Temperature-jump and potentiometric studies on recombinant wild type and Y143F and Y254F mutants of Saccharomyces cerevisiae flavocytochrome b2: role of the driving force in intramolecular electron transfer kinetics.

The kinetics of intramolecular electron transfer between flavin and heme in Saccharomyces cerevisiae flavocytochrome b2 were investigated by performing potentiometric titrations and temperature-jump experiments on the recombinant wild type and Y143F and Y254F mutants. The midpoint potential of heme was determined by monitoring redox titrations spectrophotometrically, and that of semiquinone flavin/reduced flavin (Fsq/Fred) and oxidized flavin (Fox)/Fsq couples by electron paramagnetic resonance experiments at room temperature. The effects of pyruvate on the kinetic and thermodynamic parameters were also investigated. At room temperature, pH 7.0 and I = 0.1 M, the redox potential of the Fsq/Fred, Fox/Fsq, and oxidized heme/reduced heme (Hox/Hred) couples were -135, -45, and -3 mV, respectively, in the wild-type form. Although neither the mutations nor excess pyruvate did appreciably modify the potential of the heme or that of the Fsq/Fred couple, they led to variable positive shifts in the potential of the Fox/Fsq couple, thus modulating the driving force that characterizes the reduction of heme by the semiquinone in the -42 to +88 mV range. The relaxation rates measured at 16 degreesC in temperature-jump experiments were independent of the protein concentrations, with absorbance changes corresponding to the reduction of the heme. Two relaxation processes were clearly resolved in wild-type flavocytochrome b2 (1/tau1 = 1500 s-1, 1/tau2 = 200 +/- 50 s-1) and were assigned to the reactions whereby the heme is reduced by Fred and Fsq, respectively. The rate of the latter reaction was determined in the whole series of proteins. Its variation as a function of the driving force is well described by the expression obtained from electron-transfer theories, which provides evidence that the intramolecular electron transfer is not controlled by the dynamics of the protein.

N-terminal arm exchange is observed in the 2.15 A crystal structure of oxidized nitrite reductase from Pseudomonas aeruginosa.

BACKGROUND: Nitrite reductase from Pseudomonas aeruginosa (NiR-Pa) is a dimer consisting of two identical 60 kDa subunits, each of which contains one c and one d1 heme group. This enzyme, a soluble component of the electron-transfer chain that uses nitrate as a source of energy, can be induced by the addition of nitrate to the bacterial growth medium. NiR-Pa catalyzes the reduction of nitrite (NO2-) to nitric oxide (NO); in vitro, both cytochrome c551 and azurin are efficient electron donors in this reaction. NiR is a key denitrification enzyme, which controls the rate of the production of toxic nitric oxide (NO) and ultimately regulates the release of NO into the atmosphere. RESULTS: The structure of the orthorhombic form (P2(1)2(1)2) of oxidized NiR-Pa was solved at 2.15 A resolution, using molecular replacement with the coordinates of the NiR from Thiosphaera pantotropha (NiR-Tp) as the starting model. Although the d1-heme domains are almost identical in both enzyme structures, the c domain of NiR-Pa is more like the classical class I cytochrome-c fold because it has His51 and Met88 as heme ligands, instead of His17 and His69 present in NiR-Tp. In addition, the methionine-bearing loop, which was displaced by His17 of the NiR-Tp N-terminal segment, is back to normal in our structure. The N-terminal residues (5/6-30) of NiR-Pa and NiR-Tp have little sequence identity. In Nir-Pa, this N-terminal segment of one monomer crosses the dimer interface and wraps itself around the other monomer. Tyr10 of this segment is hydrogen bonded to an hydroxide ion--the sixth ligand of the d1-heme Fe, whereas the equivalent residue in NiR-Tp, Tyr25, is directly bound to the Fe. CONCLUSIONS: Two ligands of hemes c and d1 differ between the two known NiR structures, which accounts for the fact that they have quite different spectroscopic and kinetic features. The unexpected domain-crossing by the N-terminal segment of NiR-Pa is comparable to that of 'domain swapping' or 'arm exchange' previously observed in other systems and may explain the observed cooperativity between monomers of dimeric NiR-Pa. In spite of having similar sequence and fold, the different kinetic behaviour and the spectral features of NiR-Pa and NiR-Tp are tuned by the N-terminal stretch of residues. A further example of this may come from another NiR, from Pseudomonas stutzeri, which has an N terminus very different from that of the two above mentioned NiRs.

