Biomedical subjects

Gerard W Canters

Publications and source records attributed to Gerard W Canters.

At least 19 recordsLinked to original sources

PubMed2007

Effect of the methionine ligand on the reorganization energy of the type-1 copper site of nitrite reductase.

Copper-containing nitrite reductase harbors a type-1 and a type-2 Cu site. The former acts as the electron acceptor site of the enzyme, and the latter is the site of catalytic action. The effect of the methionine ligand on the reorganization energy of the type-1 site was explored by studying the electron-transfer kinetics between NiR (wild type (wt) and the variants Met150Gly and Met150Thr) with Fe(II)EDTA and Fe(II)HEDTA. The mutations increased the reorganization energy by 0.3 eV (30 kJ mol-1). A similar increase was found from pulse radiolysis experiments on the wt NIR and three variants (Met150Gly, Met150His, and Met150Thr). Binding of the nearby Met62 to the type-1 Cu site in Met150Gly (under influence of an allosteric effector) lowered the reorganization energy back to approximately the wt value. According to XRD data the structure of the reduced type-1 site in Met150Gly NiR in the presence of an allosteric effector is similar to that in the reduced wt NiR (solved to 1.85 A), compatible with the similarity in reorganization energy.

Binding Sites↗

PubMed2006

Spectroscopic characterization of a high-potential lipo-cupredoxin found in Streptomyces coelicolor.

For many streptomycetes, a distinct dependence on the "bioavailability" of copper ions for their morphological development has been reported. Analysis of the Streptomyces coelicolor genome reveals a number of gene products encoding for putative copper-binding proteins. One of these appears as an unusual copper-binding protein with a lipoprotein signal sequence and a cupredoxin-like domain harboring a putative Type-1 copper-binding motif. Cloning of this gene from S. coelicolor and subsequent heterologous expression in Escherichia coli has allowed for a thorough spectroscopic interrogation of this putative copper-binding protein. Optical and electron paramagnetic resonance spectroscopies have confirmed the presence of a "classic" Type-1 copper site with the axial ligand to the copper a methionine. Paramagnetic NMR spectroscopy on both the native Cu(II) form and Co(II)-substituted protein has yielded active-site structural information, which on comparison with that of other cupredoxin active sites reveals metal-ligand interactions most similar to the "classic" Type-1 copper site found in the amicyanin family of cupredoxins. Despite this high structural similarity, the Cu(II)/(I) midpoint potential of the S. coelicolor protein is an unprecedented +605 mV vs normal hydrogen electrode at neutral pH (amicyanin approximately +250 mV), with no active-site protonation of the N-terminal His ligand observed. Suggestions for the physiological role/function of this high-potential cupredoxin are discussed.

Amino Acid Sequence↗

PubMed2006

Paramagnetic properties of the halide-bound derivatives of oxidised tyrosinase investigated by 1H NMR spectroscopy.

The (1)H NMR relaxation characteristics of the histidines in the oxidised type-3 copper site of tyrosinase (Ty(met)) from the bacterium Streptomyces antibioticus in the halide-bound forms (Ty(met)X with X = F(-), Cl(-), Br(-)) have been determined and analysed. The (1)H NMR spectra of the Ty(met)X species display remarkably sharp, well-resolved, paramagnetically shifted (1)H signals, which originate from the protons of the six His residues coordinated to the two Cu(II) ions in the type-3 centre. From the temperature-dependence of the (1)H paramagnetic shifts the following values for the exchange-coupling parameter -2J were determined: 260 (Ty(met)F), 200 (Ty(met)Cl) and 162 cm(-1) (Ty(met)Br). The (1)H T(1) relaxation is dipolar in origin and correlates with the Cu--H distances. Electronic relaxation times tau(S) derived from the (1)H T(1) data amount to about 10(-11) s and follow the order Ty(met)F>Ty(met)Cl>Ty(met)Br. They are two orders of magnitude shorter than the tau(S) values reported for mononuclear copper systems, in accordance with the sharpness of the (1)H signals. The results corroborate the Cu(2) bridging mode of the halide ions. On the basis of the measured hyperfine interaction constants for the ligand histidine nuclei, it is concluded that 70-80 % of the spin density in the excited triplet state resides on the two copper ions and the bridging atoms.

Bacterial Proteins↗

PubMed2006

Spin-density distribution in the copper site of azurin.

