Conformational equilibria accompanying the electron transfer between cytochrome c (P551) and azurin from Pseudomonas aeruginosa.

The redox reaction between cytochrome c (Cyt c) (P-551) and the blue copper protein azurin, both from Pseudomonas aeruginosa, was studied using the temperature-jump technique. Two relaxation times were observed in a mechanism assumed to involve three equilibria. The fast relaxation time (0.4 less than tau less than 8 ms) was ascribed to the electron exchange step. The slow relaxation time (tau congruent to 37 ms) was assigned to a conformational equilibrium of the reduced azurin that was coupled through the electron exchange step to a faster conformational equilibrium of the oxidized Cyt c (P551). But because the Cyt c (P551) isomerization, being very rapid, was uncoupled from the two slower equilibria, and was assumed to involve no spectral change, the amplitude of its relaxation time (tau congruent to 0.1 ms) would be zero. At 25 degrees C and pH 7.0 the rate constants for the oxidation and reduction of Cyt c (P551) by azurin were 6.1 X 10(6) and 7.8 X 10(6) M-1 s-1, respectively; for the formation and disappearance of the reactive conformational isomer of azurin they were 12 and 17 s-1, respectively. The rates for the Cyt c (P551) isomerization could only be estimated at approximately 10(4) s-1. The thermodynamic parameters of each reaction step were evaluated from the amplitudes of the relaxations and from Eyring plots of the rate constants. Measurements of the overall equilibrium constant showed it to be temperature independent (5-35 degrees C), i.e. deltaHtot = 0. This zero enthalpy change was found to be compatible with the enthalpies calculated for the individual steps. In the electron exchange equilibrium, the values of the activation enthalpies were two to three times higher than the values published for various low molecular weight reagents in their electron exchange with copper proteins, yet the rate of exchange between Cyt c (P551) and azurin was some hundreds of times faster. This was explained in terms of the measured positive or zero entropies of activation that could result from a high level of specificity between the proteins particularly in areas of complementary charges. The mechanism of electron transfer was considered as essentially an outer sphere reaction, of which the rate could be approximated by the Marcus theory.     

The effect of iron-hexacyanide binding on the determination of redox potentials of cytochromes and copper proteins

The midpoint redox potentials of Pseudomonas aeruginosa cytochrome c-551 and Rhodopseudomonas viridis cytochrome c₂ were measured as a function of pH in the presence of Euglena cytochrome c-558 and the results compared with those obtained in the presence of ferro-ferricyanide. The pattern of pH dependence observed for the two bacterial cytochromes was the same whether it was measured by equilibrium with another redox protein or with the inorganic redox couple. Thus, the pH dependence of redox potential is not a consequence of pH-dependent ligand binding. The midpoint potential of Ps. aeruginosa azurin was measured as a function of pH using both ferro-ferricyanide mixtures and redox equilibrium with horse cytochrome c or Rhodopseudomonas capsulata cytochrome c₂. In this case also the pattern of pH dependence obtained did not vary with the redox system used and it closely resembled that of Ps. aeruginosa cytochrome c-551. This is consistent with the observation that the equilibrium between cytochrome c-551 and azurin is relatively independent of pH. An equation was derived which described pH-dependent ligand binding and which can produce theoretical curves to fit the experimental pH dependence of redox potential for both cytochrome and azurin. However, the pronounced effect on such curves produced by varying the ligand association constants, and the insensitivity of the experimental data to changes in ionic strength, suggest that ligand binding effects do not account for the pH dependence of redox potential.     

Studies of individual carbon sites of azurin from Pseudomonas aeruginosa by natural-abundance carbon-13 nuclear magnetic resonance spectroscopy.

The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Czota of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak chi) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cgamma of titrating histidines, and of Cgamma and Czota of the two tyrosines. The resonances of Cgamma and Cdelta2 of the single tryptophan residue (and Czota of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of "solvent-exposed" tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cgamma of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 +/- 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Ndelta1-H tautomer. The Cgamma resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nepsilon2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information. (i) Peak chi arises from an amide group that probably is coordinated to the copper. (ii) The two nontitrating histidine residues are probably copper ligands, with Ndelta1 coordinated to the metal. (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either. (iv) The gamma carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.     

Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study.

The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.     

pH dependence of the reduction-oxidation reaction of azurin with cytochrome c-551: role of histidine-35 of azurin in electron transfer

A fluorescence quenching experiment confirms that in the redox reaction between cytochrome c-551 and azurin, protein complexing is negligible. Azurin-pH indicator T-jump experiments show that Pseudomonas aeruginosa (Ps.) azurin exhibits a slow time constant, tau, in its return to pH equilibrium but Alcaligenes faecalis (Alc.) azurin does not. The decrease of l/tau with increasing pH shows that the rate-determining process is a slow transformation of the imidazolium form of histidine-35 from a conformation where it cannot ionize to one in which it can. The fast relaxation time constant of the redox reaction varies little with pH, but the slow time constant increased by a factor of approximately 2.5 increasing pH between pH 5 and pH 8. The corresponding amplitudes, especially the slow one, vary with pH. On the basis of all the present evidence it is concluded that, while some differences of redox reactivity do occur on protonation, these differences are not major. In general, the two proteins cyt c-551 and azurin react with each other with rates only weakly dependent upon pH. A classical pH titration was carried out on the reduced and oxidized form of Ps. and Alc. azurin with the result that two protons were released between pH 6 and pH 8, in the former from His-35 and -83 and in the latter from His-83 and Ala-1.     

The electron transfer system of Pseudomonas aeruginosa: a study of the pH-dependent transitions between redox forms of azurin and cytochrome c551

Fast reaction kinetic experiments on the electron transfer reaction between azurin and cytochrome c₅₅₁ isolated from Pseudomonas aeruginosa confirmed the existence of two redox forms of reduced azurin previously reported. The pH dependence of the amplitudes of the relaxation processes observed in temperature jump experiments indicate that these two redox forms are in pH dependent equilibrium. The pH independence of the overall equilibrium constant indicates that redox active and inactive forms of cytochrome c₅₅₁ may also exist. Evidence that reduced cytochrome c₅₅₁ undergoes a pH transition is given by optical spectrophotometry. The nature of the transition is discussed in the context of recent nmr studies and in terms of the Marcus theory of electron transfer. The metabolic consequences of these transitions are also discussed.     

Structure and reactivity of type-I copper sites

Summary Biological electron transfer is not well understood. The question is addressed in this contribution with reference to the so-called blue copper proteins, each of which has a single copper atom at its active centre. The redox activity (as probed by the electron self exchange reaction) of the Cu centre seems not to be affected. The electron self exchange reaction is known to proceed through His-117, and the hydrophobic patch is most important in the formation of the azurin/azurin encounter complex. Ph effects have not been observed on the three-dimensional structure ofA. denitrificans azurin, which may indicate that if present at all these have no direct physiological implications. Mutants are in process of construction.     

The reactions of neuroglobin with CO: evidence for two forms of the ferrous protein

The normally hexa coordinate ferrous form of neuroglobin binds CO by replacement of the heme-linked distal histidine residue. We have studied this reaction in detail using stopped flow techniques. The reaction time courses are complex at all the wavelengths studied. Specifically the reaction with CO occurs in two temporally separable phases, each of which shows a hyperbolic dependence of rate on CO concentration, indicating they each arise from histidine replacement by CO. Analysis of the observed rates as a function of the CO concentration, measured in the pH range 6.0-8.0, allows us to determine both the rate of histidine-heme ligand binding and dissociation for each of the two forms of the protein present in solution at each pH value. The pH dependence of the histidine association and dissociation rates is complex, as are the derived equilibrium constants for distal histidine binding. The spectral change associated with each reaction phase is very similar and independent of the CO concentration, showing that the two protein forms responsible for the two observed kinetic processes are not in equilibrium on the time scale of our investigations. Our data suggests that, unlike many other heme proteins, neuroglobin displays complex reactivity with ligands in the ferrous form due to heme rotational disorder, as has previously been reported for the ferric form of the protein.     

Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives.

A novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3-D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His-N or Cys-S) and the observed rate constants of Cu(II) reduction.     