Electron-transfer properties of Pseudomonas aeruginosa [Lys44, Glu64]azurin.

In the hydrophobic patch of azurin from Pseudomonas aeruginosa, an electric dipole was created by changing Met44 into Lys and Met64 into Glu. The effect of this dipole on the electron-transfer properties of azurin was investigated. From a spectroscopic characterization (NMR, EPR and ultraviolet-visible) it was found that both the copper site and the overall structure of the [Lys44, Glu64]azurin were not disturbed by the two mutations. A small perturbation of the active site at high pH, similar to that observed for [Lys44]azurin, occurs in the double mutant. At neutral pH the electron-self-exchange rate constant of the double mutant shows a decrease of three orders of magnitude compared with the wild-type value. The possible reasons for this decrease are discussed. Electron transfer with the proposed physiological redox partners cytochrome c551 and nitrite reductase have been investigated and the data analyzed in the Marcus framework. From this analysis it is confirmed that the hydrophobic patch of azurin is the interaction site with both partners, and that cytochrome c551 uses its hydrophobic patch and nitrite reductase a negatively charged surface area for the electron transfer.

Identification of the prion protein allotypes which accumulate in the brain of sporadic and familial Creutzfeldt-Jakob disease patients.

A characteristic feature of Creutzfeldt-Jakob disease (CJD) is the accumulation in the brain of the amyloid protease-resistant protein PrPres. PrPres derives from a host-encoded, protease-sensitive isoform, PrPsen. Mutations of this protein are linked to familial variants of the disease, and the presence of a methionine or valine residue at the polymorphic position 129 may be critical in sporadic CJD cases. We found that in the brain of patients heterozygous for the mutation in which isoleucine is substituted for valine at codon 210 (Val21Olle), the PrPres is formed by both the wild-type and mutant PrPsen. We also found that in a sporadic CJD patient, who was heterozygous (Met/Val) at position 129, PrPres is also formed by both allotypes. These data associate transmissible spongiform encephalopathies with other amyloidosis, although the nature of the transmissible agent remains unsettled.

Expression and characterization of Pseudomonas aeruginosa cytochrome c-551 and two site-directed mutants: role of tryptophan 56 in the modulation of redox properties.

The gene coding for Pseudomonas aeruginosa cytochrome c-551 was expressed in Pseudomonas putida under aerobic conditions, using two different expression vectors; the more efficient proved to be pNM185, induced by m-toluate. Mature holo-(cytochrome c-551) was produced in high yield by this expression system, and was purified to homogeneity. Comparison of the recombinant wild-type protein with that purified from Ps. aeruginosa showed no differences in structural and functional properties. Trp56, an internal residue in cytochrome c-551, is located at hydrogen-bonding distance from haem propionate-17, together with Arg47. Ionization of propionate-17 was related to the observed pH-dependence of redox potential. The role of Trp56 in determining the redox properties of Ps. aeruginosa cytochrome c-551 was assessed by site-directed mutagenesis, by substitution with Tyr (W56Y) and Phe (W56F). The W56Y mutant is similar to the wild-type cytochrome. On the other hand, the W56F mutant, although similar to the wild-type protein in spectral properties and electron donation to azurin, is characterized by a weakening of the Fe-Met61 bond, as shown in the oxidized protein by the loss of the 695 nm band approx. 2 pH units below the wild-type. Moreover, in W56F, the midpoint potential and its pH-dependence are both different from the wild-type. These results are consistent with the hypothesis that hydrogen-bonding to haem propionate-17 is important in modulation of the redox properties of Ps. aeruginosa cytochrome c-551.

Electron transfer in zinc-reconstituted nitrite reductase from Pseudomonas aeruginosa.

