[Analysis of low temperature magnetic circular dichroism of high spin ferrihemoproteids in the near UV region]. / Analiz nizkotemperaturnogo magnitnogo krugovogo dikhroizma vysokospinovykh ferrigemoproteidov v blizhnei UF-oblasti.

Magnetic circular dichroism spectra of fluoride complexes of metmyoglobin, methemoglobin, and horseradish peroxidase in the region of 300-450 nm at temperatures from 300 to 2.1 K were measured and analyzed. The temperature dependence of magnetic circular dichroism in the Soret region was found to be different from that of other paramagnetic forms and from the theoretically predicted dependence. The difference is explained by the superposition of the pi-->pi*-transition of porphyrin with one (peroxidase) or two charge transfer transitions and by substantially different temperature dependences of magnetic circular dichroism for the transitions of the two types. By minimization of differences between the expected and observed temperature dependences of magnetic circular dichroism, the parameters of its temperature dependence for charge transfer transitions and the parameter D of the zero-field splitting of the electronic ground state of the heme were found. The values of D for the fluoride complexes of metmyoglobin (5.8 cm-1) and methemoglobin (6.1 cm-1) agree well with those obtained by other methods. The D value for the fluoride complex of horseradish peroxidase (8.8 cm-1) was determined for the first time.

[Electron-conformational interactions at the active site of reduced bacterial cytochrome P450cam induced by a substrate and analysis of the electron structure of heme]. / Indutsirovannye substratom élektronno-konformatsionnye vzaimodeistviia v aktivnom tsentre vostanovlennogo bakterial'nogo tsitokhroma P450cam i analiz élektronnoi struktury gema.

Magnetic circular dichroism (MCD) spectra in the Soret region (360-480 nm) of camphor-free and camphor-bound reduced bacterial cytochrome P450cam from Pseudomonas putida were recorded and analysed in the temperature range from 2 K to 290 K. The temperature dependences of the MCD intensity are qualitatively changed by binding of substrate to the enzyme. In the absence of camphor the linear increase of the MCD intensity with 1/T at T < 4.2 K gives evidence for degeneracy or near degeneracy of the ground electronic state. In the presence of substrate the degeneracy is removed and temperature profiles show saturation behaviour at T < 4.2 K and wavelength dependence of their high-temperature parts. The temperature profiles for the long-wavelength region of the Soret band have a maximum approximately at 15 K, whereas the MCD intensity increases in a monotonous manner up to saturation in the short-wavelength region. The wavelength dependence of temperature profiles gives evidence for the co-existence of two different forms of substrate-bound reduced P450cam. The following conclusions were obtained from a theoretical analysis of the temperature profiles. In the absence of substrate there are very small if any rhombic distortions at the heme iron, and a parameter D of axial zero-field splitting is negative (D = -8.3 cm-1 and -6.2 cm-1 for P450cam and P450LM2, respectively). In the presence of substrate the two forms of reduced P450cam have positive parameters D but of different values (D1 = 12 cm-1 and D2 = 28 cm-1), and there are large rhombic distortions at the heme iron. More than two-fold difference between the D values made it possible to isolate temperature-dependent contributions of the two enzyme forms from the total MCD spectra and to simulate the alterations of the MCD spectra with temperature for reduced P450cam in the presence of substrate. Taking into account the drastic effect of substrate binding on the ground electronic state of reduced P450cam one can suggest that substrate binding induces the transition of enzyme from an inactive to an active state.

[Contribution of protein conformation to stereochemistry and reactivity of the active center of heme proteins and enzymes. The existence of horseradish peroxidase conformations and their possible role in the catalysis mechanism]. / Vklad konformatsii belka v stereokhimiiu i reaktsionnuiu sposobnost' aktivnogo tsentra gemovykh belkov i fermentov. Sushchestvovanie dvukh konformatsii peroksidazy iz khrena i ikh vozmozhnaia rol' v mekhanizme kataliza.

In the spectral region 350-800 nm at 4.2 K we measured magnetic circular dichroism (MCD) spectra of the pentacoordinated complex of protcheme with 2-methylimidazole, deoxyleghemoglobin, neutral and alkaline forms of reduced horseradish peroxidase in the equilibrium states, as well as in non-equilibrium states produced by low-temperature photolysis of their carbon monoxide derivatives. Earlier the corresponding results have been obtained for myoglobin, hemoglobin and cytochromes P-450 and P-420. The energies of Fe-N (proximal His) and Fe-N(pyrroles) bonds and their changes upon ligand binding in heme proteins and enzymes were compared with those in the model heme complex thus providing conformational contribution into stereochemistry of the active site. The examples of weak and strong conformational "pressure" on stereochemistry were analysed and observed. If conformational energy contribution into stereochemistry prevails the electronic one the heme stereochemistry remains unchanged on ligand binding as it was observed for leghemoglobin and alkaline horseradish peroxidase. The change of bond energies in myoglobin and hemoglobin on ligand binding are comparable with those in protein free pentacoordinated protoheme, giving an example of weak conformational contribution to heme stereochemistry. The role of protein conformation energy in the modulation of ligand binding properties of heme in leghemoglobin relative to those in myoglobins is discussed. The most striking result were obtained in the study of reduced horseradish peroxidase in the pH region of 6.0-10.2. It was found that such different perturbations as ligand binding and heme-linked ionization of the distal amino acid residue induce identical changes in heme stereochemistry. Neither heme-linked ionization in the carbon monoxide complex nor the geometry of Fe-Co bond affect the heme local structure of photoproducts. These and other findings suggest a very low conformation mobility of horseradish peroxidase whose protein constraints appear to allow only two preferable geometries of specific amino acid residues that form the heme pocket. The role of the two tertiary structure constraints on the heme in the mechanism of horseradish peroxidase function is discussed. It is supposed that one conformation produces a heme environment suitable for two-electron oxidation of the native enzyme to compound I by hydrogen peroxide while another conformation changes the heme stereochemistry in the direction favourable for back reduction of compound I by the substrate to the resting enzyme through two one-electron steps. The switch from one tertiary structure to another is expected to be induced by substrate bind

