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P Douzou

Publications and source records attributed to P Douzou.

At least 19 recordsLinked to original sources
PubMed1995

Antagonistic effects of hydrostatic pressure and osmotic pressure on cytochrome P-450cam spin transition.

The combined effects of hydrostatic pressure and osmotic pressure, generated by polyols, on the spin equilibrium of fenchone-bound cytochrome P-450cam were investigated. Hydrostatic pressure indices a high spin to low spin transition, whereas polyols induce the reversed reaction. Of the four solutes used, glycerol, glucose, stachyose, and sucrose, only the last two would act on the spin transition by osmotic stress. The spin volume changes measured by both techniques are different, 29 and -350 ml/mol for hydrostatic pressure and osmotic pressure, respectively. It suggests that even if the two are perturbing water molecules, different properties are probed. From the volume change induced by osmotic stress, 19 water molecules are deduced that would be implicated in the spin transition of the fenchone-bound protein. This result suggests that water molecules other than the well defined ones located in the active site play a key role in modulating the spin equilibrium of cytochrome P-450cam.

Camphor 5-Monooxygenase↗
PubMed1994

Electrostatic control of the substrate access channel in cytochrome P-450cam.

Camphor binding to ferric cytochrome P-450cam is a two-step process. The first step corresponds to the diffusion of camphor into the heme pocket, and the second one corresponds to an observable spin transition of the heme iron. In this paper, electrostatic interactions that may control the opening of the structure to allow substrate access to the buried and not solvent-exposed active site were examined. The electrostatic interactions occurring at the protein surface were weakened by increasing the ionic strength of the medium with sodium salts and strengthened by decreasing the dielectric constant of the medium with ethylene glycol as a cosolvent. The results obtained with the wild-type protein were compared to those obtained with the site-directed mutant of cytochrome P-450cam in which the Arg 186-Asp 251 and Lys 178-Asp 251 salt bridges, located at the entrance of the proposed access channel, were suppressed by replacing Asp 251 with an asparagine residue. Over a range of sodium chloride concentrations from 0 to 400 mM, camphor binding is favored, as seen in the variation in the first step dissociation equilibrium constant, K1d, which decreases from 49.5 to 24 microM, respectively. Addition of ethylene glycol favors the dissociation of the substrate-bound complex. The addition of sodium to the ethylene glycol-containing samples reverses the effect of the cosolvent. Removal of the Arg 186-Asp 251 and Lys 178-Asp 251 salt bridges results in an alteration in camphor binding in which K1d is equal to 34 microM without sodium.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1994

Effects of monovalent cations on cytochrome P-450 camphor. Evidence for preferential binding of potassium.

Binding of monovalent cations of increasing ionic radius to ferric cytochrome P-450cam was measured. Potassium has the highest affinity for the cation binding site observed in the X-ray crystallographic structure with Kdcat = 12 mM, compared with the smaller cation lithium, (Kdcat = 37 mM) and the larger cation cesium (Kd cat = 20 mM). Coupling between cation binding and camphor binding is established by the observation of a linear relationship between the corresponding binding free energies. Potassium binding favours a conformational change of tyrosine 96 which increases the affinity of the protein for camphor and fully dehydrates the active site.

Binding Sites↗
PubMed1994

Osmotic regulation of gene action.

Most reactions involved in gene translation systems are ionic-dependent and may be explained in electrostatic terms. However, a number of observations of equilibria and rate processes making up the overall reactions clearly indicate that there is still an enormous gap between the rough picture of the mechanism of ionic regulation and the detailed behavior of reactions at the molecular level that hold the key to specific mechanisms. The present paper deals with possible osmotic contributions arising from the gel state of gene systems that are complementary to, and interdependent of, electrostatic contributions. This treatment, although still oversimplified, explains many previous observations by relating them to a general osmotic mechanism and suggests experimental approaches to studying the mechanisms of gene regulation in organelle-free and intact systems.

Biopolymers↗
PubMed1993

Conformational dynamics of cytochrome P-450cam as monitored by photoacoustic calorimetry.

