Reinhard Lange - Academia.edu (original) (raw)

Papers by Reinhard Lange

Journal of Biological Chemistry, 1998

The reaction of reduced NO synthase (NOS) with molecular oxygen was studied at ؊30°C. In the abse... more The reaction of reduced NO synthase (NOS) with molecular oxygen was studied at ؊30°C. In the absence of substrate, the complex formed between ferrous NOS and O 2 was sufficiently long lived for a precise spectroscopic characterization. This complex displayed similar spectral characteristics as the oxyferrous complex of cytochrome P450 ( max ‫؍‬ 416.5 nm). It then decomposed to the ferric state. The oxidation of the flavin components was much slower and could be observed only at temperatures higher than ؊20°C. In the presence of substrate (L-arginine), another, 12-nm blue-shifted, intermediate spectrum was formed. The breakdown of the latter species resulted in the production of N -hydroxy-L-arginine in a stoichiometry of maximally 52% per NOS heme. This product formation took place also in the absence of the reductase domain of NOS. Both formation of the blueshifted intermediate and of N -hydroxy-L-arginine required the presence of tetrahydrobiopterin (BH 4 ). We propose that the blue-shifted intermediate is the result of reductive activation of the oxygenated complex, and the electron is provided by BH 4 . These observations suggest that the reduction of the oxyferroheme complex may be the main function of BH 4 in NOS catalysis.

Biochimica et Biophysica Acta (BBA) - General Subjects, 2003

Annals of the New York Academy of Sciences, 2010

A wide range of parameters influence allosteric communications between the alpha- and beta-subuni... more A wide range of parameters influence allosteric communications between the alpha- and beta-subunits of the Trp synthase alpha(2)beta(2) multienzyme complex with L-Ser, including monovalent cations, pH, temperature, ligands, organic solvents, and hydrostatic pressure. The conformational change from closed to open can be monitored either by absorbance at 423 nm or fluorescence at 495 nm from the pyridoxal-5'-phosphate-L-Ser complex. Pressure perturbation was used to quantify the effects of monovalent cations, ligands, and mutations on the conformational equilibrium of Trp synthase. P-jump kinetics in the presence of Na(+), NH(4) (+), and Na(+) together with benzimidazole were also examined. The plots of lnk versus P are nonlinear and require a compressibility (beta(double dagger) (o)) term to obtain a good fit. beta(double dagger) (o) is positive for the Na(+) enzyme but negative for NH(4) (+) and Na(+) with benzimidazole. These results suggest that there is a large contribution of solvation to the kinetics of the conformational change of Trp synthase. The relaxation kinetics are also different if the P-jumps are made by increasing or decreasing pressure, suggesting that the enzyme conformations are ensembles of microstates.

Comparative Biochemistry and Physiology Part A: Physiology, 1997

ABSTRACT In this paper we review some basic facts about the reversible and irreversible effects o... more ABSTRACT In this paper we review some basic facts about the reversible and irreversible effects of high pressure on proteins. The effects include changes in intra- or intermolecular interactions (noncovalent bonds), in conformation and in solvation. Particular attention is directed to the interpretation of data where pressure-temperature dependency is an important phenomenon. Using model reactions, we have formulated a putative interpretation of physiological problems; we use these to explain how biological systems maintain activity when the temperature decreases and pressure increases, as in the case of barophilic micro-organisms in the deep sea world.

The Journal of biological chemistry, Jan 14, 2015

Protein oligomerization has been associated with a wide range of diseases. High pressure approach... more Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct β-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation...

European Journal of Biochemistry, 1977

The spin state of camphor-bound cytochrome P-450 is shown to depend largely on medium and tempera... more The spin state of camphor-bound cytochrome P-450 is shown to depend largely on medium and temperature in aqueous as well as in mixed organic buffer. At sub-zero temperatures a variation of pa,, ionic strength or camphor concentration modifies the spin equilibrium from nearly pure high-spin form to nearly pure low-spin form. Since the apparent pK, of transition is a linear function of log I, the spin state seems to be controlled by the electrostatic potential in the heme proximity. K' is found to have a specific effect on the spin state.

