The structure of NADH:ubiquinone oxidoreductase from beef-heart mitochondria. Crystals containing an octameric arrangement of iron-sulphur protein fragments

We have investigated the structure of two-dimensional crystals from preparations of NADH:ubiquinone oxidoreductase from beef-heart mitochondria. The crystal structure of these crystals was previously determined to be equivalent with two native enzyme molecules per unit cell, i.e. a p2 symmetry [Boekema, E. J., Van Heel, M. G. & Van Bruggen, E. F. J. (1984) Biochim. Biophys. Acta 787, 19-26]. However, the optical diffraction patterns of the crystals displayed a clear fourfold symmetry. A Fourier analysis carried out on the calculated diffraction pattern proved unambiguously that the crystal symmetry was p42(1)2. Following crystallographic rules the unit cell therefore contained eight identical molecules. As a consequence, only a subcomplex of the enzyme rather than the intact enzyme formed the crystal. Electron microscopy of isolated, single molecules of the iron-sulphur protein, a dissociation product of complex I, revealed the presence of square complexes with sides of approximately 15 nm. Since these complexes were indistinguishable from the building blocks (unit cells) of the two-dimensional crystals, the crystals could be composed of Fe-S protein fragments only. The nature of the fragments in the unit cell was probed by immuno-labelling with monovalent antibodies (Fab's), raised against the 75-kDa subunit from the Fe-S protein, followed by image analysis. We found at least four binding sites for the anti-(75-kDa subunit) Fab per unit cell, indicating the presence of at least four copies of the antigen. In order to account for these observations we postulate the hypothesis that the two-dimensional crystals obtained from complex I are composed of iron-sulphur protein molecules in an octameric arrangement.     

The structure of the metal-nucleotide complex substrate for yeast hexokinase

A comparison has been made of the kinetic parameters obtained with yeast hexokinase using Mg²⁺ or Ni²⁺ as metal ion activator and ATP or tubercidin-5′-triphosphate as nucleotide substrate. It is concluded that the relative specificity of the enzyme for MgATP^2? does not involve a contribution arising from an interaction of the metal ion with the purine ring of the nucleotide.     

Electron transport particles from beef-heart mitochondria as a test system for inhibitors of the respiratory chain]

Respiratory inhibitors exert a potentially biocidic action on all aerobic organisms. Therefore the systematic search for them and the elucidation of their mechanism of action is of great importance. The non-phosphorylating electron transfer particles (ETP) according to Crane et al. are a suitable test object for this aim. A standardized preparation of ETP from beef heart is described and characterized enzymatically. The ETP show a variety of advantages in comparison with other systems; their field of application is broad. Owing to the absence of bypasses of the respiration, particles from animals are more suitable than those from plants for the search for fungicides and herbicides which inhibit the respiratory chain. With relatively little expense, the two standard assay systems proposed (NADH oxidase and succinate-cytochrome c oxidoreductase) pick up with certainty all inhibitors of the main pathway of the respiratory chain; they are useful for screening studies. The system proposed is also suitable for the analysis of the exact site and the mechanism of action of respiratory inhibitors. Furthermore, ETP can be used to obtain parameters of the hydrophobicity of drugs as well as for toxicological studies.     

Molecular aspects of the adenine nucleotide carrier from mitochondria

The ADP/ATP carrier (AAC) of mitochondria is a functionally central and characteristic component of the eukaryotic cell. By linking the thermodynamically divergent metabolites in the intra- and extramitochondrial compartments, it had to evolve with the emergence of the eukaryotic cell. Because of a number of unique properties, the AAC provided advanced insight into the molecular basis of solute transport through biomembrane carriers. With highly specific and unusually large substrates, ADP and ATP, and with high-affinity inhibitors binding selectively either from the inside or the outside, the first molecular demonstration of the single-binding-center gated pore mechanism was made. This framework can only partially be interpreted with the available yet rapidly increasing structural information on the AAC. The primary structure, first established for the AAC from beef heart mitochondria, showed a relatively wide distribution of hydrophilic residues which permits assignment of only two hydrophobic transmembrane stretches. However, a striking tripartition of the primary structure into about three 100-residue-long domains allows a more significant assignment of transmembrane elements. With alignment of these three domains for maximum conservation of structurally critical residues, each domain can be assigned to have two transmembrane alpha elements between 18 and 22 residues long. The interdomain homology between these alpha regions is low. The central regions flanked by these helices contain most of the polar residues and are significantly interdomain conserved. With lysine probes the central regions are assigned to the matrix side (m-side) and the two connecting regions as well as C and N termini to the cytosolic side (c-side). Out of the central regions a loop is assumed to protrude through the membrane, probably for lining the translocation channel. This localization of a major protein mass within the membrane agrees with hydrodynamic evidence, the carrier being an oblate ellipsoid with only about 50 A along the short axis. In accordance, the loops of domains 2 and 3 are affinity labeled by azido-ADP or azido-atractylate. Primary structures of AAC from other sources (fungi, plants) also exhibit the tripartition. The interdomain conserved residues are also interspecies conserved, thus showing that they are essential. These repeat domains have probably evolved from a common gene coding for about 100 residues. Isoforms of the AAC exist, as shown by primary structure analysis of human cDNA libraries from different organs. Three different isoforms are identified in human organs.(ABSTRACT TRUNCATED AT 400 WORDS)     