Conformational transitions of cytochrome P-450cam following the dissociation of CO from the ferrous heme were investigated by using photoacoustic calorimetry. The effect of substrate association on the acoustic signal was also examined. Results show that the conformational dynamics of cytochrome P-450cam substrate-free protein occur faster than 10 ns, which is the time scale of the instrument response. The enthalpy and volume change for the dissociation reaction are 2.2 kcal mol-1 and 1.8 mL mol-1, respectively. Upon addition of camphor, the reaction is markedly slowed. An intermediate is formed whose lifetime is 130 ns at 17 degrees C. The overall enthalpy and volume changes are -15.9 kcal mol-1 and 10.3 mL mol-1, respectively. These results, together with published transient Raman spectra [Wells, A. V., Pusheng, L., Champion, P. M., Martinis, S. A., & Sligar, S. G. (1992) Biochemistry 31, 4384-4393] suggest that camphor leaves the heme pocket concomitant with the photoinduced expulsion of CO into the solvent and induces a considerable conformational change in the protein.

Calorimetry↗
PubMed1992

A critical role of protein-bound water in the catalytic cycle of cytochrome P-450 camphor.

The rates of NADH oxidation during the hydroxylation of camphor by cytochrome P-450cam were followed in the presence of co-solvents used to increase the osmotic pressure surrounding the protein-bound water. As a result, the measured Vmax decreases independently of the perturbant tested. Roughly 28 molecules of water, involved during the catalytic cycle, are deduced from the variation of Vmax as a function of osmotic pressure. These molecules, in part, could be those present in the cytochrome P-450cam-putidaredoxin interface.

Camphor 5-Monooxygenase↗
PubMed1992

Heme-pocket-hydration change during the inactivation of cytochrome P-450camphor by hydrostatic pressure.

Hydrostatic pressure has been used to convert cytochrome P-450camphor to cytochrome P-420. The latter is an inactivated but soluble and undenaturated form of cytochrome P-450camphor. Using camphor analogues as probes of the active site we show that the inactivation volume change is directly correlated to the initial degree of hydration of the heme pocket. The values range between -73 ml/mol and -197 ml/mol [Di Primo, C., Hui Bon Hoa, G., Douzou, P. & Sligar, S. G. (1990) Eur. J. Biochem. 193, 383-386] for a totally hydrated (substrate-free, low-spin, six coordinated heme iron) and a non-hydrated (camphor-bound, high-spin, five coordinated heme iron) heme pocket. These results suggest that the larger value, -197 ml/mol, for the inactivation volume change is due to a hydration change of the heme pocket resulting from the displacement of the substrate during the compression and the subsequent entrance of water molecules. Similarly, the stability of the protein against compression is correlated with water accessibility to the active site. Increase in substrate mobility by loss of specific interactions with both regions of well defined secondary structure of cytochrome P-450camphor results in an increase of water accessibility and decrease of stability. Thus for camphor and adamantanone which strongly interact with the protein and exclude water from the active site [Poulos, T. L., Finzel, B. C. & Howard, A. J. (1987) J. Mol. Biol. 195, 687-700; Raag, R. & Poulos, T. L. (1989) Biochemistry 28, 917-922] the increase in stability compared to the free protein is roughly 30 kJ/mol at 20 degrees C. With smaller substrates such as norcamphor, which loosely fits into the active site and does not completely exclude water [Raag, R. & Poulos, T. L. (1989) Biochemistry 28, 917-922], the increase in stability is only 7 kJ/mol. Finally these results suggest that cytochrome P-420 induced by hydrostatic pressure is a unique form where the active site is hydrated and camphor is displaced from its binding site.

Camphor 5-Monooxygenase↗
PubMed1990

Effect of the tyrosine 96 hydrogen bond on the inactivation of cytochrome P-450cam induced by hydrostatic pressure.