Progress in Biotechnology, 1996

... Methanol dehydrogenase proved to be extremely stable towards high pressure. The cohesion of t... more ... Methanol dehydrogenase proved to be extremely stable towards high pressure. The cohesion of the quaternary structure of this protein appears to be reinforced by high pressure. ... 3 R. Ragone, G. Colonna, C. Balestrieri, L. Servillo and G. Irace, Biochemistry 23 (1984) 1871. ...

Biochimica et biophysica acta, Jan 25, 2002

High hydrostatic pressure affects proteins, changing their intra- or intermolecular interactions,... more High hydrostatic pressure affects proteins, changing their intra- or intermolecular interactions, conformation and solvation. How to detect these changes? In this paper, via some selected examples, we show the potentiality (but also the limits) of the ultraviolet derivative spectroscopy specially adapted to high pressure experiments.

Journal of Steroid Biochemistry, 1989

The effect of Troleandomycin (TAO) and pregnenolone 16 alpha-carbonitrile (PCN) on the hepatic mi... more The effect of Troleandomycin (TAO) and pregnenolone 16 alpha-carbonitrile (PCN) on the hepatic microsomal progesterone metabolism in the rat is evaluated. Over thirteen hydroxylated progesterone derivatives are detected, including the novel 6 beta, 21-, 6 beta, 16 alpha-, 6 beta, 16 beta- and 2,21-dihydroxy derivatives, suggesting the induction of several cytochrome P-450 isozymes. PCN treatment results overall in an augmented production of progesterone metabolites whereas TAO treatment both induces and represses specific hydroxylase activities. Progesterone metabolism with purified isozymes isolated from liver microsomes from TAO and PCN treated rats differs significantly from that observed with intact microsomes, reflecting the complexity of the induction pattern of the cytochrome P-450 III family.

Although the x-ray crystallography is giving a relatively precise picture of spatial arrangement ... more Although the x-ray crystallography is giving a relatively precise picture of spatial arrangement of the majority of pro- tein structure, it is not able to give detailed information on the flexibility of the protein active site. The properties of ac- tive sites of cytochromes P450 (CYP) were supposed earlier to be relatively similar, reflecting the substrate specificities of the individual

Methods in enzymology, 2002

... High pressure: A new tool to study P450 structure and function. Frédéric Bancel, Gaston Hui B... more ... High pressure: A new tool to study P450 structure and function. Frédéric Bancel, Gaston Hui Bon Hoa, Pavel Anzenbacher, Claude Balny, Reinhard Lange. ... 252, 166 (1998). 25 j. Hudecek, E. Anzenbacherovfi, P. Anzenbacher, AW Munro, and P. Hildebrandt, Arch. Biochem. ...

European Journal of Biochemistry, 1997

A previous thermodynamic study [Lange, R., Larroque, C. & Anzenbacher, P. (1992) Eur. J. ... more A previous thermodynamic study [Lange, R., Larroque, C. & Anzenbacher, P. (1992) Eur. J. Biochem. 207, 69-73] demonstrated two conformations (A and B) of cytochrome P-450scc (SCC), the enzyme which initiates steroid biosynthesis by cleaving the side chain of cholesterol. The conformation found at the lowest temperatures (form A) displays a six-ligand high-spin heme iron [Hildebrandt, P., Heibel, G., Anzenbacher, P., Lange, R., Krüger, V. & Stier, A. (1994) Biochemistry 33, 12920-12929]. Analytical centrifugation shows that the oligomeric composition of SCC is the same for the A and the B conformers. However, as revealed by fourth-derivative ultraviolet spectroscopy, the two conformers differ in the mean environment of the tryptophan residues, which was more polar in the A form. The structural role of water in these two conformations was investigated using the pressure-jump technique under various pH, temperature and osmotic-stress conditions. Applying hydrostatic pressure to SCC induced very slow (tau >30 min) biexponential relaxation kinetics corresponding to the high-spin to low-spin transition. Analysis of the activation volumes suggested a dissociative mechanism for the A conformer (+45 ml/mol), and an associative mechanism for the B conformer (-39 ml/mol). Applying osmotic stress to the A form changed its kinetic characteristics to those of the B form. These results are consistent with a model comprising a solvent intake (ten water molecules) between the B and the A conformers and protonation of their respective high-spin states. The sixth ligand of the high-spin form in the A conformer involves a water molecule and an unknown constraining structure.

