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PubMed1984

Characterization of the Fe-S cluster in aconitase using low temperature magnetic circular dichroism spectroscopy.

Beef heart aconitase has been studied by low temperature magnetic circular dichroism (MCD) spectroscopy in the wavelength region 300 to 1900 nm. Together with parallel electron paramagnetic resonance and activity measurements, these data enable correlations between Fe-S cluster-type and enzymic activity in aconitase. In samples not exposed to extraneous Fe, the Fe-S cluster in aconitase exhibits the characteristic properties of a 3Fe center in both the as isolated and dithionite-reduced states. On the basis of the detailed form of the low temperature MCD spectra, three types of 3Fe center can be distinguished in biological samples. These are typified by the 3Fe centers in aconitase, Desulfovibrio gigas FdII, and Azotobacter crooccocum Fd. In aconitase, maximal enzymic activity is found to be associated with the build-up of [4Fe-4S]2+ clusters in good agreement with the Mössbauer studies of Kent et al. (Kent, T. A., Dreyer, J. L., Kennedy, M. C., Huynh, B. H., Emptage, M. H., Beinert, H., and Münck, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1096-1100). However, significant catalytic activity (approximately 60%) was obtained by reduction of the 3Fe center with dithionite in the absence of added Fe. The form and intensity of the resultant MCD spectrum are consistent with the majority of the Fe being in the form of reduced 3Fe clusters. The possibility that a reduced 3Fe cluster is capable of promoting partial catalytic activity in aconitase is discussed in light of these results.

Aconitate Hydratase↗
PubMed1981

The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase.

Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal Mr = 66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced alpha-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase, however, is able to transfer electrons from H2 to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially re-oxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable nor re-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57 degrees C. Purified nitrite reductase also has hydroxylamine reductase activity, and the Km for nitrite was determined to be 1.14 mM and that for hydroxylamine is 113.5 mM. The difference in Km values seems to exclude the possibility of hydroxylamine being a free intermediate in the reduction of nitrite.

Amino Acids↗
PubMedDate not supplied

[The possibility of using certain antioxidants for reducing ferriheme].

Kinetic parameters of the hemin spectral alterations occurred in reactions with sodium thiosulfate and sodium dithionite were studied depending on the reagents content as well as on the ratio between the antioxidant initial concentration and hemin. Simultaneous analysis of the kinetic parameters enabled one to suggest that hemin was reduced in reactions with sodium thiosulfate and sodium dithionite.

Antioxidants↗
PubMed1994

Mössbauer characterization of the metal clusters in Azotobacter vinelandii nitrogenase VFe protein.

The VFe protein of alternative nitrogenase, isolated from Azotobacter vinelandii, strain LS15 and designated as Av1', has been investigated by Mössbauer spectroscopy. The Mössbauer spectrum of the dithionite-reduced Av1', recorded at 4.2 K with a 60-millitesla magnetic field applied parallel to the gamma-beam, is a superposition of three spectral components: 1) a complex spectrum (the M component) with magnetic hyperfine structures attributed to the paramagnetic FeV cofactor, 2) a component (the P component) consisting of three quadrupole doublets identifiable as the Fe2+, D, and S doublets similar to those observed for the P cluster pairs in MoFe proteins, and 3) a minor (4% of total absorption) quadrupole doublet attributable to adventitiously bound iron. The observed 4.2-K parameters for the Fe2+ (delta EQ = 2.99 mm/s and delta = 0.64 mm/s), D (delta EQ = 0.75 mm/s and delta = 0.63 mm/s), and S (delta EQ = 1.2 mm/s and delta = 0.65 mm/s) iron sites and their temperature dependence are very similar to those observed for the P cluster pairs in the conventional MoFe proteins. Similar to those of the MoFe protein, strong field spectra indicate that these doublets are associated with a diamagnetic system. Their percent absorption intensities (Fe(2+)/D/S = 13.0:32.2:6.8) determined at 4.2 K after the removal of the contributions from the adventitiously bound iron are comparable to those of the P cluster pairs in MoFe proteins. These observations established that Av1' also contains P cluster pairs that are identical, in both composition and quantity, to those of the MoFe proteins; i.e. each molecule contains two P cluster pairs and each pair is formed by two Fe2+, five D, and one S iron sites. Considering that 52% absorption of the P component corresponding to two 8Fe clusters, the remaining 48% absorption determined for the M component is consistent with two 7Fe-containing FeV cofactors/molecule of Av1'. The fact that both P cluster pairs are found in the diamagnetic states implies that the S = 3/2 and S = 1/2 signals detected in earlier EPR measurements are associated with the FeV cofactor and suggests a spin mixture for the FeV cofactor in the dithionite-reduced Av1'.

