Biomedical subjects
Publications and source records attributed to R M Davydov.
Membrane fragments of St. aureus are able to synthesize up to 7 nmol/mg protein of ATP after a jump-like increase of pH value. The presence of respiratory substrates is obligatory. The effect is realized only during the jump, if pH passes of 7.4 value. ADP phosphorylation induced by pH-jump is completely inhibited by DCCD.
It was shown that ferrocytochrome P450 forms a nonequilibrium state if ferrocytochrome P450 and its complexes are reduced in freezed water-glycerol solutions by thermolysed electrons, arising during gamma-radiolysis of the matrix at 77 degrees K. Unlike the equilibrium form of ferrocytochrome P450 with the heme iron at the high-spin state the reduced nonequilibrium form of the protein contains the heme iron at a low-spin state. The absorption spectrum of ferrocytochrome P450 in the nonequilibrium state is characterized by alpha and beta-bands at 562 and 534 nm, respectively, whereas the magnetic circular dichroism spectra exhibit type A effect at 562 nm. Upon temperature increasing the nonequilibrium state is relaxed to the equilibrium one. Type 1 substrates had practically no influence on the spectral characteristic of the nonequilibrium form of ferrocytochrome P450. Binding of type 2 substrates results in an essential decrease of the intensity ratio of the alpha- and beta-bands (A alpha/A beta) and is accompanied by a red-shift of the alpha-band and corresponding magnetic circular dichroism effect. It was shown that mercaptoethanol complex of hemoglobin, formed by reduction at 77 degrees K is spectrally similar to the nonequilibrium ferrocytochrome P450 complex with type 2 substrates. From analysis of experimental data one can conclude that (i) the ligand environment of heme iron in oxidased and reduced cytochrome P450 are different; (ii) the sixth axial ligand of the heme iron in the oxidised protein is probably a water molecule (OH-) attached by a hydrogen bond to the neighbouring histidine. It is assumed that a similar nonequilibrium form of cytochrome P450 can be formed in physiological conditions.
Photoreduction of ferricytochrome P-450 with eosine radicals was studied by flash-photolysis. Reactivity of reduced protein was estimated by CO binding. It has been stated that both, at direct reduction of cytochrome P-450 and at its recombination with CO after its photodissociation of carboxycomplex the reaction consists of three stages with similar rate constants but different weight ratio of corresponding stages. This result well agrees with the supposition about non-equilibrium (in the course of reduction) distribution of cytochrome P-450 conformers.
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It has been shown that low temperatures (77 degrees K) irradiation of frozen water-glycerol solutions of oxymyoglobin and oxyhemoglobin induces kinetically stabilized nonequilibrium electronic adducts (MbO2-, HbO2-) at the expense of binding of thermolyzed electrons formed during matrix radiolysis to oxygenated hem iron. The absorption spectra of HbO2-and MbO2- have a wide band with the maximum at 545 nm and Soret's band at 421 nm. At 77 K MbO2- gives the ESR spectrum with g beta 1 = 2.203 and g beta 2 = 2.103. Unlike the latter HbO2- ESR spectrum consists of two signals g beta 1 = 2.234, g beta 2 = 2.135 and g alpha 1 = 2.195, g alpha 2 = 2.103. Two signals in HbO2- spectra are shown to be conditioned by electronic adducts of oxygenated alpha- and beta-subunits. The observed effect points to non-equivalency of O2 in alpha- and beta-subunits of oxyhemoglobin. Binding of inositolhexaphopshate to oxyhemoglobin induces changes in the electron structure of HbO2-active centres.
Low temperature (77 degrees K) absorption spectra of nonequilibrium states of cytochrome c oxidase produced by reduction of oxidases form protein by thermolysed electrons at 77 degrees K was studied. During reduction of cytochrome oxidase water-glycerol solution by thermolysed electrons at 77 degrees K a nonequilibrium reduced protein is formed. Low temperature (77 degrees K) absorption spectra of the nonequilibrium cytochrome oxidase differs from those reduced by ditionite. It was shown that the oxidation state of cytochrome a3 or addition of cytochrom c have no influence on these spectral changes. It is assumed, that the observed effects are conditioned by structural differences of reduced and oxidased cytochrome oxidase active center. Similar spectral changes were observed for cytochrome oxidase, bound to the mitochondrial membrane. At temperature increasing the low temperature reduced protein is relaxed to a corresponding equilibrium state. The spectral properties of bacterial cytochrome oxidase M. lysodeicticus do not depend on the way of reduction (by dytionite or thermolysed electrons at 77 degrees K).
It has been shown that during fast (< 1 ms) photosensitized by anthraquinone or benzophenone reduction of cytochrome c in 0.15 N NaOH water-glycerol solutions ferrocytochrome c in a nonequilibrium state with increased reactivity was formed. The rate constants for reactions of CO binding to nonequilibrium and equilibrium ferrocytochrome c are 2.10(4) M-1S-1 and 70 M-1S-1 correspondingly. Nonequilibrium cytochrome c is relaxed to corresponding equilibrium state with lambda = 4 S-1.
Nonequilibrium hemoglobin states formed at low-temperature (T = 77K) reduction of its derivatives (MetHb and HbO2) by thermolysed electrons have been studied by Mössbauer spectroscopy. Relaxation of nonequilibrium states at the samples heating was observed. Correlation between the relaxation temperatures and the changes of protein dynamic structure determined from Mössbauer data were stated.
