Electron tunneling in proteins: role of the intervening medium

Analysis of electron-transfer (ET) kinetics data obtained from experiments on Ru-modified proteins (azurin, cytochrome c, myoglobin) and the bacterial photosynthetic reaction center reveals that distant donor-acceptor electronic couplings depend upon the secondary structure of the intervening polypeptide matrix. The β-sheet azurin structure efficiently and isotropically mediates coupling with an exponential distance-decay constant of 1.1?Å^–1. The experimentally derived distance-decay constant of 1.4?Å^–1 for long-range ET in myoglobin and the reaction center suggests that hydrogen-bond couplings are weaker through α helices than across β sheets. The donor-acceptor interactions of systems with comparable tunneling energies fall into two coupling zones: the β zone (bounded by distance-decay constants of 0.9?and 1.15 Å^–1) includes all the β-sheet (azurin) couplings and all but one coupling in cytochrome c; the α zone (boundaries: 1.25 and 1.6?Å^–1) includes less strongly coupled donor-acceptor pairs in myoglobin and the reaction center as well as a relatively weakly coupled pair in cytochrome c.     

Biochemical peculiarities of benzaldehyde lyase from Pseudomonas fluorescens Biovar I in the dependency on pH and cosolvent concentration

Benzaldehyde lyase from Pseudomonas fluorescens (BAL, EC 4.1.2.38) is a versatile catalyst for stereoselective carboligations. Nevertheless, rather inconsistent data about its biochemical properties are reported in literature. In this study, the dependency of BAL activity on ionic strength, pH, and concentration of DMSO was for the first time systematically investigated and interpreted. It was found that the activity of BAL strongly depends on all three parameters, and a correlation exists between the dependency on pH and DMSO concentration. This correlation could be explained by an interaction of DMSO with an ionic amino acid in the catalytic site. A model-based analysis indicated that the pK_a of this residue shifts to the alkaline milieu upon addition of DMSO. Consequently, the optimum pH also shifts to alkaline values when DMSO is present. Potentiometric experiments confirmed that the pK_a can most probably be attributed to Glu50 which governs the activity increase of BAL on the acidic limb of its pH-activity profile. With these findings, the apparently contradicting data from literature become comprehensible and optimal reaction conditions for synthesis can easily be deduced.     

Stoichiometry of nitrite reduction catalyzed by Pseudomonas aeruginosa nitrite-reductase

The stoichiometry of the reduction of nitrite catalyzed by Pseudomonas aeruginosa nitrite-reductase (cytochrome cd₁) has been shown to yield nitrous oxide as the final product. Gas chromatography experiments demonstrated that nitric oxide is also formed as a free intermediate. A sequential formation of NO and N₂O is discussed as proposed to the parallel formation of the two products.     

The reaction between cytochrome C1 and cytochrome C

The kinetics of electron transfer between the isolated enzymes of cytochrome c₁ and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c₁ and ferricytochrome c is fast; the second-order rate constant (k₁) is 3.0 · 10⁷ M^?1 · s^?1 at low ionic strength (I = 223 mM, 10°C). The value of this rate constant decreases to 1.8 · 10⁵ M^?1 · s^?1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c₁ and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c₁ and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c₁ and ferricytochrome c and the reverse reaction are k₁ = 2.4 · 10⁵ M^?1 · s^?1 and k_?1 = 3.3 · 10⁵ M^?1 · s^?1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10°C). The ‘equilibrium’ constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c²⁺₁ + cytochrome c³⁺ai cytochrome c³⁺₁ + cytochrome c²⁺.     

Electron flow through metalloproteins

Electron transfers in photosynthesis and respiration commonly occur between metal-containing cofactors that are separated by large molecular distances. Understanding the underlying physics and chemistry of these biological electron transfer processes is the goal of much of the work in our laboratories. Employing laser flash-quench triggering methods, we have shown that 20 Å, coupling-limited Fe(II) to Ru(III) and Cu(I) to Ru(III) electron tunneling in Ru-modified cytochromes and blue copper proteins can occur on the microsecond timescale both in solutions and crystals; and, further, that analysis of these rates suggests that distant donor-acceptor electronic couplings are mediated by a combination of sigma and hydrogen bonds in folded polypeptide structures. Redox equivalents can be transferred even longer distances by multistep tunneling, often called hopping, through intervening amino acid side chains. In recent work, we have found that 20 Å hole hopping through an intervening tryptophan is several hundred-fold faster than single-step electron tunneling in a Re-modified blue copper protein.     