Azotobacter vinelandii↗
PubMed1993

Crystal structures of oxidized and reduced Azotobacter vinelandii ferredoxin at pH 8 and 6.

Crystal structures of Azotobacter vinelandii ferredoxin I (FdI) have been solved and refined at 2.2 to 1.9-A resolution at pH 8 and 6 for both the oxidized and dithionite-reduced proteins. Only the [3Fe-4S] cluster is reduced by dithionite. The four structures (denoted FdI8ox, FdI8red, FdI6ox, and FdI6red) have been compared to address three questions: the effect of reduction at pH 8, the effect of pH change on the structure, and the effect of reduction at pH 6. Comparison of the FdI8ox and FdI8red structures shows that Asp-15 changes conformation in a manner consistent with increased anionic repulsion between this residue and the reduced [3Fe-4S]0 cluster. By revealing an electrostatic interaction between Asp-15 and the [3Fe-4S] cluster, this result supports the conclusion in the accompanying paper (Shen, B., Martin, L. L., Butt, J. N., Armstrong, F. A., Stout, C. D., Jensen, G. M., Stephens, P. J., LaMar, G. N., Gorst, C. M., and Burgess, B. K. (1993) J. Biol. Chem. 268, 25928-25939) that Asp-15 participates in protonation of the reduced [3Fe-4S]0 cluster at acid pH. The [3Fe-4S]0 cluster in the FdI8red structure also displays a distinct shift within the protein as well as internal distortions when compared to the [3Fe-4S]+ cluster in the FdI8ox structure. Comparison of the FdI8ox and FdI6ox structures shows that pH change does not have any significant effect on the [3Fe-4S]+ cluster or surrounding residues. Comparison of the FdI6ox and FdI6red structures shows that reduction at pH 6 also does not have any significant effect on the [3Fe-4S] cluster or Asp-15. The absence of structural change supports the conclusion that at acid pH, the reduced [3Fe-4S] cluster is protonated, i.e. [3Fe-4S]0-H+ (Shen et al., 1993). The cluster is not shifted or distorted as in the FdI8red structure. Instead, the [3Fe-4S]o-H+ cluster FdI8red is structurally similar to the [3Fe-4S]+ cluster (FdI8ox, FdI6ox), which has the same net charge. An Asp-15-Lys-84 salt bridge is observed in all four structures, indicating that Asp-15 is ionized at pH 8 and 6. An ionized state for Asp-15 is also implied by a lack of conformational change in Lys-84; the side chain of this residue rearranges when Asp-15 is substituted with a neutral amino acid (Shen et al., 1993).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

[Dinitrosyl complexes of iron with thiol-containing ligands and their reverse conversion into nitrosothiols].

Bovine serum albumin (BSA) is capable of forming dinitrosyl complexes with iron (DNIC) containing one thiol and one nonthiol ligand and yielding an EPR signal at the following values of the g-factor: g1 = 2.046; g2 = 2.03; g3 = 2.012. During interaction with L-cysteine or N-acetyl-L-cysteine the complex symmetry increased due to the substitution within DNIC of the nonthiol ligand of BSA for cysteine; such DNIC was characterized by an EPR signal with an axial-symmetrical tensor of the g-factor. At high cysteine concentrations all of the Fe(NO)-groups appeared to be transferred from BSA to cysteine to form DNIC with cysteine yielding an EPR signal with a g-factor of g perpendicular = 2.037; g parallel = 2.012. The lifetime of DNIC-BSA was about 24 hrs, whereas that of DNIC-cysteine was less than 1 min due to cysteine oxidation in the air. In 0.5 M HCl DNIC-BSA and DNIC-cysteine were reversibly converted into appropriate nitrosothiols characterized by intensive adsorption at 340-360 nm. Upon subsequent neutralization of the solution and addition of the substituent (cysteine or dithionite) these nitrosothiols were converted into DNIC. In the absence of iron cysteine and dithionite caused reductive destruction of protein and low molecular weight nitrosothiols to liberate nitrogen oxide. This property of nitrosothiols makes them distinct from those DNIC, in which cysteine acts exclusively as a scavenger of Fe(NO)-groups by shifting the equilibrium between the protein and low molecular weight DNIC towards the latter.

