Purification, crystallization and preliminary crystallographic studies of an integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli (original) (raw)
Related papers
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2000
Crystals of cytochrome bo 3 ubiquinol oxidase from E. coli diffract X-rays to 3.5 A î and the structure determination is in progress. The limiting factor to the elucidation of the structural detail is the quality of the crystals; the diffraction spots from the crystals are diffused which leads to difficulties in processing the data beyond 4.0 A î. Weak protein^protein contacts within the crystal lattice is assumed to be the cause of this problem. To improve these contacts, we have introduced protein Z to the C-terminal end of the subunit IV of cytochrome bo 3 and expressed both proteins as a single fusion. We have successfully obtained crystals of this fusion protein. The spot shape problem has clearly been solved in the crystals of the fusion protein although further optimization is necessary to obtain higher resolution. We also discuss the potential applications of this approach to the crystallization of membrane proteins in general.
Acta Crystallographica Section D-biological Crystallography, 2003
The membrane-bound respiratory complex II, succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli, has been anaerobically expressed, then puri®ed and crystallized. The initial crystals obtained were small and diffracted poorly. In order to facilitate structure determination, rational screening and sample-quality analysis using electron microscopy was implemented. The crystals of SQR from E. coli belong to the trigonal space group R32, with unit-cell parameters a = b = 138.7, c = 521.9 A Ê , and diffract to 2.6 A Ê resolution. The optimization strategy used for obtaining well diffracting SQR crystals is applicable to a wide range of membrane proteins.
Journal of Molecular Biology, 1981
Membrane crystals have been prepared from mitochondrial ubiquinol : cytochrome c reductase by mixing the enzyme-Triton complex with phospholipid-Triton micelles and subsequently removing the Triton. The electron micrographs of the negatively stained crystals diffract to 2.5 nm, with unit cell dimensions of 13.7 nm by 174 nm. The enzyme is arranged in a two-sided plane group P22,2,, i.e. alternate molecules span the bilayer in an up and down manner. By combining tilted views of the membrane crystals, a low-resolution three-dimensional structure of t'he enzyme has been calculated. The structure shows that the enzyme is a dimer, t.he monomers being related by a S-fold axis running perpendicular to the membrane. The monomeric units of the enzyme are elongated, extending approximately 15 nm across the membrane. The protein is unequally distributed with about 3O"/b of the total mass located in the bilayer, 50% in a section which extends 7 nm from one side of the bilayer and 20% in a section which extends 3 nm from the opposite side of the bilayer. The two monomeric units are in contact only in the membraneous section. This structure is compared with a model of the enzyme which is derived from biochemical properties of the isolated subunits.
Biochemistry, 1993
The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of structurally related heme-copper respiratory oxidases. The members of this family, which also includes the aa3-type cytochrome c oxidases, contain at least two heme prosthetic groups, a six-coordinate low-spin heme, and a high-spin heme. The high-spin heme is magnetically coupled to a copper, CUB, forming a binuclear center which is the site of oxygen reduction to water. Vectorial proton translocation across the membrane bilayer appears to be another common feature of this superfamily of oxidases. It has been proposed previously that the two adjacent histidines in putative transmembrane helix VI1 (H333 and H334 in the E. coli sequence) a Abstract published in Advance ACS Abstracts, October 15, 1993. Abbreviations: CO, carbon monoxide; Cu, copper; C u -m , carbonmonoxy adduct of copper; E,, midpoint potential; EPR, electron paramagnetic resonance; Fe, heme iron; Fe--C=--O, carbonmonoxy adduct of heme; FTIR, Fourier transform infrared; Aullzand BWHH, full width of stretching frequency band at half-maximal height.
Biochemistry, 1998
Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and Cu B , whose axial ligands have been identified to be six invariant histidines. This work explored the possible roles of the aromatic amino acid residues conserved in the putative transmembrane helices (or at the boundary of the membrane) of subunit I. Sixteen aromatic amino acid residues were individually substituted by Leu, except for Tyr 61 and Trp 282 by Phe and Phe 415 by Trp. Leu substitutions of Trp 280 and Tyr 288 in helix VI, Trp 331 in loop VII-VIII, and Phe 348 in helix VIII reduced the catalytic activity, whereas all other mutations did not affect the in vivo activity. Spectroscopic analyses of the purified mutant enzymes revealed that the defects were attributable to perturbations of the binuclear center. On the basis of these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved aromatic amino acid residues in subunit I of the heme-copper terminal oxidases.
2010
Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins.
Structure of bovine cytochrome c oxidase crystallized at a neutral pH using a fluorinated detergent
Acta crystallographica. Section F, Structural biology communications, 2017
Cytochrome c oxidase (CcO) couples proton pumping to O2 reduction. Its enzymatic activity depends sensitively on pH over a wide range. However, owing to difficulty in crystallizing this protein, X-ray structure analyses of bovine CcO aimed at understanding its reaction mechanism have been conducted using crystals prepared at pH 5.7, which is significantly lower than that in the cell. Here, oxidized CcO at pH 7.3 was crystallized using a fluorinated octyl-maltoside derivative, and the structure was determined at 1.77 Å resolution. No structural differences between crystals obtained at the neutral pH and the acidic pH were detected within the molecules. On the other hand, some differences in intermolecular interactions were detected between the two types of crystal. The influence of pH on the molecular surface is likely to contribute to the pH dependency of the aerobic oxidation of ferrocytochrome c.
Microscopy, 2016
Mitochondrial cytochrome c oxidase utilizes electrons provided by cytochrome c for the active vectorial transport of protons across the inner mitochondrial membrane through the reduction of molecular oxygen to water. Direct structural evidence on the transient cytochrome c oxidase-cytochrome c complex thus far, however, remains elusive and its physiological relevant oligomeric form is unclear. Here, we report on the 2D crystallization of monomeric bovine cytochrome c oxidase with tightly bound cytochrome c at a molar ratio of 1:1 in reconstituted lipid membranes at the basic pH of 8.5 and low ionic strength.
Protein Science, 2014
The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n‐dodecyl‐β‐d‐maltoside for a precisely defined mixture of two α‐maltosides and decanoylsucrose as well as the choice of...