Gated electron transfers and electron pathways in azurin: a NMR dynamic study at multiple fields and temperatures

Dynamic properties of electron transfer pathways in a small blue copper cupredoxin are explored using an extensive 15N NMR relaxation study of reduced Pseudomonas aeruginosa azurin at four magnetic fields (500-900 MHz) and at two temperatures chosen well below the melting point of the protein. Following a careful model-free analysis, several protein regions with different dynamic regimes are identified. Nanosecond time-scale mobility characterizes various residues of the hydrophobic surface patch believed to mark the natural entry point for electrons, notably the surface-exposed copper-ligand His117. These findings are consistent with a gated electron transfer process according to the "dynamic docking" model. Residues 47-49 along intramolecular pathways of electrons show rigidity that is remarkably conserved when increasing the temperature. Three different conformational exchange processes were observed in the millisecond range, one near the only disulfide bridge in the molecule and two near the copper ion. The latter two processes are consistent with previous data such as crystal structures at various pH values and NMR relaxation dispersion experiments; they may indicate an additional gated electron transfer mechanism at slower time-scales.     

Resolution of two distinct electron transfer sites on azurin

Pseudomonas aeruginosa azurin is stoichiometrically and specifically labeled upon reduction by Cr(II)aq ions, yielding a substitution-inert Cr(III) adduct on the protein surface. We investigated the effect of this chemical modification on the reactivity of azurin with two of its presumed partners in the redox system of the bacterium. The Pseudomonas cytochrome oxidase catalyzed oxidation of reduced native and Cr(III)-labeled azurin by O2 was found to be unaffected by the modification. The kinetics of the electron exchange reaction between native or Cr(III)-labeled azurin and cytochrome c551 were studied by the temperature-jump method. Though similar chemical relaxation spectra were observed for native and modified systems, they differ quantitatively. Analysis of the concentration dependences of the relaxation times and amplitudes showed that both obey the same mechanism but that the specific reaction rates of the Cr(III)-modified protein are attenuated. This decreased reactivity of Cr(III)-labeled azurin toward one of its physiological partners suggests the involvement of the labeled region in the electron transfer reaction with cytochrome c551. Furthermore, the presence of a second active site, involved in the reduction of cytochrome oxidase, is suggested by the results.     

Equilibrium and kinetics of the reaction of Aplysia myoglobin with azide.

The present paper reports a study on the equilibria and kinetics of the acid-alkaline transition and the azide binding reaction by ferric Aplysia myoglobin. A single completely reversible spectrophotometric titration curve is found over the pH range from similar to 5 to similar to 9, with an apparent pK equals to 7.5 for the acid-alkaline transition. The kinetics of the process, followed by the temperature-jump method, gives, at pH values close to the pK of the transition, one single, well-resolved, relaxation independent of protein concentration and of type of buffer used. The pattern accords to a simple pH dependent reaction, in buffered medium, between the two forms of the protein. The results of the azide binding reaction show that the process conforms to simple equilibrium as expected for a single site protein. The méasured association constant is reported as a function of pH. The kinetics of the reaction of Aplysia metMb with N3- minus shows, on the other hand, a complex behavior. The relaxation pattern is found to strongly depend on pH and ligand concentration in such a way to suggest a linkage between ligand binding and acid-alkaline transition. The system is discussed on the basis of two simplifying conditions, i.e., at low and higher pH with respect to the pK of the acid-alkaline transition. At acid pH the reaction corresponds to a single bimolecular process as expected for a simple binding reaction; at alkaline pH, the dependence of relaxation time on ligand concentration implies the existence of a rate-limiting monomolecular step. On the basis of a reaction scheme implying that binding of the ligand can only occur through the acid (aquomet) form of the protein via the displacement of the water molecule, the experimental data are quantitatively accounted for.     

