggest a basic proton pumping mechanism in which heme a pumps protons while heme a 3 reduces O₂as proposed in 1978.

Structure of bovine cytochrome c oxidase in the ligand-free reduced state at neutral pH.

Although the enzymatic activity of cytochrome c oxidase (CcO) depends sensitively on pH over a wide range, X-ray structural analyses of bovine CcO have been conducted using crystals prepared at pH 5.7 owing to the difficulty in crystallizing this protein. Here, the structure of ligand-free reduced CcO was successfully determined at 1.99 Å resolution.

Structure of bovine cytochrome c oxidase crystallized at a neutral pH using a fluorinated detergent

Although the enzymatic activity of cytochrome c oxidase (CcO) depends sensitively on pH over a wide range, X-ray structure analyses of bovine CcO have been conducted using crystals prepared at pH 5.7 owing to difficulty in crystallizing this protein. Here, oxidized CcO at pH 7.3 was successfully crystallized using a fluorinated octyl-maltoside derivative, and the structure was determined at 1.77 Å resolution.

Low-dose X-ray structure analysis of cytochrome oxidase utilizing high-energy X-rays

Radiation damage on macromolecular crystallography (MX) appears as degradation of crystal quality (global damage) and local structural change (specific-damage) depending on accumulated dose. Although X-ray absorption by macromolecular crystals can be reduced with utilization of high-energy X-rays, reduction of diffraction intensity and lower efficiency of area detectors will be problematic in practice [1]. However, it is expected to mitigate radiation damage by utilizing a sufficiently high-efficient area detector for high-energy photons. Bovine hart cytochrome c oxidase (CcO) is an enzyme which functions in the cellular respiratory electron transport chain at inner mitochondrial membrane. It has been confirmed to bind a ligand peroxide at the reduction center consisting of a copper ion and a heme iron, by a damage-less structure analysis with femtosecond crystallography at SACLA [2]. However, on synchrotron based MX, the peroxide ion is readily reduced to waters by X-ray irradiation, and bond length between two oxygen atoms of peroxide is observed elongated according to the accumulated dose [3]. To verify the effect of radiation-damage suppression with high-energy X-rays for radiation sensitive proteins, a low-dose diffraction data collection for CcO crystals with 30 keV X-ray has been conducted at BL41XU, SPring-8. A high-sensitive pixel array detector with CdTe sensor (Pilatus 3 X CdTe, DECTRIS, Ltd.) was utilized for data collection by shutter-less helical scanning method. Controlling the average dose to be 55 kGy by translation-speed and frame-rate settings, a diffraction data set of maximum resolution up to 1.9 Å was collected (dose estimation with RADDOSE version 2). The result of structure analysis showed the ligand structure of 1.55 Å bond length, which provides the evidence of the largely suppressed radiation damage. Currently, further investigation of possibility for X-ray energy dependency of site specific damage on the reduction center, utilizing microspectroscopy is in progress. [1] Arndt et al., J. Appl. Cryst. (1984). 17, 118-119 [2] Hirata et al., Nature Methods (2014). 11, 734-736 [3] Aoyama et al., PNAS (2009). 106, 2165-2169

Fixed-target serial crystallography at SACLA

Masaki Yamamoto*1, Hideo Ago1, Kunio Hirata1,2, Keitaro Yamashita1, Go Ueno1,3, Minoru Kubo1,2, Seiki Baba4, Kazuya Hasegawa4, Takashi Kumasaka4, Atsuhiro Shimada5, Kyoko Shinzawa-Itoh5, Tomitake Tsukihara2,5,6, Shinya Yoshikawa5, Michihiro Suga7, Fusamichi Akita7, Jian-Ren Shen7 1. RIKEN SPring-8 Center, 2. Japan Science and Technology Agency (JST), PRESTO, 3. JST, CREST, 4. JASRI, 5. Univ. Hyogo, 6. Osaka Univ., 7. Okayama Univ., *yamamoto@riken.jp

Time-resolved XFEL crystallography and spectroscopy of cytochrome c oxidase

Bovine heart cytochrome c oxidase (CcO) is a large membrane enzyme (210 kDa) that catalyzes O2 reduction to water, coupled with proton pump across the mitochondrial inner membrane. The enzyme includes a hydrogen-bond network and a water channel in tandem as a pathway for proton pump (H-pathway). The O2-reduction site is composed of heme a3 and CuB, where O2 binds in the fully-reduced state (Fea3^2+, CuB^1+). After the O=O bond cleavage, CcO sequentially receives four electron equivalents from cytochrome c. Each of the electron transfer processes is coupled with pumping of one proton equivalent, during which the water channel is closed to prevent proton back leak. Recently, high-resolution X-ray structural analysis has revealed that binding of CO (O2 analogue) to Fea3 induces a bulge (unpaired backbone C=O) formation at Ser382 and Met383 in HelixX, which closes the water channel. To elucidate the coupling mechanism of ligand dynamics with water channel gating, here, we developed novel instruments for time-resolved X-ray crystallography using an X-ray free electron laser (XFEL) at SACLA and time-resolved single-crystal IR spectroscopy, and investigated the structural dynamics following CO-photolysis from Fea3. To induce the CO-photolysis, the visible light pulse was focused onto the loop-mounted crystal from two directions (from the front and back sides of the crystal), allowing us to use the large crystals with a 100% CO-photolysis efficiency. The results of timeresolved X-ray crystallography and IR spectroscopy demonstrate that CO binds to CuB transiently in the crystalline phase as in the solution phase. The communication mechanism between the Fea3-CuB site and the water channel in the H-pathway will be discussed at the presentation.

Structures of bovine cytochrome oxidase reveal proton active transports

Bovine cytochrome c oxidase (CcO) pumps four protons in each catalytic cycle through H-pathway including a hydrogen-bond network and a water channel in tandem. Protons, transferred through the water-channel from the negative side of mitochondrial inner membrane into the hydrogen-bond network, are pumped to the positive side of the membrane electrostatically by net positive charges on a heme (heme a) iron created upon electron transfer to the O2 reduction site. For blockage of backward proton leak from the hydrogen-bond network, which determines the proton-pumping direction, the water channel is closed after O2 binding to initiate proton-pump. Thus, four protons must be collected in the hydrogen-bond network before O2 binding. The X-ray structural analyses of the oxidized/reduced CcO at 1.5/1.6 Å resolution reveal a large cluster composed of ~21 water molecules and a Mg2+ site including Glu198 (Subunit II) bridging CuA and Mg2+. The cluster of the oxidized state have 20 water sites with full occupancy and two sites with partial occupies of water, while that of the reduced state have 19 water sites with full occupancy and 3 sites with partial occupancies. The carboxyl group of Glu198 changes its coordination structure to Mg2+ upon the reduction of the active centers. The cluster is tightly sealed sterically against proton exchanges with the cluster outside except for a short hydrogen-bond network connecting the cluster with H-pathway. Five proton-acceptable groups hydrogen-bonded with the cluster suggest sufficient storage capacity for four proton equivalents. The redox-coupled structural changes in the electron transfer pathway from CuA, the initial electron acceptor from cytochrome c, to heme a suggest redox-driven effective proton donations from the cluster to H-pathway, facilitated by Glu198. These results indicate that the cluster is a crucial element of the proton-pumping system of bovine CcO.