Pseudomonas aeruginosa overexpression system of nitric oxide reductase for in vivo and in vitro mutational analyses

Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N₂O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is produced from nitrite (NO₂^?) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study. Characterizing the variants of the five conserved Glu residues located around the heme/non-heme iron active center allowed us to establish how the anaerobic growth rate of cNOR-deficient strains expressing cNOR variants correlates with the in vitro enzymatic activity of the variants. Since bacterial strains require active cNOR to eliminate cytotoxic NO and to survive under denitrification conditions, the anaerobic growth rate of a strain with a cNOR variant is a good indicator of NO decomposition capability of the variants and a marker for the screening of functionally important residues without protein purification. Using this in vivo screening system, we examined the residues lining the putative proton transfer pathways for NO reduction in cNOR, and found that the catalytic protons are likely transferred through the Glu57 located at the periplasmic protein surface. The homologous cNOR expression system developed here is an invaluable tool for facile identification of crucial residues in vivo, and for further in vitro functional and structural studies.     

A predicted S-type pyocin shows a bactericidal activity against clinical Pseudomonas aeruginosa isolates through membrane damage

The nucleic acid sequence at the positions 1067817-1066321 of Pseudomonas aeruginosa PAO1 genome was predicted to encode a novel S-type pyocin, designated S5, based on the genome sequence. However, its antimicrobial spectrum, activity and mechanism have not been investigated. Herein, we report that pyocin S5 has an antimicrobial activity against seven clinical P. aeruginosa isolates (DWW3, InA, InB, In3, In4, In7, and In8). Among them, DWW3 is most sensitive with a minimum inhibitory concentration of 12.6 μg/ml and a killing percentage of 95.7 at 225 μg/ml. Further, we demonstrated that the antimicrobial mechanism of pyocin S5 is membrane damage, evidenced by the leakage of intracellular materials, the increase of membrane permeability, and cell surface disruption.     

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a³⁺a²⁺₃-CO) complex and the mixed-valence carboxycytochrome c oxidase (a³⁺a²⁺₃-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a²⁺₃-CO complex. This would confirm that the spectral properties of cytochrome a₃ are not affected by the redox state of cytochrome a and Cu_A. When the absorbance changes after photolysis of cytochrome a²⁺₃-CO with a laser flash were followed on a time scale from 1 μs to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7·10³s^?1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a₃. Concomitantly, a decrease of the 830 nm band was observed due to reduction of Cu_A. This demonstrates that in the partially reduced enzyme a pathway is present between Cu_A and the cytochrome a₃-Cu_B pair, via which electrons are transferred rapidly.     

Identification of water-conditioned Pseudomonas aeruginosa by Raman microspectroscopy on a single cell level

The identification of Pseudomonas aeruginosa from samples of bottled natural mineral water by the analysis of subcultures is time consuming and other species of the authentic Pseudomonas group can be a problem. Therefore, this study aimed to investigate the influence of different aquatic environmental conditions (pH, mineral content) and growth phases on the cultivation-free differentiation between water-conditioned Pseudomonas spp. by applying Raman microspectroscopy. The final dataset was comprised of over 7500 single-cell Raman spectra, including the species Pseudomonas aeruginosa, P. fluorescens and P. putida, in order to prove the feasibility of the introduced approach. The collection of spectra was standardized by automated measurements of viable stained bacterial cells. The discrimination was influenced by the growth phase at the beginning of the water adaptation period and by the type of mineral water. Different combinations of the parameters were tested and they resulted in accuracies of up to 85% for the identification of P. aeruginosa from independent samples by applying chemometric analysis.     

