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P Debey

Publications and source records attributed to P Debey.

At least 55 records · Page 3Linked to original sources
PubMed1983

Heme P460 of hydroxylamine oxidoreductase of Nitrosomonas. Reaction with CO and H2O2.

Hydroxylamine oxidoreductase (HAO) of Nitrosomonas catalyzes the dehydrogenation of NH2OH and subsequent addition of oxygen to form nitrite. HAO contains c hemes and the CO-binding heme P460 in a 7:1 ratio; dehydrogenation of NH2OH involves passage of electrons to P460 and then c hemes. We now report that electrons rapidly pass from c hemes of HAO to the P460 center and then to H2O2. This conclusion is supported by (a) inhibition of c heme oxidation with CO and (b) loss of H2O2-oxidizability of ferrous c hemes following specific destruction of heme P460. Reaction of ferrous P460 with H2O2 is rate-limiting. Activation of dioxygen for N-oxidation by ferrous HAO may involve the two-electron reduction of O2 by P460. The reaction of ferrous HAO with H2O2 was studied as it may reveal aspects of the mechanism of activation of dioxygen. Reaction of ferrous heme P460 with CO is slow and with low affinity as compared with other hemoproteins. Values for reaction of CO with enzyme were: k1, 1.1 X 10(-3) M-1 s-1 and Kd, 12 microM.

Carbon Monoxide↗
PubMed1982

One-electron photoreduction of bacterial cytochrome P-450 by ultraviolet light. I. Steady-state irradiations.

Bacterial ferric cytochrome P-450 can be photoreduced by ultraviolet light in presence of camphor as shown by the formation of the ferrous cytochrome P-450--CO complex absorbing at 446 nm in CO-saturated solutions. This photoreduction can only be performed with wavelengths (lambda less than 315 nm) corresponding to the absorption of light by aromatic residues and in particular, tryptophan. The primary process leading to the photoreduction is thus the photoionization of tryptophan residue(s) near the active site of the protein as deduced from competitive electron scavenging with N2O. In agreement with results obtained with chemical reduction, the quantum yield of photoreduction is proportional to the content of the high-spin state of the ferric cytochrome P-450 solution. It is also shown that the camphor concentration affects the CO binding.

Camphor↗
PubMed1982

One-electron photoreduction of bacterial cytochrome P-450 by ultraviolet light. II. A study using laser flash photolysis of the dynamics of the reduction of bacterial cytochrome P-450 in the presence of carbon monoxide. The role of the protein conformation.

The intramolecular photoreduction of the bacterial cytochrome P-450 demonstrated by the formation of the complex of reduced cytochrome m with camphor and CO has been studied by photolysis with a laser flash at 265 nm. This photoreduction of the heme follows the photoionization of a proximal aromatic amino acid residue and especially tryptophan in the nanosecond time scale as evidenced by the N2O effect on the reduction yield. However most of the hydrated electrons released in the solution do not react with the ferric porphyrin. In agreement with steady state studies, the quantum yield for formation of the complex of reduced cytochrome m with camphor and CO is proportional to the concentration of the high-spin species. The low-spin ferric porphyrin is thus not reducible by photoejected electrons. It is also shown that the growth kinetics of the transient absorbance at 446 nm characterizing the complex of reduced cytochrome m with camphor and CO correspond to a structural change affecting the protein and not to the formation of the complex of CO with the camphor-bound reduced cytochrome in which occurs on a shorter time scale in the present experimental conditions.

Carbon Monoxide↗
PubMed1980

Visible and ultraviolet spectral transitions of camphor-bound cytochrome P-450. A comprehensive study.

A systematic analysis of the low-spin to high-spin transition in the ultraviolet and visible absorption spectrum of camphor-bound cytochrome P-450 is presented. The equilibrium depends on the ionization of two groups, the deprotonation of the first one (pK1 = 5.4 at -17 degrees C) leading to an increased content of high-spin type of spectrum, that of the second one (pK2 > 6) to more low-spin type of spectrum. Both protonation processes and their thermodynamic terms are studied under various conditions of salt, solvent and temperature, including subzero temperatures. The basic mechanism of the transition is the same in aqueous and mixed organic solvent, i.e. the protonation of the first group triggers a conformational change of the whole protein. These phenomena are discussed with reference to functional conformational changes during the catalytic cycle of cytochrome P-450.

Camphor↗
PubMed1979

Testing cosolvent cryoenzymology on multi-enzyme systems.

