Takashi Tomizaki

Bio: Takashi Tomizaki is an academic researcher from Osaka University. The author has contributed to research in topics: Cytochrome c oxidase & Heme A. The author has an hindex of 4, co-authored 6 publications receiving 4230 citations. Previous affiliations of Takashi Tomizaki include Kwansei Gakuin University.

The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A.

Abstract: The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution with an R value of 19.9 percent reveals 13 subunits, each different from the other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A, and three copper, one magnesium, and one zinc. Of 3606 amino acid residues in the dimer, 3560 have been converged to a reasonable structure by refinement. A hydrogen-bonded system, including a propionate of a heme A (heme a), part of peptide backbone, and an imidazole ligand of CuA, could provide an electron transfer pathway between CuA and heme a. Two possible proton pathways for pumping, each spanning from the matrix to the cytosolic surfaces, were identified, including hydrogen bonds, internal cavities likely to contain water molecules, and structures that could form hydrogen bonds with small possible conformational change of amino acid side chains. Possible channels for chemical protons to produce H2O, for removing the produced water, and for O2, respectively, were identified.

Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 A

Abstract: The high resolution three-dimensional x-ray structure of the metal sites of bovine heart cytochrome c oxidase is reported. Cytochrome c oxidase is the largest membrane protein yet crystallized and analyzed at atomic resolution. Electron density distribution of the oxidized bovine cytochrome c oxidase at 2.8 A resolution indicates a dinuclear copper center with an unexpected structure similar to a [2Fe-2S]-type iron-sulfur center. Previously predicted zinc and magnesium sites have been located, the former bound by a nuclear encoded subunit on the matrix side of the membrane, and the latter situated between heme a3 and CuA, at the interface of subunits I and II. The O2 binding site contains heme a3 iron and copper atoms (CuB) with an interatomic distance of 4.5 A; there is no detectable bridging ligand between iron and copper atoms in spite of a strong antiferromagnetic coupling between them. A hydrogen bond is present between a hydroxyl group of the hydroxyfarnesylethyl side chain of heme a3 and an OH of a tyrosine. The tyrosine phenol plane is immediately adjacent and perpendicular to an imidazole group bonded to CuB, suggesting a possible role in intramolecular electron transfer or conformational control, the latter of which could induce the redox-coupled proton pumping. A phenyl group located halfway between a pyrrole plane of the heme a3 and an imidazole plane liganded to the other heme (heme a) could also influence electron transfer or conformational control.

Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase.

Abstract: Crystal structures of bovine heart cytochrome c oxidase in the fully oxidized, fully reduced, azide-bound, and carbon monoxide–bound states were determined at 2.30, 2.35, 2.9, and 2.8 angstrom resolution, respectively. An aspartate residue apart from the O2 reduction site exchanges its effective accessibility to the matrix aqueous phase for one to the cytosolic phase concomitantly with a significant decrease in the pK of its carboxyl group, on reduction of the metal sites. The movement indicates the aspartate as the proton pumping site. A tyrosine acidified by a covalently linked imidazole nitrogen is a possible proton donor for the O2 reduction by the enzyme.

Structure analysis of bovine heart cytochrome c oxidase at 2.8 A resolution.

Abstract: The crystal structure of bovine heart cytochrome c oxidase has been determined at 2.8 A resolution by the multiple isomorphous replacement (MIR) method with three heavy-atom derivatives. An asymmetric unit of the crystal has a molecular weight of 422 kDa. Eight heavy atoms as main sites of a CH3HgCl derivative were clearly located by solving the difference Patterson function. The electron density obtained by the MIR method was refined by density modification, consisting of solvent flattening, histogram matching and non-crystallographic symmetry averaging. The enzyme exhibits a dimeric structure in the crystal. Out of 3606 amino-acid residues in 26 subunits in the dimer, 3560 residues were located in the electron-density map. The structure was refined by X-PLOR. The final R factor and the free R factor were 0.199 and 0.252 at 2.8 A resolution, respectively. One monomer in the dimeric structure with a stronger packing interaction has a lower averaged temperature factor than the other, by 16 A2. The region \pm12 A from the centre of the transmembrane part is almost 100% \alpha-helix, despite the glycine residue content being as high as 7.1% in the transmembrane region. The residues around haem a of animals have evolved away from those of bacteria in contrast with the residues of the haem a3. The hierarchy of the structural organization of the enzyme complex has been proposed on the basis of intersubunit interactions.