Hydrophobic Surface Patches on LolA of Pseudomonas aeruginosa Are Essential for Lipoprotein Binding

Many lipoproteins reside in the outer membrane (OM) of Gram-negative bacteria, and their biogenesis is dependent on the Lol (localization of lipoproteins) system. The periplasmic chaperone LolA accepts OM-destined lipoproteins that are released from the inner membrane by the LolCDE complex and transfers them to the OM receptor LolB. The exact nature of the LolA-lipoprotein complex is still unknown. The crystal structure of Escherichia coli LolA features an open β-barrel covered by α helices that together constitute a hydrophobic cavity, which would allow the binding of one acyl chain. However, OM lipoproteins contain three acyl chains, and the stoichiometry of the LolA-lipoprotein complex is 1:1. Here we present the crystal structure of Pseudomonas aeruginosa LolA that projects clear hydrophobic surface patches. Since these patches are large enough to accommodate acyl chains, their role in lipoprotein binding was investigated. Several LolA mutant proteins were created, and their functionality was assessed by studying their capacity to release lipoproteins produced in sphaeroplasts. Interruption of the largest hydrophobic patch completely destroyed the lipoprotein-releasing capacity of LolA, while interruption of smaller patches apparently reduced efficiency. Thus, the results show a new lipoprotein transport model that places (some of) the acyl chains on the hydrophobic surface patches.     

The junctional integrity of epithelial cells is modulated by Pseudomonas aeruginosa quorum sensing molecule through phosphorylation-dependent mechanisms

In Pseudomonas aeruginosa, cell-cell communication based on acyl-homoserine lactone (HSL) quorum sensing molecules is known to coordinate the production of virulence factors and biofilms by the bacterium. Incidentally, these bacterial signals can also modulate mammalian cell behaviour. We report that 3O-C₁₂-HSL can disrupt adherens junctions in human epithelial Caco-2 cells as evidenced by a reduction of the expression and distribution of E-cadherin and β-catenin. Using co-immunoprecipitation we also found that P. aeruginosa 3O-C₁₂-HSL-treatment resulted in tyrosine hyperphosphorylation of E-cadherin, β-catenin, occludin and ZO-1. Similarly, serine and threonine residues of E-cadherin and ZO-1 became more phosphorylated after 3O-C₁₂-HSL treatment. On the contrary, occludin and β-catenin underwent dephosphorylation on serine and threonine residues after exposition of 3O-C₁₂-HSL. These changes in the phosphorylation state were paralleled by alteration in the structure of junction complexes and increased paracellular permeability. Moreover, pre-treatment of the Caco-2 cells with protein phosphatase and kinase inhibitors prevented 3O-C₁₂-HSL-induced changes in paracellular permeability and interactions between occludin-ZO-1 and the E-cadherin-β-catenin. These findings clearly suggest that an alteration in the phosphorylation status of junction proteins are involved in the changes in cell junction associations and enhanced paracellular permeability, and that bacterial signals are indeed sensed by the host cells.     

The effect of pH and ionic strength on the pre-steady-state reaction of cytochrome c and cytochrome aa3

(1) In the pH range between 5.0 and 8.0, the rate constants for the reaction of ferrocytochrome c with both the high- and low-affinity sites on cytochrome aa₃ increase by a factor of approx. 2 per pH unit. (2) The pre-steady-state reaction between ferrocytochrome c and cytochrome aa₃ did not cause a change in the pH of an unbuffered medium. Furthermore, it was found that this reaction and the steady-state reaction are equally fast in H₂O and ²H₂O. From these results it was concluded that no protons are directly involved in a rate-determining reaction step. (3) Arrhenius plots show that the reaction between ferrocytochrome c and cytochrome aa₃ requires a higher enthalpy of activation at temperatures below 20°C (15–16 kcal/mol) as compared to that at higher temperature (9 kcal/mol). We found no effect of ionic strength on the activation enthalpy of the pre-steady-state reaction, nor on that of the steady-state reaction. This suggests that ionic strength does not change the character of these reactions, but merely affects the electrostatic interaction between both cytochromes.     