A 95 GHz pulsed deuterium ENDOR study has been performed on single crystals of azurin from Pseudomonas aeruginosa selectively deuterated at the C(beta) position of the copper-coordinating cysteine 112. Complete hyperfine tensors of the two deuterium atoms have been obtained, which reveal identical isotropic parts. Analysis of the hyperfine tensors provides insight into the spin-density delocalization over the cysteine ligand. Approximately 45 % of the spin density in the paramagnetic site can be attributed to copper and 30 % to sulfur.

Anisotropy↗

PubMed2006

A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase.

The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO(-)(2)] reduction of the type-2 site precedes nitrite binding, at high [NO(-)(2)] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an approximately 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.

Alcaligenes faecalis↗

PubMed2006

A rearranging ligand enables allosteric control of catalytic activity in copper-containing nitrite reductase.

In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A(460)/A(600)=0.71) differs significantly from that of the native nitrite reductase (A(460)/A(600)=1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E(M)=312(+/-5)mV versus hydrogen) is higher than that of the native enzyme (E(M)=213(+/-5)mV). M150G has a lower catalytic activity (k(cat)=133(+/-6)s(-1)) than the wild-type nitrite reductase (k(cat)=416(+/-10)s(-1)). The binding of external ligands to M150G restores spectral properties, midpoint potential (E(M)<225mV), and catalytic activity (k(cat)=374(+/-28)s(-1)). Also the M150H (A(460)/A(600)=7.7, E(M)=104(+/-5)mV, k(cat)=0.099(+/-0.006)s(-1)) and M150T (A(460)/A(600)=0.085, E(M)=340(+/-5)mV, k(cat)=126(+/-2)s(-1)) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of copper-containing redox sites.

Alcaligenes faecalis↗

PubMed2005

Oxygen binding to tyrosinase from streptomyces antibioticus studied by laser flash photolysis.

Tyrosinases catalyze the o-hydroxylation of monophenols (monophenolase activity) and the oxidation of o-diphenols to o-quinones (diphenolase activity) and possess a dinuclear copper active site. The O2 binding kinetics of oxytyrosinase is studied by flash-photolysis measurements, and the O2 binding rate constant (kO2) is obtained as kO2 = 13 +/- 3 muM-1 s-1. Small molecules, such as carbon monoxide and p-nitrophenol (a substrate-analogue inhibitor), are demonstrated to affect O2 binding kinetics. The activation enthalpy of the rate-limiting step of O2 binding is calculated by the temperature dependence of kO2 to be 12.8 +/- 2.6 kcal/mol.

Bacterial Proteins↗

PubMed2005

A Förster-resonance-energy transfer-based method for fluorescence detection of the protein redox state.

A method for fluorescence detection of a protein's redox state based on resonance energy transfer from an attached fluorescence label to the prosthetic group of the redox protein is described and tested for proteins containing three types of prosthetic groups: a type-1 copper site (azurin, amicyanin, plastocyanin, and pseudoazurin), a heme group (cytochrome c550), and a flavin mononucleotide (flavodoxin). This method permits one to reliably distinguish between reduced and oxidized proteins and to perform potentiometric titrations at submicromolar concentrations.

Azurin↗

PubMed2005

The substrate-bound type 2 copper site of nitrite reductase: the nitrogen hyperfine coupling of nitrite revealed by pulsed EPR.

A pulsed electron paramagnetic resonance study has been performed on the type 2 copper site of nitrite reductase (NiR) from Alcaligenes faecalis. The H145A mutant, in which histidine 145 is replaced by alanine, was studied by ESEEM and HYSCORE experiments at 9 GHz on frozen solutions. This mutant contains a reduced type 1 copper site which allowed a selective investigation of the type 2 site of H145A and of its nitrite-bound form H145A (NO2(-)). The experiments yielded hyperfine and quadrupole parameters of the remote nitrogens of two of the histidines in the type 2 copper site of the protein and revealed the changes of these values induced by substrate binding (14NO2(-) and 15NO2(-)). The HYSCORE experiments displayed a signal of 15NO2(-) bound to H145A, from which hyperfine parameters of the nitrite nitrogen were estimated. The small isotropic hyperfine coupling, 0.36 MHz, of the nitrite nitrogen (14N) suggests that the substrate binds in an axial position to the copper in the type 2 site and that the molecular orbital containing the unpaired electron extends onto the substrate. This and other changes in the EPR parameters occurring after nitrite binding suggest a change in electronic structure of the site, which most likely prepares the site for the catalytic reaction. We propose that this change is essential for the reaction to occur.

Alcaligenes faecalis↗

PubMed2005

Sensitive detection of the redox state of copper proteins using fluorescence.

