Structural insights into electron transfer in caa3-type cytochrome oxidase

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36?? resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.     

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, Cu(A) and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.     

Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that catalyses the conversion of methane to methanol. Knowledge of how pMMO performs this extremely challenging chemistry may have an impact on the use of methane as an alternative energy source by facilitating the development of new synthetic catalysts. We have determined the structure of pMMO from the methanotroph Methylococcus capsulatus (Bath) to a resolution of 2.8 A. The enzyme is a trimer with an alpha3beta3gamma3 polypeptide arrangement. Two metal centres, modelled as mononuclear copper and dinuclear copper, are located in soluble regions of each pmoB subunit, which resembles cytochrome c oxidase subunit II. A third metal centre, occupied by zinc in the crystal, is located within the membrane. The structure provides new insight into the molecular details of biological methane oxidation.     

Assignment of Soret MLCT band of reduced form of copper binuclear cluster in cytochrome c oxidase film

Low concentration of dithionite results in the reduction of Cu-Cn binuclear and heine a active sites of the cytochrome c oxidase thin solid film immersed in the acidic phosphate buffer, but Fe-Cu binuclear center keeps in the oxidation state. It manifests as a negative peak at 426 nm and a positive one at -408 nln in the difference spectra induced by dithionite. The former implies decrease of the oxidized form of heme a center, that is, Fea^3 →Fea^2 . And the latter results from the contribution of metal-ligand charge transfer (MLCT) transition in the reduced binuclear Cu-Cu cluster, rather than from that of heine a center. This stronger Soret MLCT band must be helpful to overcoming the difficulty in distinguishing the weaker copper sign from the stronger one of iron when studying copper-iron protein.     

A mechanistic principle for proton pumping by cytochrome c oxidase

In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c. The electron transfer through CytcO, accompanied by proton uptake to form H2O drives the physical movement (pumping) of four protons across the membrane per reduced O2. So far, the molecular mechanism of such proton pumping driven by electron transfer has not been determined in any biological system. Here we show that proton pumping in CytcO is mechanistically coupled to proton transfer to O2 at the catalytic site, rather than to internal electron transfer. This scenario suggests a principle by which redox-driven proton pumps might operate and puts considerable constraints on possible molecular mechanisms by which CytcO translocates protons.     

Structure and mechanism of copper, zinc superoxide dismutase

Copper, zinc superoxide dismutase (SOD) catalyses the very rapid two-step dismutation of the toxic superoxide radical (O-2) to molecular oxygen and hydrogen peroxide through the alternate reduction and oxidation of the active-site copper. We report here that after refitting and further refinement of the previous 2 A structure of SOD2, analysis of the new model and its calculated molecular surface shows an extensive surface topography of sequence-conserved residues stabilized by underlying tight packing and H-bonding. There is a single, highly complementary position for O-2 to bind to both the Cu(II) and activity-important Arg 141 with correct geometry; two water molecules form a ghost of the superoxide in this position. The geometry and molecular surface of the active site, together with biochemical data, suggest a specific model for the enzyme mechanism.     

Identification of the BAL-labile factor

One of us has previously reported that treatment of the Keilin and Hartree heart-muscle preparation with 2,3-dimercaptopropanol (BAL), in the presence of air, leads to the complete inactivation of the succinate oxidase system with little if any effect on the activities of succinate dehydrogenase (until more than half the BAL was oxidized) or cytochrome c oxidase. The inactivation of the complete succinate oxidase system requires the oxidation of BAL by air in the presence of the enzyme. It is not caused by H2O2 or BAL disulphides produced during the oxidation of BAL. Spectroscopic studies identified the block as lying between cytochromes b and c. It was suggested that a BAL-labile factor is present which transfers electrons from cytochrome b to cytochrome c and which is destroyed by coupled oxidation with BAL. The factor is also required for NADH oxidation. Subsequent work showed it is not identical with cytochrome c1 (ref. 4), myoglobin present in the preparation or the antimycin-binding site. We report here that this factor is identical to the iron-sulphur protein in the central portion of the respiratory chain first identified by Rieske.     