The reaction of reduced NO synthase (NOS) with mo- lecular oxygen was studied at 230 °C. In the a... more The reaction of reduced NO synthase (NOS) with mo- lecular oxygen was studied at 230 °C. In the absence of substrate, the complex formed between ferrous NOS and O2 was sufficiently long lived for a precise spectroscopic characterization. This complex displayed similar spec- tral characteristics as the oxyferrous complex of cyto- chrome P450 (lmax 5 416.5 nm). It then decomposed

ChemInform, 2009

Physical chemistry Z 0225 Asymmetric Kinetics of Protein Structural Changes -[42 refs.]. -(MARCHA... more Physical chemistry Z 0225 Asymmetric Kinetics of Protein Structural Changes -[42 refs.]. -(MARCHAL, S.; FONT, J.; RIBO, M.; VILANOVA, M.; PHILLIPS, R. S.; LANGE*, R.; TORRENT, J.; Acc. Chem. Res. 42 (2009) 6, 778-787; INSERM, Univ. Montpellier, F-34095 Montpellier, Fr.; Eng.) -Koehler 37-262

Methods in enzymology, 2005

The role of tetrahydrobiopterin (BH4) as a cofactor in nitric oxide synthase (NOS) has been the o... more The role of tetrahydrobiopterin (BH4) as a cofactor in nitric oxide synthase (NOS) has been the object of intense research in the last few years. It was found that in addition to its established effects on the NOS heme spin state, substrate affinity, and enzyme dimerization, BH4 is required as a one-electron donor to oxyferrous [Fe(II).O2] heme that is formed as an intermediate in the catalytic cycle. Cryogenic spectroscopic techniques proved particularly useful in the identification of this role of BH4 in NO synthesis. With these methods, the mechanism of fast reactions, such as the reaction of ferrous NOS with O2, can be unraveled by lowering the reaction temperature to subzero values. This may not only reduce the rate to such an extent that the reaction can be followed on a time scale from seconds to minutes, but intermediates may be observed that do not accumulate at higher temperatures. Cryogenic ultraviolet-visible (UV-vis) and electron paramagnetic resonance spectroscopy have...

The Journal of Physical Chemistry B, 2009

In this work, we investigated the effect of pressure on the structure and stability of the recomb... more In this work, we investigated the effect of pressure on the structure and stability of the recombinant D-trehalose/D-maltose-binding protein isolated from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). The spectroscopic results obtained both in the absence and in the presence of maltose or trehalose revealed that the TMBP-Mal complex exhibits a larger structural stability under high pressure values than TMBP-Tre complex. In addition, the results also pointed out that pressure induces reversible denaturation transitions of the protein structure. By combining the fluorescence results obtained with 8-anilino-1-naphtalene sulfonate as extrinsic probe and the intrinsic indolic fluorescence of TMBP, it is evident that the protein structural changes above 400 MPa that involve the exposure to the solvent of a large portion of the hydrophobic protein domains are preceded by a partially unfolded protein structural state. The spectroscopic results have been interpreted and discussed by taking into account the X-ray structure of the protein and, in particular, the interactions of maltose and trehalose within the three-dimensional structure of TMBP.