The effects of removal of the tyrosine 96 hydrogen bond on the stability and conformational events of cytochrome P-450cam are presented in this communication. Hydrostatic pressure has been used as a tool to perturbe the structure leading to the formation of cytochrome P-420, an inactivated but soluble and undenatured form of the enzyme. We show that the spin transition of cytochrome P-450cam, which is known to be influenced by hydrostatic pressure, is affected by this single mutation. The free energy of stabilisation of native substrate-free cytochrome P-450cam is not affected by the removal of the tyrosine 96 hydrogen bond via mutagenesis to phenylalanine, whereas the substrate-bound protein shows a difference of 21 kJ/mol. These results, as well as an observed 110 ml/mol difference for the volume of the inactivation reaction between substrate-bound native and mutant proteins, have been interpreted in terms of a more hydrated heme pocket for the site-directed mutant at position 96 compared to the wild-type protein where camphor is tightly bound via the tyrosine 96 hydrogen bond and water excluded from the active site.

Camphor↗
PubMed1990

The formation of cytochrome P-450 from cytochrome P-420 is promoted by spermine.

This paper is concerned with camphor-bound bacterial cytochrome P-450 and processes that alter its spin-state equilibrium and influence its transition to the nonactive form, cytochrome P-420, as well as its renaturation to the native camphor-bound cytochrome P-450. Spermine, a polycation carrying a charge of 4 +, and potassium, a monovalent cation, were shown to differently cause an increase of high-spin content of camphor-bound cytochrome P-450. The spermine-induced spin transition saturates around 75% of the high spin; a further addition of KCl to the spermine-containing sample shifted the spin state to 95% of the high spin. The volume change of these spin transitions as measured by the use of high pressure indicated an excess of -40 mL/mol for the sample containing potassium as compared to that containing spermine. These results suggest that the proposed privileged site for potassium has not been occupied by spermine and that pressure forces both the camphor and the potassium ion from its sites, allowing solvent movement into the protein as well as ordering of solvent by the excluded camphor and potassium. Cytochrome P-420 was produced from cytochrome P-450 by hydrostatic pressure in the presence of potassium, spermine, and cysteine. Potassium cation shows a bigger effect on the stability of cytochrome P-450 than spermine or cysteine, as revealed by a higher value of the pressure of half-inactivation, P1/2, and a bigger inactivation volume change. However, potassium cation did not promote renaturation of cytochrome P-420 to cytochrome P-450 while the presence of spermine did.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1990

Mutagenesis of a single hydrogen bond in cytochrome P-450 alters cation binding and heme solvation.

Cytochrome P-450cam, a monoxygenase responsible for the regiospecific hydroxylation of camphor, binds its substrate through complimentary van der Waals contacts and the formation of a single hydrogen bond between tyrosine 96 and the ketone group of camphor. Substrate association is positively regulated through the binding of a monovalent cation and the oxidation-reduction potential modulated by the spin state of the ferric heme controlled by water access to the sixth coordination site of the iron. Removal of this single hydrogen bond via site-directed mutagenesis of tyrosine 96 to phenylalanine 96 defines this aspect of the protein structure as responsible for the linkage between cation and substrate cooperativities, the degree of spin state conversion resulting from water access via macromolecule and substrate dynamics, and suggests a specific location for the cation binding site.

Camphor↗
PubMed1989

Conformational changes of cytochromes P-450cam and P-450lin induced by high pressure.

Absorbance and fluorescence spectra of bacterial cytochrome P-450cam and cytochrome P-450lin have been studied as a function of pressure. These pressure-induced spectral perturbations fall into two categories, which are interpreted as resulting from denaturation domains and are discussed in terms of protein structural dynamics. The results presented herein support a view that these two bacterial cytochromes have large structural differences and suggest a picture in which the gellike cortex of each protein may play an essential role in stability and function.

Camphor↗
PubMed1989

Sol-gel processing of actin to obtain homogeneous glasses at low temperatures.

The lethal effects of freezing on cells are currently attributed to the crystallization of extracellular water which leaves concentrated solutions of salts, macromolecules and so forth in the extracellular space. This concentrated fluid establishes a strong osmotic gradient which draws water from the cells. Thus, a cell surrounded by ice can survive only if means can be found for reducing the osmotically driven outflow of cellular water. This is usually attempted through vitrification of the extracellular space, but may also be attained through suitable modifications of cellular plasms. Starting from microscopic observations on early rabbit embryos and related cryotolerance, we investigated purified actin solutions under similar conditions, and found that sol-gel processing could result in the formation of homogeneous glass, and through drying, give rise to monolithic solids, glasses and composites. The first process may be at least partially responsible for the induced cryotolerance of cells, while the second may be representative of new and useful biomaterials.