A highly unsaturated fatty acid predicts carbon transfer between primary producers and consumers

The factors that regulate energy transfer between primary producers and consumers in aquatic ecosystems have been investigated for more than 50 years. Among all levels of the food web (plants, herbivores, carnivores), the plant-animal interface is the most variable and least predictable link. In hypereutrophic lakes, for example, biomass and energy transfer is often inhibited at the phytoplankton-zooplankton link, resulting in an accumulation of phytoplankton biomass instead of sustaining production at higher trophic levels, such as fish. Accumulation of phytoplankton (especially cyanobacteria) results in severe deterioration of water quality, with detrimental effects on the health of humans and domestic animals, and diminished recreational value of water bodies. We show here that low transfer efficiencies between primary producers and consumers during cyanobacteria bloom conditions are related to low relative eicosapentaenoic acid (20:5omega3) content of the primary producer community. Zooplankton growth and egg production were strongly related to the primary producer 20:5omega3 to carbon ratio. This indicates that limitation of zooplankton production by this essential fatty acid is of central importance at the pelagic producer-consumer interface.     

Control of ion selectivity in potassium channels by electrostatic and dynamic properties of carbonyl ligands

Potassium channels are essential for maintaining a normal ionic balance across cell membranes. Central to this function is the ability of such channels to support transmembrane ion conduction at nearly diffusion-limited rates while discriminating for K+ over Na+ by more than a thousand-fold. This selectivity arises because the transfer of the K+ ion into the channel pore is energetically favoured, a feature commonly attributed to a structurally precise fit between the K+ ion and carbonyl groups lining the rigid and narrow pore. But proteins are relatively flexible structures that undergo rapid thermal atomic fluctuations larger than the small difference in ionic radius between K+ and Na+. Here we present molecular dynamics simulations for the potassium channel KcsA, which show that the carbonyl groups coordinating the ion in the narrow pore are indeed very dynamic ('liquid-like') and that their intrinsic electrostatic properties control ion selectivity. This finding highlights the importance of the classical concept of field strength. Selectivity for K+ is seen to emerge as a robust feature of a flexible fluctuating pore lined by carbonyl groups.     

Control of organ shape by a secreted metalloprotease in the nematode Caenorhabditis elegans.

The molecular controls governing organ shape are poorly understood. In the nematode Caenorhabditis elegans, the gonad acquires a U-shape by the directed migration of a specialized 'leader' cell, which is located at the tip of the growing gonadal 'arm'. The gon-1 gene is essential for gonadal morphogenesis: in gon-1 mutants, no arm elongation occurs and somatic gonadal structures are severely malformed. Here we report that gon-1 encodes a secreted protein with a metalloprotease domain and multiple thrombospondin type-1-like repeats. This motif architecture is typical of a small family of genes that include bovine procollagen I N-protease (P1NP), which cleaves collagen, and murine ADAMTS-1, the expression of which correlates with tumour cell progression. We find that gon-1 is expressed in two sites, leader cells and muscle, and that expression in each site has a unique role in forming the gonad. We speculate that GON-1 controls morphogenesis by remodelling basement membranes and that regulation of its activity is crucial for achieving organ shape.     

Structural basis for cofactor-independent dioxygenation in vancomycin biosynthesis

Enzyme-catalysed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.     

EMND与人血清白蛋白的分子作用机制

微波条件下,运用一锅法合成了二氢嘧啶-2-酮的衍生物—5-乙酯基-6-甲基-4-(4-硝基苯基)-3,4-二氢嘧啶-2-酮(EMND).运用同步荧光光谱、位点竞争结合实验、荧光共振能量转移(FRET)理论以及分子模拟技术研究了EMND与人血清白蛋白(HSA)的分子作用机制.实验结果表明:EMND与HSA的结合位点位于色氨酸(Trp214)附近,EMND的结合位点位于HSA的IIA亚域.通过FRET理论计算得出EMND与色氨酸残基的结合距离r=4.25nm,说明EMND与HSA之间能够发生非辐射能量转移.分子模拟表明EMND结合到HSA IIA亚域的疏水空腔,它们之间存在氢键作用,进一步印证了同步荧光及位点竞争结合实验结果.本研究对于在分子水平上理解小分子与生物大分子的相互作用本质以及设计基于二氢嘧啶-2-酮的药物等都有一定的参考作用.     