European Journal of Biochemistry, 1988

The cholesterol analogue 25-doxyl-27-nor-cholesterol (CNO), was found to be a substrate for cytoc... more The cholesterol analogue 25-doxyl-27-nor-cholesterol (CNO), was found to be a substrate for cytochrome P-450scc. Upon incubation with the cytochrome P-450scc electron transfer system, CNO is transformed to pregnenolone (Km = 33 microM, Vmax = 0.32 min-1). The pregnenolone formation from endogenous cholesterol is strongly inhibited by CNO (50% at 5 microM). It binds tightly to cytochrome P-450scc as evidenced by a reversed type I spectral absorbance change (Kd = 5.9 microM) which is paralleled by a greater hyperfine splitting of the room-temperature CNO ESR spectrum due to an enhanced probe immobilization (Kd = 1.9 microM). This finding is in accord with a rotational correlation time of about 10(-7) s, which is close to the tumbling rate of the protein. At 110 K the CNO-bound cytochrome P-450scc displays the ESR g-values gx = 2.404/2.456, gy = 2.245 and gz = 1.916; these are different from those of cholesterol-liganded cytochrome P-450scc and may thus serve as a marker for cytochrome P-450scc. Our data indicate that the stereospecificity of the cytochrome P-450scc side-chain-cleaving activity is not dependent on the nature of the cholesterol side-chain termination (C25 to C27). The substrate binding site is however rather sensitive to a modification of the side chain. The doxyl ring confers a stronger affinity of the substrate to the enzyme. Upon binding it becomes embedded in the protein matrix, and we estimate that its final position is 0.6-1.0 nm from the heme moiety.

European Journal of Biochemistry, 1979

Variations of the spin state in camphor-bound cytochrome P-450 are interpreted in the light of th... more Variations of the spin state in camphor-bound cytochrome P-450 are interpreted in the light of the polyelectrolyte theory and its implications on the microenvironment of the heme. The ratio of high-spin to low-spin iron can serve as a tool to determine the local paH in the microenvironment of a group (pK0, app approximately 5.4) which governs the spin state. The local paH depends on the electrostatic potential created by negatively charged groups (pKa = 5.6), modulated in turn by paH and by the screening effect of ionic strength. A model is given for the proton-coupled spin state change.

Journal of Biological Chemistry, 1998

The reaction of reduced NO synthase (NOS) with molecular oxygen was studied at ؊30°C. In the abse... more The reaction of reduced NO synthase (NOS) with molecular oxygen was studied at ؊30°C. In the absence of substrate, the complex formed between ferrous NOS and O 2 was sufficiently long lived for a precise spectroscopic characterization. This complex displayed similar spectral characteristics as the oxyferrous complex of cytochrome P450 ( max ‫؍‬ 416.5 nm). It then decomposed to the ferric state. The oxidation of the flavin components was much slower and could be observed only at temperatures higher than ؊20°C. In the presence of substrate (L-arginine), another, 12-nm blue-shifted, intermediate spectrum was formed. The breakdown of the latter species resulted in the production of N -hydroxy-L-arginine in a stoichiometry of maximally 52% per NOS heme. This product formation took place also in the absence of the reductase domain of NOS. Both formation of the blueshifted intermediate and of N -hydroxy-L-arginine required the presence of tetrahydrobiopterin (BH 4 ). We propose that the blue-shifted intermediate is the result of reductive activation of the oxygenated complex, and the electron is provided by BH 4 . These observations suggest that the reduction of the oxyferroheme complex may be the main function of BH 4 in NOS catalysis.

