Hoeon Kim

Crystallization and preliminary structure of beef heart mitochondrial cytochrome-bc1 complex

The method reported for isolation of ubiquinol-cytochrome-c reductase complex from submitochondrial particles was modified to yield a preparation for crystallization. The cytochrome bc1 complex was first crystallized in large thin plate form and diffracts X-rays to 7 A resolution in the presence of mother liquor. This crystalline complex was enzymatically active and contains ten protein subunits. It had 33 mol phospholipid and 0.6 mol ubiquinone per mol protein. With slightly modified crystallization conditions, different crystal forms were obtained. Crystals grown in the presence of 20% glycerol diffracted X-rays up to 2.9 A resolution using a synchrotron source. Four heavy atom derivatives have been obtained. The 3-D structure of the cytochrome bc1 complex was solved to 3.4 A resolution. Crystalline cytochrome bc1 complex is a dimer: most of the masses of core proteins I and II protrudes from the matrix side of the membrane, whereas the cytochrome b protein is located mainly within the membrane. There are 13 transmembrane helices in each monomer. Most of the mass of cytochrome c1 and iron-sulfur protein including their redox centers are located on the cytoplasmic side of the membrane. The distances between these redox centers have been determined, and several electron transfer inhibitor binding sites in the complex have been located.

ACTIVATION OF A MATRIX PROCESSING PEPTIDASE FROM THE CRYSTALLINE CYTOCHROME BC1 COMPLEX OF BOVINE HEART MITOCHONDRIA

No mitochondrial processing peptidase (MPP) activity is detected in crystalline bovine heart mitochondrial cytochrome bc 1 complex, which possesses full electron transfer activity. However, when the complex is treated with increasing concentrations of Triton X-100 at 37 °C, the electron transfer activity decreases, whereas peptidase activity increases. Maximum MPP activity is obtained when the electron transfer activity in the complex is completely inactivated with 1.5 mm of Triton X-100. This result supports our suggestion that the lack of MPP activity in the mammalian cytochrome bc 1complex is because of binding of an inhibitor polypeptide to the active site of MPP located at the interface of core subunits I and II. This suggestion is based on the three-dimensional structural information for the bc 1 complex and the sequence homology between subunits of MPP and the core subunits of the beef complex. Triton X-100, at concentrations that disrupt the structural integrity of the bc 1 complex as indicated by the loss of its electron transfer activity, weakens the binding of inhibitor polypeptide to the active site of MPP in core subunits, thus activating MPP. The Triton X-100-activated MPP is pH-, buffer system-, ionic strength-, and temperature-dependent. Maximum activity is observed with an assay mixture containing 15 mm Tris-HCl buffer at neutral pH (6.5–8.5) and at 37 °C. Activated MPP is completely inhibited by metal ion chelators such as EDTA ando-phenanthroline and partially inhibited by myxothiazol (58%), ferricyanide (28%), and dithiothreitol (81%). The metal ion chelator-inhibited activity can be partially restored by the addition of divalent cations such as Zn2+ (68%), Mg2+(44%), Mn2+ (54%), Co2+ (62%), and Fe2+ (92%), indicating that metal ion is required for MPP activity. The cleavage site specificity of activated MPP depends more on the length of amino acid sequence from the mature protein portion and less on the presequence portion, when a synthetic peptide composed of NH2-terminal residues of a mature protein and the COOH-terminal residues of its presequence is used as a substrate.

Structural basis of multifunctional bovine mitochondrial cytochrome bc1 complex

The mitochondrial cytochrome bc1 complex is a multifunctional membrane protein complex. Itcatalyzes electron transfer, proton translocation, peptide processing, and superoxide generation.Crystal structure data at 2.9 Å resolution not only establishes the location of the redox centersand inhibitor binding sites, but also suggests a movement of the head domain of the iron–sulfurprotein (ISP) during bc1 catalysis and inhibition of peptide-processing activity during complexmaturation. The functional importance of the movement of extramembrane (head) domain ofISP in the bc1 complex is confirmed by analysis of the Rhodobacter sphaeroides bc1 complexmutants with increased rigidity in the ISP neck and by the determination of rate constants foracid/base-induced intramolecular electron transfer between [2Fe–2S] and heme c1 in nativeand inhibitor-loaded beef complexes. The peptide-processing activity is activated in bovineheart mitochondrial bc1 complex by nonionic detergent at concentrations that inactivate electrontransfer activity. This peptide-processing activity is shown to be associated with subunits Iand II by cloning, overexpression and in vitro reconstitution. The superoxide-generation siteof the cytochrome bc1 complex is located at reduced bL and Q•−. The reaction is membranepotential-, and cytochrome c-dependent.

Atomic force microscopy of the three-dimensional crystal of membrane protein, OmpC porin

Three-dimensional microcrystals of OmpC osmoporin were air-dried slowly and imaged in air with an atomic force microscope (AFM). The overall structural features in AFM images are in good agreement with the X-ray diffraction data of these OmpC osmoporin crystals: monoclinic P2(1) with the unit cell constants a=117.6 Å, b=110 Å, c=298.4 Å, beta=97 degrees. Such a good correspondence between X-ray diffraction and AFM data suggests that the slow and mild air-drying of these crystals did not induce any significant alterations in the crystal lattices as expected upon crystal dehydration. At the (100) crystal face, individual trimeric protein-detergent complexes were resolved. These results show the potential for studying the molecular structure of microcrystals of integral membrane proteins. This study also suggests that the crystal grew in a fashion of rapid two-dimensional expansion along the bc plane followed by a slow deposition along the a axis, perhaps as a rate-limiting nucleation process. Thus, AFM imaging of air-dried crystals would also be of considerable use in the early stages of a project to grow large three-dimensional crystals of membrane proteins suitable for high-resolution X-ray diffraction studies.

Crystallization of OmpC osmoporin from Escherichia coli.

OmpC porin, one of the major outer-membrane proteins of Gram-negative bacteria, participates in bacterial osmoregulation by counteracting OmpF porin. Although these two osmoporins from Escherichia coli share high sequence homology, their crystallization behavior was found to be very different. OmpC could be crystallized under a variety of conditions by either microdialysis or hanging-drop methods using PEG 4000 as precipitant. The crystals belong to space group P21 with unit-cell constants a = 117.6, b = 110, c = 298.4 A, beta = 97 degrees. They diffract beyond 4 A with a rotating anode and show intense non-Bragg scattering.

The Crystal Structure of Mitochondrial Cytochrome bc 1 Complex

The cytochrome bc 1 complex (commonly known as ubiquinol-cytochrome c reductase, or complex III) is a segment of the mitochondrial respiratory chain that catalyzes antimycin-sensitive electron transfer from ubiquinol to cytochrome c (Rieske, 1967; Hatefi et al., 1962). The reaction is coupled to the translocation of protons across the mitochondrial inner membrane to generate a proton gradient and membrane potential for ATP synthesis.