ABSTRACT
The dimeric integral membrane protein nicotinamide nucleotide transhydrogenase is required for cellular regeneration of NADPH in mitochondria and prokaryotes, for detoxification and biosynthesis purposes. Under physiological conditions, transhydrogenase couples the reversible reduction of NADP+ by NADH to an inward proton translocation across the membrane. Here, we present crystal structures of the NAD(H)-binding domain I of transhydrogenase from Escherichia coli, in the absence as well as in the presence of oxidized and reduced substrate. The structures were determined at 1.9-2.0 A resolution. Overall, the structures are highly similar to the crystal structure of a previously published NAD(H)-binding domain, from Rhodospirillum rubrum transhydrogenase. However, this particular domain is unique, since it is covalently connected to the integral-membrane part of transhydrogenase. Comparative studies between the structures of the two species reveal extensively differing surface properties and point to the possible importance of a rigid peptide (PAPP) in the connecting linker for conformational coupling. Further, the kinetic analysis of a deletion mutant, from which the protruding beta-hairpin was removed, indicates that this structural element is important for catalytic activity, but not for domain I:domain III interaction or dimer formation. Taken together, these results have important implications for the enzyme mechanism of the large group of transhydrogenases, including mammalian enzymes, which contain a connecting linker between domains I and II.
Subject(s)
Escherichia coli/chemistry , NADP Transhydrogenases/chemistry , Proton Pumps/chemistry , Binding Sites , Computer Simulation , Crystallography, X-Ray , Dimerization , Models, Molecular , Protein Structure, TertiaryABSTRACT
Transhydrogenase is a proton-pumping membrane protein that is required for the cellular regeneration of NADPH. The NAD(H)-binding domain (domain I) of transhydrogenase from Escherichia coli was crystallized using the hanging-drop vapour-diffusion technique at room temperature. The crystals, which were grown from PEG 4000 and ammonium acetate in citrate buffer, belong to the triclinic space group P1, with unit-cell parameters a = 38.8, b = 66.8, c = 76.4 A, alpha = 67.5, beta = 80.8, gamma = 81.5 degrees. X-ray diffraction data were collected to 1.9 A resolution using synchrotron radiation. The crystals contain one dimer of transhydrogenase domain I per asymmetric unit.
Subject(s)
Crystallization , Escherichia coli Proteins/chemistry , NADP Transhydrogenases/chemistry , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , NAD/chemistryABSTRACT
The structure of Escherichia coli succinate dehydrogenase (SQR), analogous to the mitochondrial respiratory complex II, has been determined, revealing the electron transport pathway from the electron donor, succinate, to the terminal electron acceptor, ubiquinone. It was found that the SQR redox centers are arranged in a manner that aids the prevention of reactive oxygen species (ROS) formation at the flavin adenine dinucleotide. This is likely to be the main reason SQR is expressed during aerobic respiration rather than the related enzyme fumarate reductase, which produces high levels of ROS. Furthermore, symptoms of genetic disorders associated with mitochondrial SQR mutations may be a result of ROS formation resulting from impaired electron transport in the enzyme.
Subject(s)
Escherichia coli/enzymology , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Reactive Oxygen Species/metabolism , Succinate Dehydrogenase/chemistry , Succinate Dehydrogenase/metabolism , Aerobiosis , Anaerobiosis , Binding Sites , Crystallography, X-Ray , Dinitrophenols/chemistry , Dinitrophenols/pharmacology , Electron Transport , Electron Transport Complex II , Flavin-Adenine Dinucleotide/metabolism , Heme/chemistry , Models, Molecular , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/genetics , Mutation , Oxidation-Reduction , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/genetics , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , Succinate Dehydrogenase/antagonists & inhibitors , Succinate Dehydrogenase/genetics , Succinic Acid/metabolism , Superoxides/metabolism , Ubiquinone/chemistry , Ubiquinone/metabolismABSTRACT
The membrane-bound respiratory complex II, succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli, has been anaerobically expressed, then purified 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, and diffract to 2.6 A resolution. The optimization strategy used for obtaining well diffracting SQR crystals is applicable to a wide range of membrane proteins.
Subject(s)
Escherichia coli/enzymology , Multienzyme Complexes/chemistry , Oxidoreductases/chemistry , Succinate Dehydrogenase/chemistry , Anisotropy , Coloring Agents , Crystallization , Crystallography, X-Ray , Electron Transport Complex II , Microscopy, Electron , Multienzyme Complexes/isolation & purification , Oxidoreductases/isolation & purification , Succinate Dehydrogenase/isolation & purification , UltracentrifugationABSTRACT
The structure of cytochrome c oxidase from Rhodobacter sphaeroides has been solved at 2.3/2.8A (anisotropic resolution). This high-resolution structure revealed atomic details of a bacterial terminal oxidase including water molecule positions and a potential oxygen pathway, which has not been reported in other oxidase structures. A comparative study of the wild-type and the EQ(I-286) mutant enzyme revealed structural rearrangements around E(I-286) that could be crucial for proton transfer in this enzyme. In the structure of the mutant enzyme, EQ(I-286), which cannot transfer protons during oxygen reduction, the side-chain of Q(I-286) does not have the hydrogen bond to the carbonyl oxygen of M(I-107) that is seen in the wild-type structure. Furthermore, the Q(I-286) mutant has a different arrangement of water molecules and residues in the vicinity of the Q side-chain. These differences between the structures could reflect conformational changes that take place upon deprotonation of E(I-286) during turnover of the wild-type enzyme, which could be part of the proton-pumping machinery of the enzyme.