Biochimica et Biophysica Acta (BBA) - General Subjects, 2003

Annals of the New York Academy of Sciences, 2010

A wide range of parameters influence allosteric communications between the alpha- and beta-subuni... more A wide range of parameters influence allosteric communications between the alpha- and beta-subunits of the Trp synthase alpha(2)beta(2) multienzyme complex with L-Ser, including monovalent cations, pH, temperature, ligands, organic solvents, and hydrostatic pressure. The conformational change from closed to open can be monitored either by absorbance at 423 nm or fluorescence at 495 nm from the pyridoxal-5'-phosphate-L-Ser complex. Pressure perturbation was used to quantify the effects of monovalent cations, ligands, and mutations on the conformational equilibrium of Trp synthase. P-jump kinetics in the presence of Na(+), NH(4) (+), and Na(+) together with benzimidazole were also examined. The plots of lnk versus P are nonlinear and require a compressibility (beta(double dagger) (o)) term to obtain a good fit. beta(double dagger) (o) is positive for the Na(+) enzyme but negative for NH(4) (+) and Na(+) with benzimidazole. These results suggest that there is a large contribution of solvation to the kinetics of the conformational change of Trp synthase. The relaxation kinetics are also different if the P-jumps are made by increasing or decreasing pressure, suggesting that the enzyme conformations are ensembles of microstates.

Comparative Biochemistry and Physiology Part A: Physiology, 1997

ABSTRACT In this paper we review some basic facts about the reversible and irreversible effects o... more ABSTRACT In this paper we review some basic facts about the reversible and irreversible effects of high pressure on proteins. The effects include changes in intra- or intermolecular interactions (noncovalent bonds), in conformation and in solvation. Particular attention is directed to the interpretation of data where pressure-temperature dependency is an important phenomenon. Using model reactions, we have formulated a putative interpretation of physiological problems; we use these to explain how biological systems maintain activity when the temperature decreases and pressure increases, as in the case of barophilic micro-organisms in the deep sea world.

The Journal of biological chemistry, Jan 14, 2015

Protein oligomerization has been associated with a wide range of diseases. High pressure approach... more Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct β-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation...

European Journal of Biochemistry, 1977

The spin state of camphor-bound cytochrome P-450 is shown to depend largely on medium and tempera... more The spin state of camphor-bound cytochrome P-450 is shown to depend largely on medium and temperature in aqueous as well as in mixed organic buffer. At sub-zero temperatures a variation of pa,, ionic strength or camphor concentration modifies the spin equilibrium from nearly pure high-spin form to nearly pure low-spin form. Since the apparent pK, of transition is a linear function of log I, the spin state seems to be controlled by the electrostatic potential in the heme proximity. K' is found to have a specific effect on the spin state.

Progress in Biotechnology, 1996

... Methanol dehydrogenase proved to be extremely stable towards high pressure. The cohesion of t... more ... Methanol dehydrogenase proved to be extremely stable towards high pressure. The cohesion of the quaternary structure of this protein appears to be reinforced by high pressure. ... 3 R. Ragone, G. Colonna, C. Balestrieri, L. Servillo and G. Irace, Biochemistry 23 (1984) 1871. ...

Biochimica et biophysica acta, Jan 25, 2002

High hydrostatic pressure affects proteins, changing their intra- or intermolecular interactions,... more High hydrostatic pressure affects proteins, changing their intra- or intermolecular interactions, conformation and solvation. How to detect these changes? In this paper, via some selected examples, we show the potentiality (but also the limits) of the ultraviolet derivative spectroscopy specially adapted to high pressure experiments.

Journal of Steroid Biochemistry, 1989

The effect of Troleandomycin (TAO) and pregnenolone 16 alpha-carbonitrile (PCN) on the hepatic mi... more The effect of Troleandomycin (TAO) and pregnenolone 16 alpha-carbonitrile (PCN) on the hepatic microsomal progesterone metabolism in the rat is evaluated. Over thirteen hydroxylated progesterone derivatives are detected, including the novel 6 beta, 21-, 6 beta, 16 alpha-, 6 beta, 16 beta- and 2,21-dihydroxy derivatives, suggesting the induction of several cytochrome P-450 isozymes. PCN treatment results overall in an augmented production of progesterone metabolites whereas TAO treatment both induces and represses specific hydroxylase activities. Progesterone metabolism with purified isozymes isolated from liver microsomes from TAO and PCN treated rats differs significantly from that observed with intact microsomes, reflecting the complexity of the induction pattern of the cytochrome P-450 III family.