[Electron structure of the heme of reduced cytochrome P450 and P420 from the data of low-temperature magnetic circular dichroism]. / Elektronnaia struktura gema vosstanovlennykh tsitokhromov P450 i P420 po dannym nizkotemperaturnogo magnitnogo krugovogo dikhroizma.

Magnetic circular dichroism spectra (MCD) of reduced cytochromes P450 and P420 from rabbit liver microsomes have been recorded and analyzed for the 350-600 nm spectral region in the temperature interval from 2 to 290 K. The shape, intensity and temperature dependence of the MCD of reduced P450 in the Soret region are quite different from that of other high-spin ferrous hemoproteins, whose heme iron is coordinated to the imidazole of histidine (deoxymyoglobin, deoxyhemoglobin, reduced peroxidase and cytochrome c oxidase). Assuming that in the reduced P450 as well as in its CO-complex the protein-derived ligand is mercaptide (RS-) the differences can be explained by the existence of two electronic transitions in the Soret region: the common for hemoproteins pi----pi porphyrin transition and sulfur to porphyrin charge-transfer transition, p+(Sp)----eg (pi). The unusual spectral characteristics of the CO-complex of P450 have been ascribed earlier to strong configurational interaction of these two transitions. From the similarities of the Soret MCD and their temperature dependences for the reduced P420 and for other high-spin ferrous hemoproteins one can conclude that heme iron of the reduced P420 is high-spin and is coordinated to the imidazole of histidine. The zero-field splitting parameter D of the spin Hamiltonian has been estimated from the MCD temperature dependences. The obtained splitting of approximately 30 cm-1 for P450 and of approximately 10 cm-1 for P420 exceeds that for myoglobin and hemoglobin (approximately 5 cm-1).

[Comparison of the heme electron state of reduced cytochrome P450 and P420 in equilibrium and non-equilibrium protein conformations. The nature of the protein heme ligand]. / Sravnenie élektronnykh sostoianii gema vosstanovlennykh tsitokhromov P450 i P420 v ravnovesnoi i neravnovesnoi konformatsii belka. Priroda belkovogo liganda gema.

Magnetic circular dichroism (MCD) spectra of reduced cytochromes P450 and P420 in equilibrium and non-equilibrium protein conformations are compared at 4.2 K for the 350-800 spectral region. Non-equilibrium forms have been produced by photolysis of CO-complexes at 4.2 K. The differences between MCD spectra of proteins in equilibrium and non-equilibrium conformations, in particular for the visible region, show clearly the structural changes in the heme iron coordination sphere to occur on ligand binding. The comparison of the Soret MCD spectra of reduced proteins in their equilibrium and non-equilibrium forms with those of other high-spin ferrous hemoproteins suggest that mercaptide (RS-) is the protein ligand of the heme iron in reduced P450, as well as in its CO-complex, and that imidazole of histidine is the fifth ligand of the iron both in reduced P420 and its CO-complex. The thermal recombination of the photoproducts with CO have been studied. When temperature rises from 4.2 to 77 K for two hours both proteins have similar temperature characteristics during the recombination processes. The recombination begins at T approximately equal to 10 K and is completed at approximately equal to 50 K. The temperature at which half of the total photolyzed molecules are restored to the CO-form is equal to 25 K. For products of photolysis of CO-complexes of myoglobin and hemoglobin under the same heating conditions these temperatures are equal to 35 and 23 K respectively. Thus, the photoproducts of P450, P420 and hemoglobin have similar parameters of low-temperature recombination and the kinetics of this process is faster than for photodissociated myoglobin.

[Electron structure of the active center of paramagnetic hemoproteins from the data of low-temperature magnetic circular dichroism. High-spin ferric derivatives]. / Elektronnoe stroenie aktivnogo tsentra paramagnitnykh gemoproteinov po dannym nizkotemperaturnogo magnitnogo krugovogo dikhroizma. Vysokospinovye ferriproizvodnye.