The structure of DAPI bound to DNA

The structure of the DNA fluorochrome 4'-6-diamidine-2-phenyl indole (DAPI) bound to the synthetic B-DNA oligonucleotide C-G-C-G-A-A-T-T-C-G-C-G has been solved by single crystal x-ray diffraction methods, at a resolution of 2.4 A. The structure is nearly isomorphous with that of the native DNA molecule alone. With one DAPI and 25 waters per DNA double helix, the residual error is 21.5% for the 2428 reflections above the 2-sigma level. DAPI inserts itself edgewise into the narrow minor groove, displacing the ordered spine of hydration. DAPI and a single water molecule together span the four AT base pairs at the center of the duplex. The indole nitrogen forms a bifurcated hydrogen bond with the thymine O2 atoms of the two central base pairs, as with netropsin and Hoechst 33258. The preference of all three of these drugs for AT regions of B-DNA is a consequence of three factors: (1) The intrinsically narrower minor groove in AT regions than in GC regions of B-DNA, leading to a snug fit of the flat aromatic drug rings between the walls of the groove. (2) The more negative electrostatic potential within the minor groove in AT regions, attributable in part to the absence of electropositive-NH2 groups along the floor of the groove, and (3) The steric advantage of the absence of those same guanine-NH2 groups, thus permitting the drug molecule to sink deeper into the groove. Groove width and electrostatic factors are regional, and define the relative receptiveness of a section of DNA since they operate over several contiguous base pairs. The steric factor is local, varying from one base pair to the next, and hence is the means of fine-tuning sequence specificity.     

The structure of gelsolin bound to ATP

Calcium activation of the actin-modifying properties of gelsolin is sensitive to ATP. Here, we show that soaking calcium-free gelsolin crystals in ATP-containing media results in ATP occupying a site that spans the two pseudosymmetrical halves of the protein. ATP binding involves numerous polar and hydrophobic contacts and is identical for the two copies of gelsolin related by non-crystallographic symmetry within the crystal. The gamma-phosphate of ATP participates in several charge-charge interactions consistent with the preference of gelsolin for ATP, as a binding partner, over ADP. In addition, disruption of the ATP-binding site through Ca2+ activation of gelsolin reveals why ATP binds more tightly to the inactive molecule, and suggests how the binding of ATP may modulate the sensitivity of gelsolin to calcium ions. Similarities between the ATP and PIP2 interactions with the C-terminal half of gelsolin are evident from their overlapping binding sites and in that both molecules bind more tightly in the absence of calcium ions. We propose a model for how PIP2 may bind to calcium-free gelsolin based on the ATP-binding site.     

The amino acid sequence of GTP:AMP phosphotransferase from beef-heart mitochondria. Extensive homology with cytosolic adenylate kinase

The amino acid sequence of GTP:AMP phosphotransferase (AK3) from beef-heart mitochondria has been determined, except for one segment of about 33 residues in the middle of the polypeptide chain. The established sequence has been unambiguously aligned to the sequence of cytosolic ATP:AMP phosphotransferase (AK1) from pig muscle, allowing for six insertions and deletions. With 30% of all aligned residues being identical, the homology between AK3 and AK1 is well established. As derived from the known three-dimensional structure of AK1, the missing segment is localized at a small surface area of the molecule, far apart from the active center. The pattern of conserved residues demonstrates that earlier views on substrate binding have to be modified. The observation of three different consecutive N-termini indicates enzyme processing.     

The structure of a high fidelity DNA polymerase bound to a mismatched nucleotide reveals an "ajar" intermediate conformation in the nucleotide selection mechanism

To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established "open" and "closed" states. In this "ajar" conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation.     

Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria.

Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O₂^? at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H₂O₂ at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H₂O₂ was essentially produced by disproportionation of O₂^?, which constitutes the precursor of H₂O₂. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O₂^? and 0.63 mol of H₂O₂/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O₂ to O₂^?, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H₂O₂ generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H₂O₂ generation. The efficiency of added quinones as peroxide generators decreased in the order Q₁ > Q₀ > Q₂ > Q₆ = Q₁₀, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymatically by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O₂^? and H₂O₂ during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q₀H₂), benzoquinol, duroquinol and menadiol generated O₂^? with k₃ values of 0.1 to 1.4 m^? · s^?1 and H₂O₂ with k₄ values of 0.009 to 4.3 m^?1 · s^?1.