Acetylcysteine↗
PubMed1993

Small-angle x-ray scattering studies of the iron-molybdenum cofactor from Azotobacter vinelandii nitrogenase.

The nitrogenase enzyme complex, consisting of the molybdenum-iron protein and the iron protein, plays a critical role in the biological reduction of dinitrogen to ammonia (nitrogen fixation). The nitrogen-fixing site within the molybdenum-iron protein is an iron-molybdenum-sulfur cofactor (FeMoco) of roughly 1000-2000 Dalton mass. Structural aspects of FeMoco have been determined by spectroscopic and more recently by crystallographic studies. In order to determine the radius of gyration (Rg) of isolated FeMoco, we have performed small-angle x-ray scattering studies of FeMoco in N-methylformamide solution, in the absence of the molybdenum-iron protein. Model compounds of known structure have also been examined in similar solvents, N,N-dimethylformamide and acetonitrile, as controls and for calibration purposes. The Rg values obtained for the models are in good agreement with calculations based upon their respective crystal structures. However, the Rg obtained for FeMoco clearly indicates that the cofactor is not monomeric in solution, but rather aggregated and possibly polydisperse. Further, Rg values were also measured after addition of thiol, dithionite, and thiol and dithionite, to the FeMoco samples. The results indicate, surprisingly, that oxidation state and putative thiol coordination have no detectable effect on the aggregation behavior of FeMoco in solution, as determined by these measurements.

Azotobacter vinelandii↗
PubMed1999

Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: elimination of labeling artifacts.

Reliable protocols were established for investigating asymmetric distributions of 6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-caproyl (C6NBD) phospholipids in the plasma membrane of boar sperm cells under physiological conditions. A method based on fluorescence resonance energy transfer was used to ensure that incorporation of the fluorescent phospholipids into the sperm proceeded via monomeric transfer. The total amount of incorporated phospholipid fluorescence and the proportion of translocated phospholipid fluorescence were determined by flow cytometric analysis before, and after, dithionite destruction of outer leaflet fluorescence. Catabolism of incorporated fluorescent phospholipids was blocked with phenylmethylsulfonyl fluoride. Membrane-damaged cells were detected with impermeant DNA stains, thereby enabling their exclusion from subsequent analyses of the flow cytometric data, whence it could be demonstrated that the labeled phospholipids were incorporated only via the outer plasma membrane leaflet in living sperm cells. Phospholipid uptake and internalization was followed at 38 degrees C. After 1 hr of labeling, about 96% of the incorporated C6NBD-phosphatidylserine, 80% of C6NBD-phosphatidylethanolamine, 18% of C6NBD-phosphatidylcholine, and 4% of C6NBD-sphingomyelin were found to have moved across the plasma membrane bilayer to the interior of the spermatozoa. These inward movements of fluorescent phospholipids were ATP-dependent and could be blocked with sulfhydryl reagents. Movements from the inner to the outer leaflet of the sperm plasma membrane were minimal for intact fluorescent phospholipids, but were rapid and ATP-independent for fluorescent lipid metabolites. The described method enables, for the first time, assessment of changes in lipid asymmetry under fertilizing conditions.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

Thermodynamic control of electron transfer rates in multicentre redox proteins.

In the analysis of kinetic data from multicentre redox proteins, it is essential to distinguish between the observable macroscopic rate constants and the structurally relevant microscopic properties. This distinction is complicated by the existence of interactions between centres. The problem is illustrated by the case of two interacting redox centres and generalised for the analysis of stopped-flow kinetic data for the reduction of cytochrome c(3), in which four redox centres and at least one proteolytic centre are mutually interacting. It is shown that fast intramolecular electron transfer, which is typical of many multicentre redox proteins, and, where present, fast proton exchange, ensure that only N rate constants can be measured for a protein with N redox centres. The equations that relate the observable macroscopic rate constants to the microscopic rate constants of individual centres depend on a set of parameters that can be approximated by using the Marcus theory of electron transfer together with a set of reasonable assumptions. The results are tested by fitting experimental data for the reduction of cytochrome c(3) by sodium dithionite, including its pH dependence.

Binding Sites↗
PubMed1991

Interaction of benzanthrone with cytochrome P450: altered patterns of hepatic xenobiotic metabolism in rats.