1. The catalytic cycle of the haem-containing nitrite reductase (NIR) from Pseudomonas aeruginosa involves electron transfer between the two prosthetic groups of the enzyme, the c-haem and the d1-haem; this reaction was shown to be slow by stopped-flow analysis. The recombinant enzyme, expressed in Pseudomonas putida, contains the c-haem but no d1-haem; we have reconstituted this protein with Zn-protoporphyrin IX in the place of the d1-haem. 2. Photoexcitation of Zn-NIR is followed by electron transfer from the triplet excited state of the Zn-porphyrin to the oxidized c-haem, with a rate constant of 7 x 10(5) s-1; since the intermediate with reduced c-haem is not significantly populated, we conclude that the back reaction is probably as fast. 3. Even taking into account that in the native NIR the driving force is close to zero, the rate constant for the c-->d1 electron transfer, estimated from our experiments, is still much higher than that observed by stopped flow (k = 0.3 s-1) using reduced azurin as the electron donor. This finding may be a direct kinetic indication that reduction of the d1-haem is associated with a substantial reorganization of the co-ordination of the metal, as shown by spectroscopy of the oxidized and reduced NIR.

Isolation and characterization of the d1 domain of Pseudomonas aeruginosa nitrite reductase.

Proteolitic digestion of nitrite reductase from Pseudomonas aeruginosa allows to obtain and purify a domain containing only the d1 heme and constituted by two noncovalently bound peptides. This d1 domain catayzes oxygen consumption, and binds carbon monoxide with a kinetic constant slightly higher than the parental dimeric holoenzyme. The capacity to oxidize the physiological substrate, cytochrome c551, is lost, even when the proteolytic c heme domain is added to this reaction mixture. This finding suggests that the two domains do not have a significant affinity for each other, and are kept together only by being part of the same polypeptide.

Monomeric Pseudomonas aeruginosa nitrite reductase: preparation, characterization, and kinetic properties.

Monomeric nitrite reductase in an active form has been prepared by controlled succinylation of the dimeric native enzyme of Pseudomonas aeruginosa and subsequent purification. The monomeric enzyme has an optical spectrum indistinguishable from that of the native enzyme. On the other hand, circular dichroic spectra in the heme and peptide absorption regions show differences with respect to the dimer that indicate that the chemical modification and/or the dissociation into monomers somewhat perturb the chromophores' environment and the secondary structure. The (negatively charged) monomer is unable to oxidize its physiological substrates, azurin and cytochrome c551. This loss of activity is not due to monomerization, but is linked to the total net charge of the succinylated molecule, which interestingly enough acquires the ability to oxidize efficiently eukaryotic cytochrome c (which is not a substrate of the native dimeric enzyme). Stopped-flow studies show that the reduced monomer reacts with oxygen with a kinetic pattern similar to that shown by the dimeric enzyme. However, a higher reaction rate in the bimolecular binding of oxygen and a much higher oxygen affinity than for the native enzyme are observed. The evidence reported in this paper indicates that the dimeric state of Pseudomonas nitrite reductase is not a prerequisite for the ferrocytochrome c-oxygen oxidoreductase activity of this enzyme.

Reaction of the Hansenula anomala flavocytochrome b2 and cytochrome b2 core with inorganic outer sphere redox compounds.

The oxidation of reduced cytochrome b2 core and flavocytochrome b2 by three inorganic outer sphere compounds, Fe(CN)6(3-), Co(phen)3(3+) and Mn(CyDTA) (H2O)-, has been studied by stopped-flow. The reaction with Fe(CN)6(3-) is very rapid; the second order rate constants at 10 degrees C (pH 7) and I = 0.02 M are k = 1 x 10(8) M-1 s-1 and 1 x 10(7) M-1 s-1 for cytochrome b2 core and flavocytochrome b2, respectively. The reaction between cytochrome b2 core and Co(phen)3(3+), too fast at pH 7.0, has been characterized at 10 degrees C and pH 4.0; the second order rate constant is k = 2 x 10(7) M-1 s-1 and becomes 4 x 10(8) M-1 s-1 at pH 6.5. The reaction between flavocytochrome b2 and Co(phen)3(3+) has a second order rate constant k = 2 x 10(7) M-1 s-1 at pH 7.0, 10 degrees C. The oxidation of both proteins by Mn(CyDTA)(H2O)- is characterized by a second order rate constant k = 2.8 x 10(6) M-1 s-1 and 2.3 x 10(5) M-1 s-1 for cytochrome b2 core and flavocytochrome b2, respectively, at pH 7.0 and 10 degrees C. The reactivity of the b2 heme towards the outer sphere oxidants is higher than that reported for heme c in bacterial and eukaryotic cytochrome c. The larger delta E and the larger accessibility of the b2 heme can account for this result. The flavodehydrogenase domain seems to modulate the electron transfer also to these inorganic compounds, as found previously in the case of macromolecular electron acceptors.