Actinin↗
PubMed1988

Biophysical chemical aspects of cellular cryobehavior.

Freezing tolerance and resistance in nature are among the most important and challenging aspects of biochemical adaptation to extreme environments. Some biochemical strategies are known but their mechanism is still poorly understood. Cryopreservation of cells and tissues of sensitive organisms is still generally based on physical chemistry rather than on biophysical chemical mechanisms. This paper describes the main aspects of these problems and features new trends in their study.

Adaptation, Physiological↗
PubMed1987

Thermodynamics of the two step formation of horseradish peroxidase compound I.

The effects of temperature (20 to -38 degrees C), pressure (normal pressures to 1.2 kbar) and solvent (water, 60% DMSO and 50% methanol) on the reaction of hydrogen peroxide or ethyl peroxide with horseradish peroxidase were studied. The formation of compound I was followed at 403 nm in a stopped flow apparatus adapted for high pressure and low temperature work. As with the alkaline form (Job and Dunford 1978), the neutral form of the peroxidase binds peroxide substrates in two steps. It was the combined use of organic solvents and low temperatures which revealed saturation kinetics: (Formula: see text) compound I, where E = horseradish peroxidase and S peroxide substrate. In water and organic solvents at temperatures above -10 degrees C, K1 was too small and k2 too large to be measured, here K1 X k2 was obtained. k-2 was too small for measurement under all conditions. Whereas K1 was insensitive to the peroxide substrate and solvent composition, k2 was very sensitive. The thermodynamic parameters delta H, delta S and delta V for K1 and k2 were obtained under different experimental conditions and the data are interpreted within the available thermodynamic theories.

Cold Temperature↗
PubMed1987

Biological macromolecules as gels: functional similarities.

Functional biological macromolecules arising from folding, cross-connection, and solvation of long-chain biopolymers forming three-dimensional networks can be compared to gels. Both involve identical internal competitive forces that are selectively influenced by external conditions and conspire to adjust conformations and modulate functions. In spite of important differences in size, chemical composition, polymer density, and configuration, biological macromolecules indeed manifest some of the essential physical-chemical properties of gels. This result represents presumptive evidence for common underlying mechanisms in functional molecules and gels. Thus, the present and highly perfectible model explains why and how, depending on initial conditions, a system may respond differently to an external parameter and similarly to different parameters. Moreover, the fact that any localized change in one of the competitive forces gives rise to a pressure in the system as a whole provides one explanation for the mechanism of the transmission of information.

Biopolymers↗
PubMed1987

[Labile behavior of gels and possible biological implications].

Gelated networks of biopolymers submitted to thermal cycles may undergo phase-transitions and phase-separations, and their cryobehavior is chiefly associated with such phenomena. The properties unveiled with models on a macroscopic scale may have implications in the cryobehavior of living systems while the lability of biopolymer gels under the influence of other external conditions may be involved in a number of biological functions.

Biopolymers↗
PubMed1987

[Mechanisms common to biological macromolecules and gels].

Functional Biological macromolecules arising from folding, cross-connection and solvation of long chain biopolymers forming three-dimensional networks may be regarded as Gels. Both involve identical internal competitive forces that are selectively influenced by external conditions and conspire to adjust conformations and modulate activities. In spite of important differences in size, chemical composition, polymer bonding, density and configuration, biological macromolecules indeed manifest some of the essential physical-chemical properties of gels when involved in equilibria and rate processes. This result represents a presumptive evidence for common underlying mechanisms in functional molecules and gels. Thus, the present and highly perfectible model explains why and how, depending on initial conditions, a system may respond differently to an external parameter, and similarly to different parameters. Moreover, the fact that any localized change in one of the competitive forces gives to a pressure in the system as a whole provides an explanation for the mechanism of the transmission of information.

Biopolymers↗