Fluid mixed solvents appear to supply an effective tool for investigation of enzyme catalysis at subzero temperatures. Labile reaction intermediates may be stabilized, characterized and separated. Cycling of reaction processes and side reactions can be slowed or stopped to permit single turnover of individual molecular events, and kinetic response to temperature and other physical parameters can provide dynamic and thermodynamic analysis of single enzyme systems. Multienzyme systems can furnish reliable probes of potential, limitations and possible procedural artefacts of the method. Amply explored examples are supplied by the studies of two components of the soluble camphor hydroxylase and the hepatic R450LM2 in solution and in the subcellular microsomal organelles, to reveal labile single enzyme compounds and multienzyme processes.

Cold Temperature↗
PubMed1979

Fast photochemical reactions of cytochrome P450 at subzero temperatures.

Several reactions of the cytochrome P450 multi-step cycle have been studied by fast light activation combined with subzero temperatures. A flash device was adapted to an Aminco-Chance DW 2 spectrophotometer equipped for subzero temperature thermostatisation. The first electron can be introduced into the cycle by non specific reducing agents such as reduced flavin mononucleotide (FMNH2) or methylviologen radical (MV.). This first reduction remains a fast process even at subzero temperatures. The oxy-compound Fe2+-O2 can thus be formed either directly from Fe2+ or via the photodissociation of the carboxy-ferro adduct. Fe2+-O2 is stable at subzero temperatures towards spontaneous autoxidation as well as further reduction by FMNH2 or MW.. In addition, the recombination of CO after flash photodissociation of Fe2+-CO was used to study in more details the specific behaviors of the purified microsomal cytochrome.

Bacterial Proteins↗
PubMed1979

Spin transition of camphor-bound cytochrome P-450. 1. local paH and electrostatic interactions.

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.

Camphor↗
PubMed1979

Spin transition of camphor-bound cytochrome P-450. 2. Kinetics following rapid changes of the local paH at sub-zero temperatures.

The kinetics of the spin transition in the heme iron of the cytochrome P-450 substrate complex have been observed by stopped-flow measurements between 4 degrees C and -27 degrees C. Large displacements in the spin equilibrium are induced by small changes in the concentration of hydrogen or potassium ions. The kinetic and thermodynamic data indicate that the spin transition is rate-limited by conformational changes of the protein. The spin transition appears to be governed by the local paH, modulated in turn by external factors: a satisfying kinetic analysis is attained only by accounting for the difference between local paH (paH, in) and bulk paH (paH, out), as described in the preceding paper. Indeed, the low-spin to high-spin rate constant obeys a local paH titration curve with a pK = 5.4 +/- 0.1 at -17 degrees C.

Camphor↗
PubMed1979

A thermodynamic-kinetic analysis of the cytochrome P-450 heme pocket.

Spin state changes in the iron center of cytochrome P-450 during the catalytic cycle suggest alterations in the heme environment that insure proper substrate binding, an increase in redox potential, the formation of an active Fe-O complex, and the attack on the substrate. We used the spin state changes of the iron following physico-chemical perturbations, as an intrinsic probe of discrete changes around the heme, or of larger ones in the protein conformation. These environmental perturbations included temperature, solvent, substrate, and ionic environment. Aqueous and hydro-organic buffers provide complementary data and interpretations; the mixed solvent accommodates temperatures suitable for direct reaction rate measurements and amplified low to high spin transition. The results suggest that the group determining the heme spin state is influenced by the electrostatic potential created by several negative charges near the heme; the modulation of the spin state by various factors reflects the modulation of the electrostatic potential and of the internal paH value. Conformational changes of the whole protein are also indicated by the large entropy terms and their variation with experimental conditions.

Bacteria↗
PubMed1978

Supercooled water as medium for enzyme reactions at subzero temperatures.

A water-in-oil emulsion technique was employed to investigate enzyme-catalyzed reactions at sub-zero temperatures in the supercooled liquid state. The results obtained with a monooxygenase (bacterial cytochrome P-450) clearly indicate the potentialities as well as the technical problems of the procedure which might be successfully used to investigate enzyme systems sensitive to cosolvents.

Cold Temperature↗
PubMed1978

Biochemical effects on radioprotective agents on the liver microsomal hydroxylating system: in vitro studies.

The action of two radioprotectors--cysteamine and cystamine--on the liver microsomal multi-enzyme hydroxylating system, a key stem in drug and biological compounds metabolism, has been studied. Their effects have been systematically analysed at the level of individual enzyme activities and global functions. The two compounds are quite inactive on NADPH and NADH cytochrome c reductase activities, but slightly denature the cytochrome P450 into cytochrome P420. Furthermore, they do inhibit to some extent (30 per cent at 10(-2) M) the rate of codeine hydroxylation and totally suppress ((at 10(-2) M) the NADPH-induced lipid peroxidation which occurs during enzymatic functioning. These results are discussed in the light of the toxicity of radioprotectors.

Animals↗