Crystallization and preliminary X-ray analysis of a new crystal form of nitrite reductase from Pseudomonas aeruginosa.

Nitrite reductase from Pseudomonas aeruginosa (EC 1.9.3.2), a redox enzyme synthesized by the bacterium grown in the presence of nitrate, is a soluble dimer of two identical subunits of 60 kDa, each containing one c and one d1 haem as prosthetic groups. A new crystal from of the Ps. aeruginosa nitrite reductase in the oxidized state, suitable for X-ray structure determination, has been obtained by vapour diffusion at 20 degrees C, in the presence of 10% polyethylene glycol 4000, 50 mM Tris-HCl (pH 8.7), 400 mM NaCl and at a protein concentration of 14 mg/ml. The crystals are dark green elongated tetragonal prisms of dimensions 1.5 mm x 0.2 mm x 0.2 mm for the largest ones. These crystals are tetragonal with space group P4(1(3))2(1)2 and cell dimensions a = b = 128.2 A, c = 172.6 A. They diffract at least up to 2.8 A. Assuming a dimer in the asymmetric unit, the VM value is 2.95 A3/Da (58% of solvent).

Pseudomonas aeruginosa nitrite reductase (or cytochrome oxidase): an overview.

The biochemistry and molecular biology of nitrite reductase, a key enzyme in the dissimilatory denitrification pathway of Ps aeruginosa which reduces nitrite to NO, is reviewed in this paper. The enzyme is a non-covalent homodimer, each subunit containing one heme c and one heme d1. The reaction mechanisms of nitrite and oxygen reduction are discussed in detail, as well as the interaction of the enzyme with its macromolecular substrates, azurin and cytochrome c551. Special attention is paid to new structural information, such as the chemistry of the d1 prosthetic group and the primary sequence of the gene and the protein. Finally, results on the expression both in Ps aeruginosa and in heterologous systems are presented.

Expression in Escherichia coli of the flavin and the haem domains of Hansenula anomala flavocytochrome b2 (flavodehydrogenase and b2 core) and characterization of the recombinant proteins.

The flavin and haem domains of Hansenula anomala flavocytochrome b2 have been independently expressed in Escherichia coli. The flavin domain activity, studied only in the total cellular extract, owing to its instability, has characteristics very similar to those of the flavin domain obtained by proteolysis. The haem domain (r-core) has been purified to homogeneity and characterized in detail from spectroscopic and functional points of view. Spectral differences with respect to the domain produced by proteolysis (p-core) were found using resonance Raman and c.d. spectroscopy and have been interpreted in terms of changes in haem-protein interactions. However, this structural difference is functionally silent, since the r-core is able to reduce cytochrome c with the same efficiency as the proteolytic domain.

The nitrite reductase gene of Pseudomonas aeruginosa: effect of growth conditions on the expression and construction of a mutant by gene disruption.

The expression of nitrite reductase has been tested in a wild-type strain of Pseudomonas aeruginosa (Pao1) as a function of nitrate concentration under anaerobic and aerobic conditions. Very low levels of basal expression are shown under non-denitrifying conditions (i.e. absence of nitrate, in both aerobic and anaerobic conditions); anaerobiosis is not required for high levels of enzyme production in the presence of nitrate. A Pseudomonas aeruginosa strain, mutated in the nitrite reductase gene, has been obtained by gene replacement. This mutant, the first of this species described up to now, is unable to grow under anaerobic conditions in the presence of nitrate. The anaerobic growth can be restored by complementation with the wild-type gene.

Expression of Pseudomonas aeruginosa nitrite reductase in Pseudomonas putida and characterization of the recombinant protein.