The reaction between reduced azurin and oxidized cytochrome c peroxidase from Pseudomonas aeruginosa

The reaction between ferric Pseudomonas cytochrome c peroxidase and reduced azurin was investigated by static titration, rapid scan, and stopped flow techniques as well as circular dichroism measurements. Kinetics of the reduction of the enzyme under pseudo-first order conditions reveals a biphasic logarithmic curve indicating that the reaction between enzyme and azurin is complex and comprises of two reactions, one rapid and a slower one. The relative portion of the fast phase from the overall reaction increases with increasing azurin concentration. Kinetic results can be explained by postulating the presence of two different enzyme forms which are slowly interconvertible. Both enzymatic forms form a complex with reduced azurin. The electron transfer between proteins occurs within the molecular complex of azurin and the peroxidase.     

The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins.

An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.     

Some effects of low pH on chloride exchange in human red blood cells

In order to test the range of pH values over which the titratable carried model for inorganic anion exchange is valid, chloride self-exchange across human red blood cells was examined between pH 4.75 and 5.7 at 0 decrees c. It was found that chloride self-exchange flux had a minimum near pH 5 and increased again with further increase in hydrogen ion activity. The Arrhenius activation energy for chloride exchange was greatly reduced at low pH values. The chloride flux at pH 5.1 did not show the saturation kinetics reported at higher pH values but was proportional to the value of the chloride concentration squared. In addition, the extent of inhibition of chloride self-exchange flux by phloretin was reduced at low pH. Our interpretation of these findings is that the carrier-mediated flux becomes a progressively smaller fraction of the total flux at lower pH values and that a different transport mode requiring two chloride ions to form the permeant species and having a low specificity and temperature dependence becomes significant below pH5. A possible mechanism for this transport is that chloride crosses red cell membranes as dimers of HCl at these very low pH values.     

The kinetics of electron transfer between pseudomonas aeruginosa cytochrome c-551 and its oxidase.

The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.     

Photophysics of metalloazurins

The fluorescence lifetimes of Cu(II), Cu(I), Ag(I), Hg(II), Co(II), and Ni(II) azurin Pae from Pseudomonas aeruginosa and Cu(II), Cu(I), and Hg(II) azurin Afe from Alcaligenes faecalis were measured at 295 K by time-correlated single-photon counting. In addition, fluorescence lifetimes of Cu(II) azurin Pae were measured between 30 and 160 K and showed little change in value. Ultraviolet absorption difference spectra between metalloazurin Pae and apoazurin Pae were measured, as were the fluorescence spectra of metalloazurins. These spectra were used to determine the spectral overlap integral required for dipole-dipole resonance calculations. All metalloazurins exhibit a reduced fluorescence lifetime compared to their respective apoazurins. Forster electronic energy transfer rates were calculated for both metalloazurin Pae and metalloazurin Afe derivatives; both enzymes contain a single tryptophyl residue which is located in a different position in the two azurins. These azurins have markedly different fluorescence spectra, and electronic energy transfers occur from these two tryptophyl sites with different distances and orientations and spectral overlap integral values. Intramolecular distances and orientations were derived from an X-ray crystallographic structure and a molecular dynamic simulation of the homologous azurin Ade from Alcaligenes denitrificans, which contains both tryptophyl sites. Assignments were made of metal-ligand-field electronic transitions and of transition dipole moments and directions for tryptophyl residues, which accounted for the observed fluorescence quenching of Hg(II), Co(II), and Ni(II) azurin Pae and Cu(II) and Hg(II) azurin Afe. The fluorescence of azurin Pae is assigned as a 1Lb electronic transition, while that of azurin Afe is 1La. The marked fluorescence quenching of Cu(II) azurin Pae and Cu(I) azurin Pae and Afe is less well reproduced by our calculations, and long-range oxidative and reductive electron transfer, respectively, are proposed as additional quenching mechanisms. This study illustrates the application of Forster electronic energy transfer calculations to intramolecular transfers in structurally well characterized molecular systems and demonstrates its ability to predict observed fluorescence quenching rates when the necessary extensive structural, electronic transition assignment, and spectroscopic data are available. The agreement between Forster calculations and quenching rates derived from fluorescence lifetime measurements suggests there are limited changes in conformation between crystal structure and solution structures, with the exception of the tryptophyl residue of azurin Afe, where a conformation derived from a molecular simulation in water was necessary rather than that found in the crystal structure.     