Benzanthrone, an anthraquinone dye intermediate, is commonly used for the synthesis of a number of polycyclic vat and disperse dyes. Our prior studies have shown that benzanthrone can be metabolized by rat hepatic microsomal cytochrome P450 (P450) (Biochem. Int., 18, 1989, 1237). In this study, the interaction of benzanthrone with rat hepatic microsomal P-450 and its effect on xenobiotic metabolism have been investigated. Parenteral administration of benzanthrone (40 mg/kg body weight) for 3, 7, or 21 days caused no change in the relative body weight or organ weight of rats. The levels of P450 were found to be reduced (33%-50%) in all the benzanthrone-exposed animals at all the time periods. In vitro addition of benzanthrone caused a spectral change with oxidized P450 and concentration-dependent reduction in the carbon monoxide spectrum of dithionite-reduced P450. The addition of benzanthrone to hepatic microsomes prepared from phenobarbital-treated rats resulted in spectral changes characterized by an absorbance maximum at 397 nm indicative of type I binding. In vitro addition of benzanthrone showed a concentration-dependent inhibition of hepatic aminopyrine N-demethylase (APD) and ethoxyresorufin-O-deethylase (ERD) activities with respective I50 values of 9.5 x 10(-4) and 8.0 x 10(-5) M. However, the inhibition of aryl hydrocarbon hydroxylase (AHH) even at the highest concentration of benzanthrone (10(-2) M), was of the order of only 29%. In vivo administration of benzanthrone also led to the inhibition of APD, AHH, and ERD activities at all treatment times although the magnitude of inhibition was of a lower order.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Catalytic activity of cytochrome P4501A2 in reconstituted system with Emulgen 913.

Physicochemical properties and catalytic activity of cytochrome P4501A2 in the reconstituted system with Emulgen 913 were studied. The formation of cytochrome P4501A2 monomers was shown by gel filtration at an Emulgen concentration of 8 g/liter. The catalytic activity of the monomeric monooxygenase system was low. The maximum rate of the 7-ethoxyresorufin O-deethylation reaction, 150 pmol resorufin min-1 nmol-1 cytochrome P4501A2, was observed at an Emulgen concentration of 0.1 g/liter when cytochrome P4501A2 pentamers predominated. The effects of Emulgen on cytochrome P4501A2 cumene hydroperoxide-dependent peroxidase activity, on its affinity to substrate and to cytochrome b5, and on the rate constants of dithionite-dependent reduction were insignificant. Study of the NADPH-dependent reduction of cytochrome P4501A2 in the reconstituted system showed that the rate constant and reduction level of cytochrome P4501A2 were always higher when the reaction was initiated by NADPH than when it was initiated by NADPH-cytochrome P450 reductase. This indicated that the reduction reaction initiated by the reductase was limited by a step in the cytochrome P4501A2 and reductase interaction. Correlation between 7-ethoxyresorufin O-deethylase activity and reduction level of cytochrome P4501A2 was found.

Animals↗
PubMed1995

Reductant-independent ATP hydrolysis catalyzed by homologous nitrogenase proteins from Azotobacter vinelandii and heterologous crosses with Clostridium pasteuranium.

Reductant-independent ATPase activity was initiated and studied for Azotobacter vinelandii and Clostridium pasteuranium nitrogenase proteins (Av1, Cp1 and Av2, Cp2, 1 designating the iron molybdenum protein and 2 the iron protein) and their heterologous crosses by two methods: (1) allowing dithionite to be depleted from a normal assay in the presence of substrate levels of MgATP and (2) using reduced but reductant-free nitrogenase proteins in the presence of substrate levels of MgATP. In both cases, at a 1:1 protein ratio, MgATP is converted initially to MgADP with a specific activity of 400-500 nmol MgATP hydrolyzed/min.mg Av1, but in slower steps the MgADP is converted to AMP and, after 12 h, AMP is ultimately converted to adenosine. This reactivity requires the presence of both proteins, increases with increasing Av2/Av1 ratio, and is not a result of unique redox states of either protein. For Av1-Av2, ATP hydrolysis in the absence of Mg2+ occurred at nearly the same rate as reductant-dependent MgATP hydrolysis. Reductant-independent ATPase activity also occurred for the Av1-Cp2 and Cp1-Av2 heterologous crosses and was 2-fold and 18-fold slower than the Av1-Av2 or Cp1-Cp2 combinations. In both cases further hydrolysis of MgADP to AMP and AMP to adenosine occurred. A unique nucleotide hydrolysis system is apparently operating in the complex formed between the two nitrogenase proteins in the absence of reductant. The relationship between the reductant-independent and reductant-dependent activities of nitrogenase catalysis is explored.