Although the x-ray crystallography is giving a relatively precise picture of spatial arrangement ... more Although the x-ray crystallography is giving a relatively precise picture of spatial arrangement of the majority of pro- tein structure, it is not able to give detailed information on the flexibility of the protein active site. The properties of ac- tive sites of cytochromes P450 (CYP) were supposed earlier to be relatively similar, reflecting the substrate specificities of the individual

Methods in enzymology, 2002

... High pressure: A new tool to study P450 structure and function. Frédéric Bancel, Gaston Hui B... more ... High pressure: A new tool to study P450 structure and function. Frédéric Bancel, Gaston Hui Bon Hoa, Pavel Anzenbacher, Claude Balny, Reinhard Lange. ... 252, 166 (1998). 25 j. Hudecek, E. Anzenbacherovfi, P. Anzenbacher, AW Munro, and P. Hildebrandt, Arch. Biochem. ...

European Journal of Biochemistry, 1997

A previous thermodynamic study [Lange, R., Larroque, C. & Anzenbacher, P. (1992) Eur. J. ... more A previous thermodynamic study [Lange, R., Larroque, C. & Anzenbacher, P. (1992) Eur. J. Biochem. 207, 69-73] demonstrated two conformations (A and B) of cytochrome P-450scc (SCC), the enzyme which initiates steroid biosynthesis by cleaving the side chain of cholesterol. The conformation found at the lowest temperatures (form A) displays a six-ligand high-spin heme iron [Hildebrandt, P., Heibel, G., Anzenbacher, P., Lange, R., Krüger, V. & Stier, A. (1994) Biochemistry 33, 12920-12929]. Analytical centrifugation shows that the oligomeric composition of SCC is the same for the A and the B conformers. However, as revealed by fourth-derivative ultraviolet spectroscopy, the two conformers differ in the mean environment of the tryptophan residues, which was more polar in the A form. The structural role of water in these two conformations was investigated using the pressure-jump technique under various pH, temperature and osmotic-stress conditions. Applying hydrostatic pressure to SCC induced very slow (tau >30 min) biexponential relaxation kinetics corresponding to the high-spin to low-spin transition. Analysis of the activation volumes suggested a dissociative mechanism for the A conformer (+45 ml/mol), and an associative mechanism for the B conformer (-39 ml/mol). Applying osmotic stress to the A form changed its kinetic characteristics to those of the B form. These results are consistent with a model comprising a solvent intake (ten water molecules) between the B and the A conformers and protonation of their respective high-spin states. The sixth ligand of the high-spin form in the A conformer involves a water molecule and an unknown constraining structure.

The reaction of reduced NO synthase (NOS) with mo- lecular oxygen was studied at 230 °C. In the a... more The reaction of reduced NO synthase (NOS) with mo- lecular oxygen was studied at 230 °C. In the absence of substrate, the complex formed between ferrous NOS and O2 was sufficiently long lived for a precise spectroscopic characterization. This complex displayed similar spec- tral characteristics as the oxyferrous complex of cyto- chrome P450 (lmax 5 416.5 nm). It then decomposed

ChemInform, 2009

Physical chemistry Z 0225 Asymmetric Kinetics of Protein Structural Changes -[42 refs.]. -(MARCHA... more Physical chemistry Z 0225 Asymmetric Kinetics of Protein Structural Changes -[42 refs.]. -(MARCHAL, S.; FONT, J.; RIBO, M.; VILANOVA, M.; PHILLIPS, R. S.; LANGE*, R.; TORRENT, J.; Acc. Chem. Res. 42 (2009) 6, 778-787; INSERM, Univ. Montpellier, F-34095 Montpellier, Fr.; Eng.) -Koehler 37-262