Structural Studies on the Pseudomonas aeruginosa Sialidase-Like Enzyme PA2794 Suggest Substrate and Mechanistic Variations

Pseudomonas aeruginosa encodes an enzyme (PA2794) that is annotated as a sialidase (or neuraminidase), as it possesses three bacterial neuraminidase repeats that are a signature of nonviral sialidases. A recent report showed that when the gene encoding this sialidase is knocked out, this led to a reduction in biofilm production in the lungs of mice, and it was suggested that the enzyme recognizes pseudaminic acid, a sialic acid analogue that decorates the flagella of Pseudomonas, Helicobacter, and Campylobacter species. Here, we present the crystal structure of the P. aeruginosa enzyme and show that it adopts a trimeric structure, partly held together by an immunoglobulin-like trimerization domain that is C-terminal to a classical β-propeller sialidase domain. The recombinant enzyme does not show any sialidase activity with the standard fluorogenic sialic-acid-based substrate. The proposed active site contains certain conserved features of a sialidase: a nucleophilic tyrosine with its associated glutamic acid, and two of the usual three arginines that interact with the carboxylic acid group of the substrate, but is missing the first arginine and the aspartic acid that acts as an acid/base in all sialidases studied to date. We show, by in silico docking, that the active site may accommodate pseudaminic acid but not sialic acid and that this is due, in part, to a phenylalanine in the hydrophobic pocket that selects for the alternative stereochemistry of pseudaminic acid at C5 compared to sialic acid. Mutation of this phenylalanine to an alanine converts the enzyme into a sialidase, albeit a poor one, which we confirm by kinetics and NMR, and this allowed us to probe the function of other amino acids. We propose that a histidine plays the role of the acid/base, whose state is altered through a charge-relay system involving a novel His-Tyr-Glu triad. The location of this relay system precludes the presence of one of the three arginines usually found in a sialidase active site.     

Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa

Background

Prokaryotic lectins offer significant advantages over eukaryotic lectins for the development of enhanced glycoselective tools. Amenability to recombinant expression in Escherichia coli simplifies their production and presents opportunities for further genetic manipulation to create novel recombinant prokaryotic lectins (RPLs) with altered or enhanced carbohydrate binding properties. This study explored the potential of the α-galactophilic PA-IL lectin from Pseudomonas aeruginosa for use as a scaffold structure for the generation of novel RPLs.

Method

Specific amino acid residues in the carbohydrate binding site of a recombinant PA-IL protein were randomly substituted by site-directed mutagenesis. The resulting expression clones were then functionally screened to identify clones expressing rPA-IL proteins with altered carbohydrate binding properties.

Results

This study generated RPLs exhibiting diverse carbohydrate binding activities including specificity and high affinity for β-linked galactose and N-acetyl-lactosamine (LacNAc) displayed by N-linked glycans on glycoprotein targets. Key amino acid substitutions were identified and linked with specific carbohydrate binding activities. Ultimately, the utility of these novel RPLs for glycoprotein analysis and for selective fractionation and isolation of glycoproteins and their glycoforms was demonstrated.

Conclusions

The carbohydrate binding properties of the PA-IL protein can be significantly altered using site-directed mutagenesis strategies to generate novel RPLs with diverse carbohydrate binding properties.

General significance

The novel RPLs reported would find a broad range of applications in glycobiology, diagnostics and in the analysis of biotherapeutics. The ability to readily produce these RPLs in gram quantities could enable them to find larger scale applications for glycoprotein or biotherapeutic purification.     

Electron transfer patterns of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c₄ by [Co(bipy)₃]^2+/3+ (bipy = 2,2′-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both “cooperative” intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c₄ is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c₄ for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less “ballistic” modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c₄ function.     