The temperature dependences of magnetic circular dichroism (MCD) spectra of the high-spin fluoride complexes of metmyoglobin, methemoglobin and horse-radish ferriperoxidase have been studied and analysed in spectral region 250-1100 nm at temperatures from 2 to 300 K. Upon lowering of the temperature the increase of the MCD intensity was observed over all spectral regions studied, indicating the presence of the so called C-type effects. In contrast to other paramagnetic hemoproteins studied previously the significant changes of the shape of MCD spectra of fluoride complexes in the near UV B (pi--pi*)-band (Soret band) was observed at the lowest temperatures. The changes are explained by the superposition of effects of two types: the usual C-type effects which have the shape of absorption dispersion and of the derivative shape CA-type effects. The CA-type effects in the Soret band were analysed in terms of the theoretical model suggested previously. The data are in qualitative agreement with the prediction of the model. New information on the energies of the 4E and 4A2 excited states of the heme iron, on the orbital contribution into ground state paramagnetism and on the value of the pi--d pi-splitting, typical for B(pi--pi*)-state and ground Kramers doublet, were obtained on the basis of quantitative analysis of the MCD temperature dependences. The parameter D of zero-field splitting was estimated from MCD spectra and its value (6 cm-1) agrees well with that obtained by other methods.

[Study of the complex of myoglobin with nicotinic acid by the method magnetic circular dichroism. Nature of the double hemochromogen]. / Issledovanie kompleksa mioglobina s nikotinovoi kislotoi metodom magnitnogo krugovogo dikhroizma. K voprosu o prirode dvoinogo gemokhromogena.

The complex formed by myoglobin and nicotinic acid exhibits unusual spectral properties. Instead of the usual two bands in the visible region the complex shows four bands assigned to the so called twin hemochromogen. Some attempts were previously made to clarify the nature of the twin hemochromogen, but the interpretation given was somewhat doubtful. We have shown that the combination of two spectral methods, namely magnetic circular dichroism and absorption spectra, give evidence that unusual absorption spectrum of the myoglobin complex with nicotinic acid is not attributed neither to the presence of the other hemochromogen nor to the soft vibrations but is due to the strong splitting of the pure electronic Q00 band into two Qox and Q0y bands. The splitting is caused by the distortion of heme structure by its asymmetrical environment.

[Absorption and magnetic circular dichroism spectra of heme proteins in nonequilibrium states. IV. Cytochrome c and its derivatives]. / Spektry pogloshcheniia i magnitonogo krugovogo dikhroizma gemsoderzhashchikh blekov v neravnovesnykh sostoianiiakh. IV. Tsitokhrom s i ego proizvodnye.

Absorption and magnetic circular dichroism spectra (77 degrees K) of nonequilibrium cytochrome c and its derivatives reduced by thermolysed electrons was studied. The low temperature spectral characteristics of reduced cytochrome c (pH 7.0, 1.6 and 10.0), dimer, carboxymetylated and formylated derivatives at netral and acid pH, also fluoride complexes differ from the characteristics of equilibrium reduced forms. The temperature increase (up to 177 degrees K) induces relaxation of nonequilibrium states. These effects are due to structural differences in the heme vicinity of the reduced and oxidized forms.

[Absorption and magnetic circular dichroism spectra of nonequilibrium states of hemoproteins. III. Complexes of peroxidase]. / Spektry pogloshcheniia i magnitnogo krurovogo dikhroizma gemsoderzhashchikh belkov v neravnovesnykh sostoianiiakh. III. Kompleksy peroksidazy.

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.

[Effect of porphyrin ring ligands on the affinity of heme iron to axial ligands]. / Vliia nie ligandov porfirinovogo kol'tsa na srodstvo zheleza gema k akshal'nym ligandam.

To clarify the influence of protein surrounding on the heme reactivity in heme proteins the effect of interaction between a porphyrin ring and pi-acceptor molecule, 1,2,4-trimethyl-pyridinium (TMP), on the affinity of deuteroheme to axial ligands (imidazole and cyanide) has been studied as a model system. It is shown that TMP induces the fourfold decrease in equilibrium constant of imidazole to deuteroheme. From the analysis of the two stages for cyanide binding it is concluded that TMP decreases the binding constant of the first cyanide by 40 times and does not apparently influence the second ligand binding. The effect of TMP on the reactivity of deuteroheme to axial ligands is interpreted as a result of a decrease in the electron density on the iron orbitals which is due to the altered pi-eleectron density in the porphyrin pi-system through the donor-acceptor interaction with TMP molecules. The possible significance of the contacts between the porphyrin and neighboring amino acid residues in determining heme affinity to axial ligands is discussed.

[Structure and functions of hemoglobin]. / Struktura i funktsii gemoglobina

The present status of our knowledge of different levels of hemoglobin molecule structural organization and of the conformation changes accompanying a hemoglobin action is reviewed. The main functional properties of hemoglobin such as cooperative effects in oxygen binding. CO2 transport, protons and organic phosphates effects on oxygen affinity are described on molecular ground. The description is based on the data obtained by different physical and chemical methods especially by X-ray analysis. The application of some mathematical models of cooperative effects in enzymes to hemoglobin is discussed.