Subject(s)
Electron Transport Complex IV/chemistry , Electron Transport Complex IV/genetics , Mutation/genetics , Rhodobacter sphaeroides/enzymology , Binding Sites , Catalysis , Copper/metabolism , Crystallography, X-Ray , Electron Transport Complex IV/metabolism , Hydrogen Bonding , Ligands , Models, Molecular , Oxidation-Reduction , Oxygen/metabolism , Protein Conformation , Protein Subunits , Protons , Rhodobacter sphaeroides/genetics , Water/metabolism , Xenon/metabolismABSTRACT
The structure of the membrane protein formate dehydrogenase-N (Fdn-N), a major component of Escherichia coli nitrate respiration, has been determined at 1.6 angstroms. The structure demonstrates 11 redox centers, including molybdopterin-guanine dinucleotides, five [4Fe-4S] clusters, two heme b groups, and a menaquinone analog. These redox centers are aligned in a single chain, which extends almost 90 angstroms through the enzyme. The menaquinone reduction site associated with a possible proton pathway was also characterized. This structure provides critical insights into the proton motive force generation by redox loop, a common mechanism among a wide range of respiratory enzymes.
Subject(s)
Escherichia coli/enzymology , Formate Dehydrogenases/chemistry , Proton-Motive Force , Binding Sites , Catalysis , Catalytic Domain , Cell Membrane/enzymology , Crystallography, X-Ray , Electron Transport , Formate Dehydrogenases/metabolism , Formates/metabolism , Guanine Nucleotides/chemistry , Guanine Nucleotides/metabolism , Hydrogen Bonding , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Membrane Potentials , Models, Molecular , Nitrate Reductases/chemistry , Nitrate Reductases/metabolism , Oxidation-Reduction , Protein Conformation , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits , Protons , Pterins/chemistry , Pterins/metabolism , Vitamin K 2/chemistry , Vitamin K 2/metabolismABSTRACT
A membrane protein complex, succinate dehydrogenase (SQR) from Escherichia coli has been purified and crystallised. This enzyme is composed of four subunits containing FAD, three iron-sulphur clusters and one haem b as prosthetic groups. The obtained crystals belong to the hexagonal space group P6(3) with the unit-cell dimensions of a=b=123.8 A and c=214.6 A. An asymmetric unit of the crystals contains one SQR monomer (M(r) 120 kDa). A data set is now available at 4.0 A resolution with 88.1% completeness and 0.106 R(merge). We have obtained a molecular replacement solution that shows sensible molecular packing, using the soluble domain of E. coli QFR (fumarate reductase) as a search model. The packing suggests that E. coli SQR is a crystallographic trimer rather than a dimer as observed for the E. coli QFR.
Subject(s)
Escherichia coli/enzymology , Multienzyme Complexes/isolation & purification , Oxidoreductases/isolation & purification , Succinate Dehydrogenase/isolation & purification , Crystallography , Electron Transport Complex II , Flavin-Adenine Dinucleotide/chemistry , Fumarates/chemistry , Fumarates/metabolism , Heme/chemistry , Intracellular Membranes/enzymology , Iron-Sulfur Proteins/chemistry , Membrane Proteins/chemistry , Models, Molecular , Multienzyme Complexes/chemistry , Oxidoreductases/chemistry , Quinone Reductases/chemistry , Quinone Reductases/isolation & purification , Succinate Dehydrogenase/chemistry , Succinic Acid/chemistry , Succinic Acid/metabolismABSTRACT
A membrane-protein complex, formate dehydrogenase-N from Escherichia coli, has been purified and crystallized. This molybdenum-containing enzyme, composed of alpha, beta and gamma subunits, is the major electron donor to the nitrate respiratory chain of E. coli. The formate dehydrogenase-N crystals belong to the cubic space group P2(1)3, with unit-cell parameters a = b = c = 203 A. An asymmetric unit of the crystals is assumed to contain one formate dehydrogenase-N monomer (MW 170 kDa). One data set to 1.6 A resolution, with 342 711 independent observations (94.4% complete) and an R(merge) of 0.08, has been collected from a single crystal. This is the highest resolution data set reported for a membrane-protein complex to date.