Adenosine Triphosphate↗
PubMed1996

Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport.

Various authors have suggested that nitric oxide (.NO) exerts cytotoxic effects through the inhibition of cellular respiration. Indeed, in intact cells .NO inhibits glutamate-malate (complex I) as well as succinate (complex II)-supported mitochondrial electron transport, without affecting TMPD/ascorbate (complex IV)-dependent respiration. However, experiments in our lab using isolated rat heart mitochondria indicated that authentic .NO inhibited electron transport mostly by reversible binding to the terminal oxidase, cytochrome a3, having a less significant effect on complex II- and no effect on complex I-electron transport components. The inhibitory action of .NO was more profound at lower oxygen tensions and resulted in differential spectra similar to that observed in dithionite-treated mitochondria. On the other hand, continuous fluxes of .NO plus superoxide (O.(2)(-)), which lead to formation of micromolar steady-state levels of peroxynitrite anion (ONOO-), caused a strong inhibition of complex I- and complex II-dependent mitochondrial oxygen consumption and significantly inhibited the activities of succinate dehydrogenase and ATPase, without affecting complex IV-dependent respiration and cytochrome c oxidase activity. In conclusion, even though nitric oxide can directly cause a transient inhibition of electron transport, the inhibition pattern of mitochondrial respiration observed in the presence of peroxynitrite is the one that closely resembles that found secondary to .NO interactions with intact cells and strongly points to peroxynitrite as the ultimate reactive intermediate accounting for nitric oxide-dependent inactivation of electron transport components and ATPase in living cells and tissues.

Adenosine Triphosphatases↗
PubMed1994

A continuous, spectrophotometric activity assay for nitrogenase using the reductant titanium(III) citrate.

A continuous, spectrophotometric assay for determining electron transfer rates through nitrogenase during substrate reduction reactions was developed. The assay takes advantage of the facts that Ti(III) citrate can serve as a reductant for nitrogenase-catalyzed reduction reactions and that oxidation of Ti(III) citrate to Ti(IV) citrate results in a dramatic change in its absorption spectrum. Ti(III) citrate supported nitrogenase-catalyzed substrate (e.g., H+ or acetylene) reduction reactions at about the same rate as that supported by the reductant dithionite (S2O4(2-)). In addition, Ti(III) citrate had an absorption maximum centered at 325 nm, while oxidized Ti(IV) citrate had a much lower absorption in this wavelength region. An absorption coefficient for Ti(III) citrate of 0.73 mM-1.cm-1 at 340 nm was determined by titration with redox dyes with known absorption coefficients. Using this experimentally determined absorption coefficient, we developed an assay that provides a convenient way to determine electron transfer rates through nitrogenase in real time by spectrophotometrically following the oxidation of Ti(III) citrate to Ti(IV) citrate. Average electron transfer rates of 3749 +/- 218 nmol of electrons transferred.min-1.mg iron protein-1 for H+ reduction were determined using this assay which are directly comparable to the rates calculated from fixed time point, gas chromatographic assays of H2 formation. The utility of the Ti(III) citrate assay for nitrogenase is discussed and demonstrated using the nitrogenase inhibitors MgADP, CN-, and NO.

Azotobacter vinelandii↗
PubMed1993

Paradoxical stabilization of the neutral flavin semiquinone of xanthine dehydrogenase at high pH.

The pH dependence of the behavior of chicken liver xanthine dehydrogenase in the course of reductive titrations with sodium dithionite has been examined. Below pH 8.5, the behavior of xanthine dehydrogenase is similar to that of the much better understood milk xanthine oxidase, with the amount of neutral semiquinone accumulating transiently in the course of the titration increasing somewhat as the pH decreases. At pH 10, however, an anomalously large accumulation of the neutral semiquinone is observed by both UV/visible and EPR spectroscopy. Treatment of xanthine dehydrogenase with the thiol reagent iodoacetamide significantly diminishes the ability of the enzyme to stabilize the neutral flavin semiquinone at high pH. These data are consistent with the presence of a protein thiol in the immediate vicinity of the flavin, whose ionization above pH 8.5 results in thermodynamic stabilization of the neutral flavin semiquinone over the anionic form.