Kinetics of cytochrome P-450 reduction with methylviologen radicals was investigated by flash-photolysis. Formation of carboxycomplex of reduced protein and effect of temperature and substrates of the I type on this reaction were studied. It has been shown that CO addition to protein proceeds in two stages. With temperature increase (20-45 degrees C) and when benzphetamine or hexabarbital are added to solution the weight of the first (faster) stage increases. Such regularity is easily explained by the presence in solution of two conformers of oxidized cytochrome P-450 (with different reactivity) whose equilibrium depends on temperature and presence of the substrate.
Using flash photolysis, horseradish peroxidase rebinding of carbon monoxide has been observed in the temperature range between 20 and -70 degrees C. A lag-period of about 50 ms immediately after photodissociation takes place. This lag-period is assumed to be due to the formation of the intermediate protein form with the low reactivity. Following rebinding is bimolecular process with the rate constant 4 x 10(3) M-1 S-1 usually observed for the peroxidase reaction with carbon monoxide. For some peroxidase molecules (about 10 per cent) rebinding of carbon monoxide is CO-concentration independent monomolecular process, which implies, apparently, ligands in these molecules do not leave the protein after photodissociation.
Absorption and magnetic circular dichroism spectra of nonequilibrium states of hemoglobin and its derivatives formed by reduction oxidased forms of hemoproteins by thermalysed electrons at 77 degrees K were studied. Mixtures of low spin and high spin ferroforms were observed for nonequilibrium hemoglobin and its complexes with inosithexaphosphate and fluorine. The content of the high spin form increasing as follows: hemoglobin, complex with inosithexaphosphate, complex with fluorine. Only low spin forms were found for cyanide and azide complexes of hemoglobin reduced at low temperature. The spectral differences of nonequilibrium low spin ferroforms were supposed to be due to the presence of different ligands in the coordination sphere of the heme iron. The alpha-band splitting was observed for the nonequilibrium imidazole complex of hemoglobin. This effect was explained by a lowe-ring of the active centre's symmetry. The temperature relaxation of all nonequilibrium systems was investigated.
Reduction of frozen water-glycerol solutions of methemoglobin by thermolysed electrons at 77 degrees K has been studied by Mossbauer spectroscopy. The formation of nonequilibrium hemoglobin states with two slightly different low-spin ferroforms was observed. The latter is explained by the presence of two ferroforms in initial methemoglobin. There was observed relaxation of the conformation - nonequilibrium states up to equilibrium hemoglobin with high-spin Fe(II) at the sample disfreezing.
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Under gamma-irradiation of frozen water--glycerol solutions of ferrocytochrome b5 at 77 degrees K ferrocytochrome b5 in a non-equilibrium state is formed. Its absorption and MCD spectra are displaced by 1,5 nm to the long wave region. When the temperature is increased to 140 degrees K the non-equilibrium form of reduced protein is relaxes. A comparison of optical characteristics of ferrocytochrome b5 in equilibrium and non-equilibrium states shows that the structure of the protein active centre slightly depends on the state of metal oxidation. It is concluded on the basis of earlier published data on low temperature reduction of different electron transport proteins that a small effect of the oxidation state of a metal ion on the active centre structure is a common property of all the electron transport proteins. This phenomenon makes these systems an effective carrier of electrons.
Absorption and magnetic circular dichroism spectra of non-equilibrium states of myoglobin and its complexes formed by reduction oxidased forms of proteins by thermalysed electrons at 77 degrees K were studied. Mixtures of high spin and low spin ferroforms were observed for nonequilibrium states of myoglobin and its complex with fluorine, the content of the high spin form is larger in the complex. Two intense peaks were found in the alpha-band region of absorption spectra of myoglobin and its spectra with F-, OH- and imidazole. This effect is due to lowering of the active centre's symmetry. Similarity of spectral characteristics of low spin ferroforms of these complexes was explained by the strong influence of distal histidine. The low temperature reduction of azide and cyanide complexes of myoglobin led to formation of nonequilibrium low spin ferroforms whose spectra demonstrate the presence of N3- and CN- in heme iron's coordination sphere. The temperature relaxation of all nonequilibrium systems were investigated.
Absorption and magnetic curcular dichroism spectra of nonequilibrium states of peroxidase and its complexes with F-, N3-, CN- produced by reduction of oxidased forms of proteins by thermalysed electrons at 77 degrees K were studied. Mixtures of high spin and low spin ferroforms were found in nonequilibrium states of peroxidase and complexes with F- and N3-, the content of the high spin ferroform increasing as follows: N3- complex less than peroxidase less than fluorine complex. Only low spin ferroforms was found after low temperature reduction of the cyanide complex. The existence of the low spin ferroform in equilibrium states of peroxidase and its complex with F- was explained by location of iron near the porphyrine plane. In the case of azide and cyanide complexes the existence of the low spin form is due to the presence of these ligands in heme iron's coordination sphere. The temperature relaxation of all nonequilibrium forms was investigated and a possible mechanism of the process is proposed.
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The reduction of nitroxide radicals by rho-phenylene diamine was studied in H2O, various organic solvents in liposome suspensions and oleic acid emulsion. Some new parameters, particularly the presence of acids, appeared to effect the rate of the process. The results obtained are consistent with the hypothesis that the electron transport in coupled membranes leads to the production of undissociated acids thus supplying protons for the ATP synthesis.