Properties of the detergent solubilised cytochrome c oxidase (cytochrome cbb(3)) purified from Pseudomonas stutzeri.

Cytochrome cbb(3) is a cytochrome c-oxidising isoenzyme that belongs to the superfamily of respiratory haem/copper oxidases. We have developed a purification method yielding large amounts of pure cbb(3) complex from the soil bacterium Pseudomonas stutzeri. This cytochrome cbb(3) complex consists of three subunits (ccoNOP) in a 1:1:1 stoichiometry and contains two b-type and three c-type haems. The protein complex behaves as a monomer with an overall molecular weight of 114.0+/-8.9 kDa and a s(0)(20,w) value of 8.9+/-0.3 S as determined by analytical ultracentrifugation. Crystals diffracting to 5.0 A resolution have been grown by the vapour diffusion sitting drop method to an average size of 0.1 x 0.1 x 0.3 mm. This is the first crystallisation report of a (cbb(3))-type oxidase.

pH-induced conformational transition in the soluble CuA domain of Paracoccus denitrificans cytochrome oxidase.

The pH-induced conformational transition in the CuA domain of subunit II of cytochrome oxidase of Paracoccus denitrificans (PdII) has been investigated using various spectroscopic and stopped-flow kinetic methods. UV-visible absorption and circular dichroism studies showed that an increase in pH from 6 to 10 leads to a conformation change with pK(a) = 8.2 associated with the CuA site of the protein. The secondary structure of the protein was, however, shown to remain unchanged in these two conformational states. Thermal and urea-induced unfolding studies showed that the "low-pH" conformation is more stable compared to the "high-pH" conformation of the protein. Moreover, the overall stability of the protein was found to decrease on reduction of the metal centers in the low-pH form, while the oxidation state of the metal centers did not have any significant effect on the overall stability of the protein in the high-pH form. Stopped-flow pH-jump kinetic studies suggested that the conformational transition is associated with a slow deprotonation step followed by fast conformational equilibrium. The results are discussed in the light of understanding the pH-induced conformational change in the beta-barrel structure of the protein and its effect on the coordination geometry of the metal site.

The active site of the bacterial nitric oxide reductase is a dinuclear iron center.

A novel, improved method for purification of nitric oxide reductase (NOR) from membranes of Paracoccus denitrificans has been developed. The purified enzyme is a cytochrome bc complex which, according to protein chemical and hydrodynamic data, contains two subunits in a 1:1 stoichiometry. The purified NorBC complex binds 0.87 g of dodecyl maltoside/g of protein and forms a dimer in solution. Similarly, it is dimeric in two-dimensional crystals. Images of these crystals have been processed at 8 A resolution in projection to the membrane. The NorB subunit is homologous to the main catalytic subunit of cytochrome oxidase and is predicted to contain the active bimetallic center in which two NO molecules are turned over to N2O. Metal analysis and heme composition implies that it binds two B-type hemes and a nonheme iron but no copper. NorC is a membrane-anchored cytochrome c. Fourier transform infrared spectroscopy shows that carbon monoxide dissociates from the reduced heme in light and associates with another metal center which is distinct from the copper site of heme/copper oxidases. Electron paramagnetic resonance spectroscopy reveals that NO binds to the reduced enzyme under turnover conditions giving rise to signals near g = 2 and g = 4. The former represents a typical nitrosyl-ferroheme signal whereas the latter is a fingerprint of a nonheme iron/NO adduct. We conclude that the active site of NOR is a dinuclear iron center.

Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 A resolution.

The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms. These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient. Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily. To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy. The crystals diffract beyond 5 A. A projection map was calculated to a resolution of 6 A. All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex. The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I. It also indicates subtle differences which are due to the distinct subunit composition. This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals.

Structural and functional analysis of aa3-type and cbb3-type cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in proton-pump design.

In Paracoccus denitrificans the aa3-type cytochrome c oxidase and the bb3-type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOOP, and demonstrate that it encodes an alternative cbb3-type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen-sensing mechanism. This view is corroborated by the observation that the ccoNOOP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR-binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa3, bb3 and cbb3 make up the complete set of terminal oxidases in P. denitrificans. Proton-translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H+/e- coupling efficiencies of the cbb3-type oxidase under certain conditions. Sequence alignment suggests that many residues that have been proposed to constitute the chemical and pumped proton channels in cytochrome aa3 (and probably also in cytochrome bb3) are not conserved in cytochrome cbb3. It is concluded that the design of the proton pump in cytochrome cbb3 differs significantly from that in the other oxidase types.

Purification and two-dimensional crystallization of bacterial cytochrome oxidases.