Electron transfer between horse heart and Candida krusei cytochromes c in the free and bound states

Electron transfer between horse heart and Candida krusei cytochromes c in the free and phosvitin-bound states was examined by difference spectrum and stopped-flow methods. The difference spectra in the wavelength range of 540–560 nm demonstrated that electrons are exchangeable between the cytochromes c of the two species. The equilibrium constants of the electron transfer reaction for the free and phosvitin-bound forms, estimated from these difference spectra, were close to unity at 20°C in 20 mM Tris-HCl buffer (pH 7.4). The electron transfer rate for free cytochrome c was (2–3) · 10⁴ M^?1 · s^?1 under the same conditions. The transfer rate for the bound form increased with increase in the binding ratio at ratios below half the maximum, and was almost constant at higher ratios up to the maximum. The maximum electron exchange rate was about 2 · 10⁶ M^?1 · s^?1, which is 60–70 times that for the free form at a given concentration of cytochrome c. The activation energy of the reaction for the bound cytochrome c was equal to that for the free form, being about 10 kcal/mol. The dependence of the exchange rate on temperature, cytochrome c concentration and solvent viscosity suggests that enhancement of the electron transfer rate between cytochromes c on binding to phosvitin is due to increase in the collision frequency between cytochromes c concentrated on the phosvitin molecule.     

Insights into the effect of detergents on the full-length rhomboid protease from Pseudomonas aeruginosa and its cytosolic domain

Rhomboids comprise a family of intramembrane serine proteases that catalyze the cleavage of transmembrane segments within the lipid membrane to achieve a wide range of biological functions. A subset of bacterial rhomboids possesses an N-terminal cytosolic domain that appears to enhance proteolytic activity via an unknown mechanism. Structural analysis of a full-length rhomboid would provide new insights into this mechanism, an objective that solution NMR has the potential to realize. For this purpose we purified the rhomboid from Pseudomonas aeruginosa in a range of membrane-mimetic media, evaluated its functional status in vitro and investigated the NMR spectroscopic properties of these samples. In general, NMR signals could only be observed from the cytosolic domain, and only in detergents that did not support rhomboid activity. In contrast, media that supported rhomboid function did not show these resonances, suggesting an association between the cytosolic domain and the protein-detergent complex. Investigations into the ability of the isolated cytosolic domain to bind detergent micelles revealed a denaturing interaction, whereas no interaction occurred with micelles that supported rhomboid activity. The cytosolic domain also did not show any tendency to interact with lipid bilayers found in small bicelles or vesicles made from Escherichia coli phospholipid extracts. Based on these data we propose that the cytosolic domain does not interact with the lipid membrane, but instead enhances rhomboid activity through interactions with some other part of the rhomboid, such as the catalytic core domain.     

Identification of Pseudomonas aeruginosa in water-bottling plants on the basis of procedures included in ISO 16266:2006

ISO 16266:2006 provides a standardized procedure for the isolation and identification of Pseudomonas aeruginosa in waters. In some cases the method described in this ISO is not conclusive enough to confirm or discard the presence of this opportunistic human pathogen. In this study the capacity of the procedure described in ISO 16266:2006 to identify presumptive P. aeruginosa isolates was evaluated.Forty-one presumptive P. aeruginosa strains, previously isolated from water-bottling plants following ISO 16266:2006, were submitted to all the tests recommended by ISO 16266:2006 (Cetrimide agar with nalidixic acid, King B agar, Acetamide broth and Oxidase test). Additional tests that have been widely used for the identification of P. aeruginosa were also performed (Asparagine broth and King A agar). Furthermore, we also conducted the non-compulsory ISO 16266:2006 assay to study the capacity of the strains to grow at 4 °C and 42 °C. Finally, all the strains were biochemically phenotyped with PhP-48 plates (Bactus AB, Sweden) and API 20NE galleries (Biomérieux, France), and their 16 rRNA gene was sequenced.ISO 16266:2006 correctly identified 27 out of 29 genotypically confirmed P. aeruginosa isolates, although two false negative identifications were obtained.Growth in Asparagine broth should be discarded as a confirmative test as it showed false negatives and false positives. In contrast, API 20NE galleries correctly identified all the confirmed isolates.King A medium and growth tests at 4 °C and 42 °C correctly discriminated all the studied strains, even the two that were not identified with the basic ISO 16266:2006 tests.Given that King A medium and growth tests at 4 °C and 42 °C are straightforward, rapid, and inexpensive, it is strongly recommended that they be used for routine confirmation of P. aeruginosa when applying ISO 16266:2006.