Acoustic levitation and rotation of thin films and their application for room temperature protein crystallography

Abstract: Abstract Acoustic levitation has attracted attention in terms of chemical and biochemical analysis in combination with various analytical methods because of its unique container-less environment for samples that is not reliant on specific material characteristics. However, loading samples with very high viscosity is difficult. To expand the scope, we propose the use of polymer thin films as sample holders, whereby the sample is dispensed on a film that is subsequently loaded onto an acoustic levitator. When applied for protein crystallography experiments, rotation controllability and positional stability are important prerequisites. We therefore study the acoustic levitation and rotation of thin films with an aspect ratio (the diameter-to-thickness ratio) of 80–240, which is an order of magnitude larger than those reported previously. For films with empirically optimized shapes, we find that it is possible to control the rotation speed in the range of 1–4 rotations per second while maintaining a positional stability of 12 ± 5 µm. The acoustic radiation force acting on the films is found to be a factor of 26–30 higher than that for same-volume water droplets. We propose use cases of the developed films for protein crystallography experiments and demonstrate data collections for large single crystal samples at room temperature.

Multicopper Oxidases and Oxygenases

Abstract: Copper is an essential trace element in living systems, present in the parts per million concentration range. It is a key cofactor in a diverse array of biological oxidation-reduction reactions. These involve either outer-sphere electron transfer, as in the blue copper proteins and the Cu{sub A} site of cytochrome oxidase and nitrous oxide redutase, or inner-sphere electron transfer in the binding, activation, and reduction of dioxygen, superoxide, nitrite, and nitrous oxide. Copper sites have historically been divided into three classes based on their spectroscopic features, which reflect the geometric and electronic structure of the active site: type 1 (T1) or blue copper, type 2 (T2) or normal copper, and type 3 (T3) or coupled binuclear copper centers. 428 refs.

Cell biology and molecular basis of denitrification.

Abstract: Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

Structural and Functional Aspects of Metal Sites in Biology

Abstract: For present purposes, a protein-bound metal site consists of one or more metal ions and all protein side chain and exogenous bridging and terminal ligands that define the first coordination sphere of each metal ion. Such sites can be classified into five basic types with the indicated functions: (1) structural -- configuration (in part) of protein tertiary and/or quaternary structure; (2) storage -- uptake, binding, and release of metals in soluble form: (3) electron transfer -- uptake, release, and storage of electrons; (4) dioxygen binding -- metal-O{sub 2} coordination and decoordination; and (5) catalytic -- substrate binding, activation, and turnover. The authors present here a classification and structure/function analysis of native metal sites based on these functions, where 5 is an extensive class subdivided by the type of reaction catalyzed. Within this purview, coverage of the various site types is extensive, but not exhaustive. The purpose of this exposition is to present examples of all types of sites and to relate, insofar as is currently feasible, the structure and function of selected types. The authors largely confine their considerations to the sites themselves, with due recognition that these site features are coupled to protein structure at all levels. In themore » next section, the coordination chemistry of metalloprotein sites and the unique properties of a protein as a ligand are briefly summarized. Structure/function relationships are systematically explored and tabulations of structurally defined sites presented. Finally, future directions in bioinorganic research in the context of metal site chemistry are considered. 620 refs.« less

Proton-Coupled Electron Transfer

Abstract: ▪ Abstract Proton-coupled electron transfer (PCET) is an important mechanism for charge transfer in a wide variety of systems including biology- and materials-oriented venues. We review several are...