The blue copper protein azurin from Pseudomonas aeruginosa has been covalently labelled with the fluorescing dye Cy5. The optical spectrum of the azurin changes markedly with its redox state. These changes are reflected in the fluorescence intensity of the dye through fluorescence resonance energy transfer (FRET). This provides a sensitive way to monitor biological redox events. The method shown to work in the nanomolar range of protein concentrations, can be easily extended into the sub-nanomolar regime and holds promise for single-molecule detection.

Azurin↗

PubMed2005

Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase.

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The temperature-induced changes of the copper centres are characterized by optical spectroscopy and electron paramagnetic resonance spectroscopy, and by establishing the thermal stability by differential scanning calorimetry. The calorimetric profile of the enzyme shows a single endothermic peak with maximum heat absorption at T(m) approximately 100 degrees C, revealing an exceptional thermal stability. The thermal transition is irreversible and the scan rate dependence of the calorimetric trace indicates that the denaturation of NiR is kinetically controlled. The divergence of the activation energy values determined by different methods is used as a criterion for the inapplicability of the one-step irreversible model. The best fit of the DSC profiles is obtained when the classical Lumry-Eyring model, N<-->U-->F, is considered. The simulation results indicate that the irreversible step prevails on the reversible one. Moreover, it is found that the conformational changes within the type-1 copper environments precede the denaturation of the whole protein. No evidence of protein dissociation within the temperature range investigated was observed.

Alcaligenes faecalis↗

PubMed2005

Ligand loop effects on the free energy change of redox and pH-dependent equilibria in cupredoxins probed on amicyanin variants.

In this work, we have determined the thermodynamic parameters of the reduction of four different variants of Thiobacillus versutus amicyanin by electrochemical techniques. In addition, the thermodynamic parameters were determined of the low-pH conformational change involving protonation of the C-terminal histidine ligand and the concomitant dissociation of this histidine from the Cu(I) ion. In these variants, the native C-terminal loop containing the Cys, His, and Met copper ligands has been replaced with the corresponding polypeptide segments of Pseudomonas aeruginosa azurin, Populus nigra plastocyanin, Alcaligenes faecalis S-6 pseudoazurin, and Thiobacillus ferrooxidans rusticyanin. For the reduction reaction, each loop invariably holds an entropic "memory" of the mother protein. The thermodynamics of the low-pH transition vary in a fashion that is species-dependent. When present, the memory effect again shows a large entropic component. In particular, loop elongation tends to favor the formation of the Cu(I)-His bond (hence disfavors His protonation, yielding lower pK(a) values) probably due to an increased flexibility of the loop in the reduced state. Overall, it appears that both reduction and low-pH transition are loop-responsive processes. The spacing between the ligands mostly affects the change in the conformational freedom that accompanies the reaction.

Amino Acid Substitution↗

PubMed2005

The oxidation state of a protein observed molecule-by-molecule.

We report the observation of the redox state of the blue copper protein azurin on the single-molecule level. The fluorescence of a small fluorophore attached to the protein is modulated by the change in absorption of the copper center via fluorescence resonance energy transfer (FRET). In our model system, the fluorescence label Cy5 was coupled to azurin from Pseudomonas aeruginosa via cysteine K27C. The Cy5 fluorescence was partially quenched by the absorption of the copper center of azurin in its oxidized state. In the reduced state, absorption is negligible, and thus no quenching occurs. We report on single-molecule measurements, both in solution by using fluorescence correlation spectroscopy (FCS) combined with fluorescence intensity distribution analysis (FIDA), and on surfaces by using wide-field fluorescence microscopy.

Absorption↗

PubMed2005

Sensing nitrite through a pseudoazurin-nitrite reductase electron transfer relay.

Nitrite is converted to nitric oxide by haem or copper-containing enzymes in denitrifying bacteria during the process of denitrification. In designing an efficient biosensor, this enzymic turnover must be quantitatively assessed. The enzyme nitrite reductase from Alcaligenes faecalis contains a redox-active blue copper centre and a nonblue enzyme-active copper centre. It can be covalently tethered to modified gold-electrode surfaces in configurations in which direct electron transfer is possible. A surface cysteine mutant of the enzyme can be similarly immobilised on bare electroactive gold substrates. Under such circumstances, however, electron transfer cannot be effectively coupled with substrate catalytic turnover. In using either the natural redox partner, pseudoazurin, or ruthenium hexammine as an "electron-shuttle" or "conduit" between enzyme and a peptide-modified electrode surface, the coupling of electron transfer to catalysis can be utilised in the development of an amperometric nitrite sensor.