The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre

Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H(2) into protons and electrons at high rates. Those hydrogenases maintaining their activity in the presence of O(2) are considered to be central to H(2)-based technologies, such as enzymatic fuel cells and for light-driven H(2) production. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations, the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O(2) has remained unclear. Here we present the crystal structure of an O(2)-tolerant [NiFe]-hydrogenase from the aerobic H(2) oxidizer Ralstonia eutropha H16 at 1.5?? resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H(2)-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of H(2) oxidation and as an electron-delivering device upon O(2) attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials. The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical H(2)-converting catalysts that are capable of cycling H(2) in air.     

Crystal structure of a copper-transporting PIB-type ATPase

Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu(+)-ATPase, in a copper-free form, as determined by X-ray crystallography at 3.2 ? resolution. The structure indicates a three-stage copper transport pathway involving several conserved residues. A PIB-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a putative copper entry point at the intracellular interface. Comparisons to Ca(2+)-ATPase suggest an ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site. The structure also provides a framework to analyse missense mutations in the human ATP7A and ATP7B proteins associated with Menkes' and Wilson's diseases.     

Crystal structure of thymine DNA glycosylase conjugated to SUMO-1

Members of the small ubiquitin-like modifier (SUMO) family can be covalently attached to the lysine residue of a target protein through an enzymatic pathway similar to that used in ubiquitin conjugation, and are involved in various cellular events that do not rely on degradative signalling via the proteasome or lysosome. However, little is known about the molecular mechanisms of SUMO-modification-induced protein functional transfer. During DNA mismatch repair, SUMO conjugation of the uracil/thymine DNA glycosylase TDG promotes the release of TDG from the abasic (AP) site created after base excision, and coordinates its transfer to AP endonuclease 1, which catalyses the next step in the repair pathway. Here we report the crystal structure of the central region of human TDG conjugated to SUMO-1 at 2.1 A resolution. The structure reveals a helix protruding from the protein surface, which presumably interferes with the product DNA and thus promotes the dissociation of TDG from the DNA molecule. This helix is formed by covalent and non-covalent contacts between TDG and SUMO-1. The non-covalent contacts are also essential for release from the product DNA, as verified by mutagenesis.     

Regions of variant histone His2AvD required for Drosophila development.

One way in which a distinct chromosomal domain could be established to carry out a specialized function is by the localized incorporation of specific histone variants into nucleosomes. H2AZ, one such variant of the histone protein H2A, is required for the survival of Drosophila melanogaster, Tetrahymena thermophila and mice (R. Faast et al., in preparation). To search for the unique features of Drosophila H2AZ (His2AvD, also referred to as H2AvD) that are required for its essential function, we have performed amino-acid swap experiments in which residues unique to Drosophila His2AvD were replaced with equivalently positioned Drosophila H2A.1 residues. Mutated His2AvD genes encoding modified versions of this histone were transformed into Drosophila and tested for their ability to rescue null-mutant lethality. We show that the unique feature of His2AvD does not reside in its histone fold but in its carboxy-terminal domain. This C-terminal region maps to a short alpha-helix in H2A that is buried deep inside the nucleosome core.     

Copper metallothionein, a copper-binding protein from Neurospora crassa

Copper is an essential constituent of many proteins which participate in biologically important reactions. In contrast to iron, where different metal storage and transport proteins have been extensively characterised, the existence of copper proteins serving such functions is still a matter of controversy. Studies on the biosynthesis of tyrosinase from Neurospora crassa with respect to the copper status of this fungus have shown that this organism accumulates copper with the concomitant synthesis of a small molecular weight copper-binding protein. This protein is now shown to have a striking sequence homology to the zinc- and cadmium-containing metallothioneins from vertebrates. Growth experiments suggest that this molecule fulfills several important physiological functions in this organism such as copper storage, copper detoxification and provision of copper for tyrosinase.     

Cu-HMS分子筛的合成及对芳烃氧化反应的催化

采用中性模板法于室温下合成了含铜的分子筛Cu-HMS,并以该分子筛为多相催化剂,对温和条件下异丙苯的氧化进行了研究.结果表明,以硝酸铜为铜源的Cu-HMS-3催化剂具有很高的催化活性,异丙苯转化率和过氧化氢异丙苯选择性都很高,分别达39.6%和98.5%.催化剂可以回收使用.另外,以Cu-HMS-3为催化剂对甲苯和乙苯的氧化进行了研究,发现在本实验条件下,甲苯未发生反应,3种芳烃的活性顺序为甲苯乙苯<异丙苯.