A thermodynamic description of the binding of iron to ferrioxamine B in aqueous solutions

The iron exchange reaction between nitrilotriacetate (NTA) and ferrioxamine B (DFO) has been investigated with titration calorimetry. Using characterization models for the initial and final states of the reaction solution based on the determination of the mole fractions of the individual species as a function of pH, the concentration of each participating species was calculated (B. L. Gould, 1980, Masters Thesis, Utah State University, Logan). The contribution of each component reaction to the overall exchange at various pH values was used to determine the enthalpy change for each of these individual reactions. The values determined for the enthalpy and entropy changes are shown below. An analysis of the thermodynamic parameters of the iron exchange system indicated the following interactions among the hydroxamate groups of the ferrioxamine B: (1) The ionization of the individual hydroxamate protons occurs independently with the same enthalpy change for each ionization, and (2) the heat of binding a hydroxamate group to the iron is dependent on the number of previously bound hydroxamates.     

Determinants of intra versus intermolecular self-association within the regulatory domains of Rlk and Itk

A protein fragment from the Tec family member Rlk (also known as Txk) containing a single proline-rich ligand adjacent to a Src homology 3 (SH3) domain has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Analysis of the concentration dependence of the chemical shifts, NMR linewidths and self-diffusion coefficients reveal that the Rlk fragment dimerizes in solution. Mutation of two critical prolines in the proline-rich ligand abolishes dimerization. Furthermore, analysis of the extrapolated chemical shifts at infinite dilution reveal that intramolecular binding of the proline-rich ligand to the SH3 domain is disfavored. This is in contrast to the corresponding fragment of Itk, for which the proline-rich ligand/SH3 interaction occurs exclusively in an intramolecular fashion and no intermolecular binding is observed. Comparison of the Itk and Rlk sequences reveals that Rlk contains five fewer residues than Itk in the linker region between the proline-rich ligand and the SH3 domain. To assess whether linker length is a molecular determinant of intra- versus intermolecular self-association, we varied the length of the linker in both Rlk and Itk and analyzed the resulting variants by NMR. Intramolecular binding in Itk is reduced by shortening the linker and conversely a longer linker between the proline-rich ligand and the SH3 domain in Rlk enhances intramolecular self-association. Association constants for the binding of peptides corresponding to the proline-rich ligand with their respective SH3 domains were also measured by NMR. The protein/peptide data combined with the association constants for binding of each proline-rich peptide to the corresponding SH3 domain provide an explanation for the opposing modes of self-association within the otherwise closely related Rlk and Itk proteins.     

Electron transfer from quinohemoprotein alcohol dehydrogenase to blue copper protein azurin in the alcohol oxidase respiratory chain of Pseudomonas putida HK5.

A blue copper protein was purified together with a type II quinohemoprotein alcohol dehydrogenase (ADH IIB) from the soluble fraction of Pseudomonas putida HK5 grown on n-butanol. The purified blue copper protein was shown to be azurin, on the basis of several properties such as its absorption maximum (623 nm), its low molecular mass (17 500 Da), its acidic nature (pI of 4.1), its relatively high redox potential (306 mV), the presence of an intramolecular disulfide bond, and N-terminal amino acid sequence homology with respect to azurins from other sources, especially from P. putida NCIB 9869 and Pseudomonas fluorescens. Direct electron transfer from ADH IIB to azurin was shown to occur at a rate of 48-70 s-1. The apparent Km value of ADH IIB for azurin, determined by steady-state kinetics, was decreased several-fold by increasing the ionic strength. Furthermore, the extent of fluorescence quenching of ADH IIB due to the interaction with azurin was increased by increasing the ionic strength, but the binding constant for binding between ADH IIB and azurin was unchanged. The redox potential of azurin was increased 12 mV by incubation with ADH but not vice versa. Furthermore, the redox potential gap between ADH and azurin was increased from 102 to 126 mV by increasing the ionic strength. It is conceivable that a hydrophobic interaction is involved in the electron transfer between both proteins, and it is also suggested that the electron transfer may occur by a freely reversible on and off binding process but may not be related to the global binding process of both proteins. Thus, the results presented here strongly suggest that azurin works as an electron-transfer mediator in a PQQ-dependent alcohol oxidase respiratory chain in P. putida HK5.