Methods in enzymology, 2005

The role of tetrahydrobiopterin (BH4) as a cofactor in nitric oxide synthase (NOS) has been the o... more The role of tetrahydrobiopterin (BH4) as a cofactor in nitric oxide synthase (NOS) has been the object of intense research in the last few years. It was found that in addition to its established effects on the NOS heme spin state, substrate affinity, and enzyme dimerization, BH4 is required as a one-electron donor to oxyferrous [Fe(II).O2] heme that is formed as an intermediate in the catalytic cycle. Cryogenic spectroscopic techniques proved particularly useful in the identification of this role of BH4 in NO synthesis. With these methods, the mechanism of fast reactions, such as the reaction of ferrous NOS with O2, can be unraveled by lowering the reaction temperature to subzero values. This may not only reduce the rate to such an extent that the reaction can be followed on a time scale from seconds to minutes, but intermediates may be observed that do not accumulate at higher temperatures. Cryogenic ultraviolet-visible (UV-vis) and electron paramagnetic resonance spectroscopy have...

The Journal of Physical Chemistry B, 2009

In this work, we investigated the effect of pressure on the structure and stability of the recomb... more In this work, we investigated the effect of pressure on the structure and stability of the recombinant D-trehalose/D-maltose-binding protein isolated from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). The spectroscopic results obtained both in the absence and in the presence of maltose or trehalose revealed that the TMBP-Mal complex exhibits a larger structural stability under high pressure values than TMBP-Tre complex. In addition, the results also pointed out that pressure induces reversible denaturation transitions of the protein structure. By combining the fluorescence results obtained with 8-anilino-1-naphtalene sulfonate as extrinsic probe and the intrinsic indolic fluorescence of TMBP, it is evident that the protein structural changes above 400 MPa that involve the exposure to the solvent of a large portion of the hydrophobic protein domains are preceded by a partially unfolded protein structural state. The spectroscopic results have been interpreted and discussed by taking into account the X-ray structure of the protein and, in particular, the interactions of maltose and trehalose within the three-dimensional structure of TMBP.

European Journal of Biochemistry, 1988

The cholesterol analogue 25-doxyl-27-nor-cholesterol (CNO), was found to be a substrate for cytoc... more The cholesterol analogue 25-doxyl-27-nor-cholesterol (CNO), was found to be a substrate for cytochrome P-450scc. Upon incubation with the cytochrome P-450scc electron transfer system, CNO is transformed to pregnenolone (Km = 33 microM, Vmax = 0.32 min-1). The pregnenolone formation from endogenous cholesterol is strongly inhibited by CNO (50% at 5 microM). It binds tightly to cytochrome P-450scc as evidenced by a reversed type I spectral absorbance change (Kd = 5.9 microM) which is paralleled by a greater hyperfine splitting of the room-temperature CNO ESR spectrum due to an enhanced probe immobilization (Kd = 1.9 microM). This finding is in accord with a rotational correlation time of about 10(-7) s, which is close to the tumbling rate of the protein. At 110 K the CNO-bound cytochrome P-450scc displays the ESR g-values gx = 2.404/2.456, gy = 2.245 and gz = 1.916; these are different from those of cholesterol-liganded cytochrome P-450scc and may thus serve as a marker for cytochrome P-450scc. Our data indicate that the stereospecificity of the cytochrome P-450scc side-chain-cleaving activity is not dependent on the nature of the cholesterol side-chain termination (C25 to C27). The substrate binding site is however rather sensitive to a modification of the side chain. The doxyl ring confers a stronger affinity of the substrate to the enzyme. Upon binding it becomes embedded in the protein matrix, and we estimate that its final position is 0.6-1.0 nm from the heme moiety.

European Journal of Biochemistry, 1979

Variations of the spin state in camphor-bound cytochrome P-450 are interpreted in the light of th... more Variations of the spin state in camphor-bound cytochrome P-450 are interpreted in the light of the polyelectrolyte theory and its implications on the microenvironment of the heme. The ratio of high-spin to low-spin iron can serve as a tool to determine the local paH in the microenvironment of a group (pK0, app approximately 5.4) which governs the spin state. The local paH depends on the electrostatic potential created by negatively charged groups (pKa = 5.6), modulated in turn by paH and by the screening effect of ionic strength. A model is given for the proton-coupled spin state change.