Azurin↗

PubMed2005

Interaction between the type-3 copper protein tyrosinase and the substrate analogue p-nitrophenol studied by NMR.

The interaction of the monooxygenating type-3 copper enzyme Tyrosinase (Ty) from Streptomyces antibioticus with its inhibitor p-nitrophenol (pnp) was studied by paramagnetic NMR methods. The pnp binds to oxidized Ty (Ty(met)) and its halide (F(-), Cl(-)) bound derivatives with a dissociation constant in the mM range. The Cu(2) bridging halide ion is not displaced upon the binding of pnp showing that the pnp does not occupy the Cu(2) bridging position. The binding of pnp to Ty(met) or Ty(met)Cl leads to localized changes in the type-3 (Cu-His(3))(2) coordination geometry reflecting a change in the coordination of a single His residue that, still, remains coordinated to Cu. The binding of pnp to Ty(met)Cl causes a decrease in the Cu(2) magnetic exchange parameter -2J from 200 cm(-)(1) in the absence to 150 +/- 10 cm(-)(1) in the presence of pnp. From the (1)H and (2)D NMR relaxation parameters of pnp bound to Ty(met), a structural model of pnp coordination to the Ty type-3 center could be derived. The model explains the absence of hydroxylase activity in the closely related type-3 copper protein catechol oxidase. The relevance of the experimental findings toward the Ty catalytic mechanism is discussed.

Copper↗

PubMed2005

Calculation of the redox potential of the protein azurin and some mutants.

Azurin from Pseudomonas aeruginosa is a small 128-residue, copper-containing protein. Its redox potential can be modified by mutating the protein. Free-energy calculations based on classical molecular-dynamics simulations of the protein and from mutants in aqueous solution at different pH values were used to compute relative redox potentials. The precision of the free-energy calculations with the lambda coupling-parameter approach is evaluated as function of the number and sequence of lambda values, the sampling time and initial conditions. It is found that the precision is critically dependent on the relaxation of hydrogen-bonding networks when changing the atomic-charge distribution due to a change of redox state or pH value. The errors in the free energies range from 1 to 10 k(B)T, depending on the type of process. Only qualitative estimates of the change in redox potential by protein mutation can be obtained.

Amino Acid Substitution↗

PubMed2005

The effects of ligand exchange and mobility on the peroxidase activity of a bacterial cytochrome c upon unfolding.

The effect on the heme environment upon unfolding Paracoccus versutus ferricytochrome c-550 and two site-directed variants, K99E and H118Q, has been assessed through a combination of peroxidase activity increase and one-dimensional NMR spectroscopy. At pH 4.5, the data are consistent with a low- to high-spin heme transition, with the K99E mutation resulting in a protein with increased peroxidase activity in the absence of or at low concentrations of denaturant. Furthermore, the mobility of the polypeptide chain at pH 4.5 for the wild-type protein has been monitored in the absence and presence of denaturant through heteronuclear NMR experiments. The results are discussed in terms of local stability differences between bacterial and mitochondrial cytochromes c that are inferred from peroxidase activity assays. At pH 7.0, a mixture of misligated heme states arising from protein-based ligands assigned to lysine and histidine is detected. At low denaturant concentrations, these partially unfolded misligated heme forms inhibit the peroxidase activity. Data from the K99E mutation at pH 7.0 indicate that K99 is not involved in heme misligation, whereas histidine coordination is proven by the data from the H118Q variant.

Amino Acid Substitution↗

PubMed2005

Solution structure of the second PDZ domain of the neuronal adaptor X11alpha and its interaction with the C-terminal peptide of the human copper chaperone for superoxide dismutase.

Protection against reactive oxygen species is provided by the copper containing enzyme superoxide dismutase 1 (SOD1). The copper chaperone CCS is responsible for copper insertion into apo-SOD1. This role is impaired by an interaction between the second PDZ domain (PDZ2alpha) of the neuronal adaptor protein X11alpha and the third domain of CCS (McLoughlin et al. (2001) J. Biol. Chem., 276, 9303-9307). The solution structure of the PDZ2alpha domain has been determined and the interaction with peptides derived from CCS has been explored. PDZ2alpha binds to the last four amino acids of the CCS protein (PAHL) with a dissociation constant of 91 +/- 2 microM. Peptide variants have been used to map the interaction areas on PDZ2alpha for each amino acid, showing an important role for the C-terminal leucine, in line with canonical PDZ-peptide interactions.

Adaptor Proteins, Signal Transducing↗