Animals↗
PubMed1996

Electron paramagnetic resonance (EPR) studies on hydrogenase-1 (HYD1) purified from a mutant strain (AP6) of Escherichia coli enhanced in HYD1.

Hydrogenase-1 (HYD1), overexpressed by twofold, has been purified to homogeneity and to a high specific activity from a mutant strain (AP6) of Escherichia coli which lacks hydrogenase-2. Plasma emission spectroscopy indicated that 0.93 atom of nickel and 11.4 iron atoms were present in HYD1. EPR studies on the as isolated HYD1 detected a complex 3Fe-4S signal and a Ni(III) species. Reduction with hydrogen gas caused disappearance of both the 3Fe-4S cluster and initial Ni(III) signals. At the same time the EPR signature (small g = 2.19 signal) of the activated hydrogenase appeared. The detection of a 4Fe-4S cluster signal was noted. Reduction of HYD1 with sodium dithionite caused all nickel signals to disappear. The 4Fe-4S complex intensity was slightly increased. The EPR responses in the three oxidation-reduction states are consistent with other known (NiFe)-hydrogenases.

Dithionite↗
PubMed2002

A physiochemical mechanism of hemozoin (beta-hematin) synthesis by malaria parasite.

Malaria parasite homogenate, the lipid extracts, and an unsaturated fatty acid, linoleic acid, which have been shown to promote beta-hematin formation in vitro, were used to investigate the mechanism of hemozoin biosynthesis, a distinct metabolic function of the malaria parasite. In vitro beta-hematin formation promoted by Plasmodium yoelii homogenate, the lipid extracts, and linoleic acid were blocked by ascorbic acid, reduced glutathione, sodium dithionite, beta-mercaptoethanol, dithiothreitol, and superoxide dismutase. Oxidized glutathione did not show any effect. Preoxidized preparations of the lipids extracts or the P. yoelii homogenate failed to catalyze beta-hematin formation. Depletion of oxygen in the reaction mixtures also inhibited the lipid-catalyzed beta-hematin formation. Under the reaction conditions similar to those used for the in vitro beta-hematin formation assay, the antioxidants and reducing agents mentioned above, except the DTT and beta-mercaptoethanol, did not cause degradation of heme. beta-Hematin formation was also inhibited by p-aminophenol, a free radical chain reaction breaker. Hemozoin biosynthesis within the digestive vacuoles of the malaria parasite may be a lipid-catalyzed physiochemical reaction. An oxidative mechanism may be proposed for lipid-mediated beta-hematin formation, which may be mediated by generation of some free radical intermediates of heme.

Aminophenols↗
PubMed1975

Biochemical characterization of highly purified cytochrome P-450 and other components of the mixed function oxidase system of liver microsomal membranes.

Cytochrome P-450 has been purified from liver microsomes of phenobarbital-induced rabbits in the presence of ionic and nonionic detergents to concentrations over 17 nmoles per mg of protein. The purified cytochrome P-450 LM gives a single major band on SDS-polyacrylamide gel electrophoresis representing about 90 per cent of the total protein. The polypeptide chain has a molecular weight of about 49,000 daltons. NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-induced rats in the presence of ionic and nonionic detergents to a stage where it catalyzes the reduction of 33,000 nmoles of cytochrome c per min per mg of protein. The ratio of activities toward cytochrome P-450 and cytochrome c is constant throughout purification. The purified reductase contains equimolar amounts of FMN and FAD and gives a single major band on SDA-polyacrylamide gel electrophoresis accounting for about 70 per cent of the total protein; the molecular weight is about 80,000 daltons. The purified cytochrome P-450 is free of cytochrome b5 but contains another electron acceptor, provisionally called Factor C, which is equivalent in amount to the heme present. Two electrons are taken up per molecule of cytochrome P-450 from dithionite or from NADPH in the presence of catalytic amounts of the reductase, and both electrons are readily transferred from the reduced cytochrome P-450 to molecular oxygen or artificial electron acceptors. The reconstituted enzyme system containing purified cytochrome P-450, purified NADPH-cytochrome P-450 reductase, and phosphatidylcholine retains the ability to catalyze the hydroxylation of drugs, fatty acids, hydrocarbons, and aniline in the presence of NADPH and molecular oxygen.

Anaerobiosis↗