Nitrite reductase from Pseudomonas aeruginosa has been successfully expressed in Pseudomonas putida. The purified recombinant enzyme contains haem c but no haem d1. Nonetheless, like the holoenzyme from Ps. aeruginosa, it is a stable dimer (molecular mass 120 kDa), and electron transfer to oxidized azurin is biphasic and follows bimolecular kinetics (k1 = 1.5 x 10(5) and k2 = 2.2 x 10(4) M-1.s-1). Unlike the chemically produced apoenzyme, recombinant nitrite reductase containing only haem c is water-soluble, stable at neutral pH and can be quantitatively reconstituted with haem d1, yielding a holoenzyme with the same properties as that expressed by Ps. aeruginosa (namely optical and c.d. spectra, molecular mass, cytochrome c551 oxidase activity and CO-binding kinetics).

Measurement of the concentration of amphotericin B in brain tissue of scrapie-infected hamsters with a simple and sensitive method.

A simple, sensitive, and reproducible assay for the measurement of the amphotericin B concentration in tissue extracts was developed by using the fourth derivative of the absorption spectrum of amphotericin B between wavelengths of 330 and 430 nm. The amphotericin B concentration in spleen and brain was proportional to the total amount administered. The amphotericin B concentration in the brain was highly correlated with the increase in the mean incubation period of intracerebrally scrapie-infected hamsters.

Involvement of the hydrophobic patch of azurin in the electron-transfer reactions with cytochrome C551 and nitrite reductase.

The electron-transfer reactions of site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c551 and nitrite reductase were investigated by temperature-jump and stopped-flow experiments. In the hydrophobic patch of azurin Met44 was replaced by Lys, and in the His35 patch His35 was replaced by Phe, Leu and Gln. Both patches were previously thought to be involved in electron transfer. 1H-NMR spectroscopy revealed only minor changes in the three-dimensional structure of the mutants compared to wild-type azurin. Observed changes in midpoint potentials could be attributed to electrostatic effects. The slow relaxation phase observed in temperature-jump experiments carried out on equilibrium mixtures of wild-type azurin and cytochrome c551 was definitively shown to be due to a conformational relaxation involving His35. Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions with both cytochrome c551 and nitrite reductase.

The reaction of Pseudomonas nitrite reductase and nitrite. A stopped-flow and EPR study.

The reaction between reduced Pseudomonas nitrite reductase and nitrite has been studied by stopped-flow and rapid-freezing EPR spectroscopy. The interpretation of the kinetics at pH 8.0 is consistent with the following reaction mechanism (where k1 and k3 much greater than k2). [formula: see text] The bimolecular step (Step 1) is very fast, being lost in the dead time of a rapid mixing apparatus; the stoichiometry of the complex has been estimated to correspond to one NO2- molecule/heme d1. The final species is the fully reduced enzyme with NO bound to heme d1; and at all concentrations of nitrite, there is no evidence for dissociation of NO or for further reduction of NO to N2O. Step 2 is assigned to an internal electron transfer from heme c to reduced NO-bound heme d1 occurring with a rate constant of 1 s-1; this rate is comparable to the rate of internal electron transfer previously determined when reducing the oxidized enzyme with azurin or cytochrome c551. When heme d1 is NO-bound, the rate at which heme c can accept electrons from ascorbate is remarkably increased as compared to the oxidized enzyme, suggesting an increase in the redox potential of the latter heme.

Silver binding to Pseudomonas aeruginosa azurin.

The interaction between azurin and silver ions was investigated, by means of ultraviolet, fluorescence and atomic absorption spectroscopies, as a function of the redox state of the protein. The Ag(I) ion has a very low affinity for oxidized azurin. Interestingly, the affinity is much higher for reduced azurin; in this case Ag(I) completely displaces the Cu(I) ion from the native binding site. The effect is very specific for silver ions since other ions, such as Hg(II), Ni(II) and Cd(II), do not produce the same effect. Treatment of reduced and oxidized azurin with excess Ag(I) (2-8-fold stoichiometric) shows that there is a second binding site for silver ions on the protein which can also bind Cu(II) and Hg(II) with comparable affinities.

Nitrite reductase from Pseudomonas aeruginosa: sequence of the gene and the protein.

The gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino-terminus of the mature protein is located at Lys-26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60,204 Da. The c-heme-binding domain, which contains the only two Cys of the molecule, is located at the amino-terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c-heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data.