A novel strategy which employes chromatography on an immobilized metal ion has been developed for the purification of bacterial cytochrome c and quinol oxidases. Many bacterial oxidase complexes appear to have a natural affinity to bind to the chelated copper ion. A combination of three different chromatographic principles (anion exchange, metal-affinity and gel filtration) makes an effective tool chest for the preparation of homogeneous and protein-chemically pure bacterial oxidases. These preparations have been used for two-dimensional crystallization. Until now, crystals have been obtained using the Paracococcus denitrificans and Rhodobacter sphaeroides cytochrome aa3 and the Escherichia coli cytochrome bo. The crystals diffract to approximately 2.5 nm in negative stain and have potential for further structural studies.

A second terminal oxidase in Sulfolobus acidocaldarius.

We previously found that the soxABCD operon encodes a quinol oxidase complex in Sulfolobus acidocaldarius and this enzyme was purified and characterized. In this study, we have used a cloning procedure based on the conservation of oxidase sequences and the polymerase chain reaction to isolate a new gene (soxM) encoding a subunit of another terminal oxidase. This terminal oxidase is a fusion between two central components of cytochrome oxidases, subunits I and III. soxM forms a transcriptional unit which is expressed under heterotrophic growth conditions. The corresponding protein was detected by direct protein sequencing in a preparation enriched with a cytochrome absorbing light at 562 nm. This preparation contains a terminal oxidase which is able to oxidize the artificial substrate N,N,N',N'-tetramethyl-p-phenylenediamine. This preparation also contains SoxC, a protein homologous to the mitochondrial cytochrome b, and a Rieske iron-sulphur center. We suggest that SoxM is the core component of a second terminal oxidase complex and that this complex may share a subunit (SoxC) with the SoxABCD complex.

The purified SoxABCD quinol oxidase complex of Sulfolobus acidocaldarius contains a novel haem.

A respiratory quinol oxidase complex that is encoded by the soxABCD operon has been purified from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The enzyme was solubilized with dodecyl maltoside and purified in the presence of this detergent and ethylene glycol. The complex is hydrodynamically homogeneous and contains at least five different polypeptides. In addition to the major subunits SoxA, SoxB and SoxC, it has two small polypeptides. One of these is the translation product of a short open reading frame (now called the soxD gene) at the end of the operon. The optical and electron paramagnetic resonance spectra of the SoxABCD complex have been characterized. It probably contains four A-type haems which are bound to SoxB and SoxC. The structure of these haems is not identical to haem A. The novel haem As has a 2-hydroxyethyl geranylgeranyl in position 2 of the porphyrin ring whereas haem A has the related farnesyl-containing side-chain.

Worldwide initiation of holocene marine deltas by deceleration of sea-level rise.

Radiocarbon-dated deltaic sequences of Holocene age from different parts of the world began to accumulate within a restricted time range, from about 8500 to 6500 years ago. Evaluation of major delta processes indicates that deceleration in sea-level rise was the key factor in Holocene delta formation. Within many deltas, there is as much as a 2000-year age range between basal deposits in seaward and landward cores. This age difference records the progressive landward migration of near mean sea-level depositional environments during the lower to mid-Holocene. Establishment of a chronostratigraphic framework for Holocene delta development provides a fundamental global baseline for distinguishing sea-level change from vertical land motion by tectonism and isostasy, and for evaluating rates of future marine incursion into low-lying deltas.

Nile delta: recent geological evolution and human impact.

Few countries in the world are as dependent on water from a single source as Egypt. The natural Nile cycle of flow and sediment discharge has been disrupted by human intervention, including closure of the High Aswan Dam; this intervention has resulted in a series of responses that now threaten the northern Nile delta. Erosion, salinization, and pollution are inducing a marked decline in agricultural productivity and loss of land and coastal lagoons at a time when the population is expanding exponentially. Geological analyses of radiocarbon-dated cores across the northern delta are used to interpret the interaction of sea-level changes, climatic oscillations, subsidence, and transport processes during the past 35,000 years. Recognition of long-term trends of these natural factors provides a basis to evaluate the profound impact of human activity and to assess future changes in the Nile delta ecosystem.

Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E. coli cytochrome o quinol oxidase complex.

The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.

Purification of rabbit liver aldehyde oxidase by affinity chromatography on benzamidine sepharose 6B.

Highly purified rabbit liver aldehyde oxidase was prepared in high yield using affinity chromatography on Benzamidine Sepharose 6B. Rabbit liver was homogenised, heat treated and ammonium sulphate was added to the supernatant to give a crude preparation of the enzyme. Aliquots of the crude preparation were chromatographed on a Benzamidine Sepharose 6B column at pH 9 and the aldehyde oxidase was eluted by a benzamidine containing buffer. This single affinity step resulted in a 38-fold increase in purity over the crude preparation with an 84% recovery of enzyme activity. Further purification on a Mono Q ion-exchange column gave an additional 1.7-fold increase in specific activity to yield a highly purified preparation of the enzyme. The new method described is considerably simpler and faster than ones hitherto employed and gives a much better yield of the highly purified enzyme.