Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes

An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65–70% of the rate with NADH. Added cytochrome b₅ stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b₅ and cytochrome b₅ reductase determine the rate of methemoglobin reduction in hemolysates.     

Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes.

An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65-70% of the rate with NADH. Added cytochrome b5 stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b5 and cytochrome b5 reductase determine the rate of methemoglobin reduction in hemolysates.     

Studies on methemoglobin reductase. Immunochemical similarity of soluble methemoglobin reductase and cytochrome b5 of human erythrocytes with NADH-cytochrome b5 reductase and cytochrome b5 of rat liver microsomes.

An antibody preparation elicited against purified, lysosomal-solubilized NADH-cytochrome b₅ reductase from rat liver microsomes was shown to interact with methemoglobin reductase of human erythrocytes by inhibiting the rate of erythrocyte cytochrome b₅ reduction by NADH. The ferricyanide reductase activity of the enzyme was not inhibited by the antibody, suggesting that the inhibition of methemoglobin reductase activity may be due to interference with the binding of cytochrorme b₅ to the flavoprotein. Under conditions of limiting concentrations of flavoprotein, the antibody inhibited the rate of methemoglobin reduction in a reconstituted system consisting of homogeneous methemoglobin reductase and cytochrome b₅ from human erythrocytes. This inhibition was due to the decreased level of reduced cytochrome b₅ during the steady state of methemoglobin reduction while the rate of methemoglobin reduction per reduced cytochrome b₅ stayed constant, suggesting that the enzyme was not concerned with an electron transport between the reduced cytochrome b₅ and methemoglobin.An antibody to purified, trypsin-solubilized cytochrome b₅ from rat liver microsomes was shown to inhibit erythrocyte cytochrome b₅ reduction by methemoglobin reductase and NADH to a lesser extent than microsomal cytochrome b₅ preparations from rat liver (trypsin solubilized or detergent solubilized) and pig liver (trypsin solubilized). The results presented establish that soluble methemoglobin reductase and cytochrome b₅ of human erythrocytes are immunochemically similar to NADH-cytochrome b₅ reductase and cytochrome b₅ of liver microsomes, respectively.     

The oxidation-reduction characteristics of cytochrome b5 in hen liver microsomes.

Hen liver microsomes contained 0.20 nmol of cytochromeb₅ per mg of protein. Upon addition of NADH about 95% cytochrome b₅ was reduced very fast with a rate constant of 206 s^?1When ferricyanide was added to the reaction system the cytochrome stayed in the oxidized form until the ferricyanide reduction was almost completed. The reduced cytochrome b₅ in microsomes was oxidized very rapidly by ferricyanide. The rate constant of 4.5 × 10⁸m^?1 s^?1, calculated on the basis of assumption that ferricyanide reacts directly with the cytochrome, was found to be more than 100 times higher than that of the reaction between ferricyanide and soluble cytochrome b₅. To explain the results, therefore, the reverse electron flow from cytochrome b₅ to the flavin coenzyme in microsomes was assumed.By three independent methods the specific activity of the microsomes was measured at about 20 nmol of NADH oxidized per s per mg of protein and it was concluded that the reduction of the flavin coenzyme of cytochrome b₅ reductase by NADH is rate-limiting in the NADH-cytochrome b₅ and NADH-ferricyanide reductase reactions of hen liver microsomes. In the NADH-ferricyanide reductase reaction the apparent Michaelis constant for NADH was 2.8 μm and that for ferricyanide was too low to be measured. In the NADH-cytochrome c reductase reaction the maximum velocity was 2.86 nmol of cytochrome c reduced per s per mg of protein and the apparent Michaelis constant for cytochrome c was 3.8 μm.     

Reconstitution of the reduced nicotinamide adenine dinucleotide phosphate- and reduced nicotinamide adenine dinucleotide-peroxidase activities from solubilized components of rat liver microsomes.

The liver microsomal enzyme system that catalyzes the oxidation of NADPH by organic hydroperoxides has been solubilized and resolved by the use of detergents into fractions containing NADPH-cytochrome c reductase, cytochrome P-450 (or P-448), and microsomal lipid. Partially purified cytochromes P-450 and P-448, free of the reductase and of cytochrome b₅, were prepared from liver microsomes of rats pretreated with phenobarbital (PB) and 3-methylcholanthrene (3-MC), respectively, and reconstituted separately with the reductase and lipid fractions prepared from PB-treated animals to yield enzymically active preparations functional in cumene hydroperoxide-dependent NADPH oxidation. The reductase, cytochrome P-450 (or P-448), and lipid fractions were all required for maximal catalytic activity. Detergent-purified cytochrome b₅ when added to the complete system did not enhance the reaction rate. However, the partially purified cytochrome P-450 (or P-448) preparation was by itself capable of supporting the NADPH-peroxidase reaction but at a lower rate (25% of the maximal velocity) than the complete system. Other heme compounds such as hematin, methemoglobin, metmyoglobin, and ferricytochrome c could also act as comparable catalysts for the peroxidation of NADPH by cumene hydroperoxide and in these reactions, NADH was able to substitute for NADPH. The microsomal NADH-dependent peroxidase activity was also reconstituted from solubilized components of liver microsomes and was found to require NADH-cytochrome b₅ reductase, cytochrome P-450 (or P-448), lipid, and cytochrome b₅ for maximal catalytic activity. These results lend support to our earlier hypothesis that two distinct electron transport pathways operate in NADPH- and NADH-dependent hydroperoxide decomposition in liver microsomes.     

Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5 as electron carriers in NADH-supported cytochrome P-450 -dependent enzyme activities in liver microsomes

The role of NADH-cytochrome b₅ reductase and cytochrome b₅ as electron carriers in NADH-supported electron transport reactions in rat liver microsomes has been examined by measuring three enzyme activities: NADH-cytochrome P-450 reductase, NADH-peroxidase, and NADH-cytochrome c reductase. The first two reactions are known to involve the participation of an NADH-specific reductase and cytochrome P-450 whereas the third requires the reductase and cytochrome b₅. Antibody prepared against NADH-cytochrome b₅ reductase markedly inhibited the NADH-peroxidase and NADH-cytochrome c reductase activities suggesting the involvement of this NADH-specific reductase in these reactions. Liver microsomes prepared from phenobarbital-pretreated rats were digested with subtilisin to remove cytochrome b₅ and the submicrosomal particles were collected by centrifugation. The specific content of cytochrome b₅ in the digested particles was about 5% of that originally present in liver microsomes and all three enzyme activities showed similar decreases whereas NADH-ferricyanide reductase activity (an activity associated with the flavoenzyme NADH-cytochrome b₅ reductase) remained virtually unchanged. Binding of an excess of detergent-purified cytochrome b₅ to the submicrosomal particles at 37 °C for 20 min followed by centrifugation and enzymic measurements revealed a striking increase in the three enzyme activities. Further evidence for cytochrome b₅ involvement in the NADH-peroxidase reaction was the marked inhibition by antibody prepared against the hemoprotein. These results suggest that in microsomal NADH-supported cytochrome P-450-dependent electron transport reactions, cytochrome b₅ functions as an intermediate electron carrier between NADH-cytochrome b₅ reductase and cytochrome P-450.     

Human erythrocytes exposure to juglone leads to an increase of superoxide anion production associated with cytochrome b5 reductase uncoupling

Cytochrome b₅ reductase is an enzyme with the ability to generate superoxide anion at the expenses of NADH consumption. Although this activity can be stimulated by cytochrome c and could participate in the bioenergetic failure accounting in apoptosis, very little is known about other molecules that may uncouple the function of the cytochrome b₅ reductase. Naphthoquinones are redox active molecules with the ability to interact with electron transfer chains. In this work, we made an inhibitor screening against recombinant human cytochrome b₅ reductase based on naphthoquinone properties. We found that 5-hydroxy-1,4-naphthoquinone (known as juglone), a natural naphthoquinone extracted from walnut trees and used historically in traditional medicine with ambiguous health and toxic outcomes, had the ability to uncouple the electron transfer from the reductase to cytochrome b₅ and ferricyanide. Upon complex formation with cytochrome b₅ reductase, juglone is able to act as an electron acceptor leading to a NADH consumption stimulation and an increase of superoxide anion production by the reductase. Our results suggest that cytochrome b₅ reductase could contribute to the measured energetic failure in the erythrocyte apoptosis induced by juglone, that is concomitant with the reactive oxygen species produced by cytochrome b₅ reductase.     

Purification and Characterization of Microsomal Cytochrome b(5) and NADH Cytochrome b(5) Reductase from Pisum sativum

In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b₅ and NADH-cytochrome b₅ reductase from the microsomal fraction of Pisum sativum. The cytochrome b₅ component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The amino-terminal amino acid sequence of the cytochrome b₅ component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer.     

Studies on NADH-cytochrome b5 reductase activities in hemolysates of human and rabbit red cells by isoelectric focusing

NADH-cytochrome $\text{b}{}_5$ reductase activities in hemolysates of young and old human erythrocytes, and in hemolysates of rabbit reticulocytes and erythrocytes were measured after the separation of the enzyme from the bulk of hemoglobin only by isoelectric focusing. In any cases, a single main peak of the enzyme activity was detected after the electrophoresis in the fraction with pH 6.8 and 8.3 for human and rabbit red cells, respectively. The rabbit enzyme showed more than 30 times higher enzyme activity than that of human erythrocytes under the standard assay conditions. Significant differences of Micahelis constants for cytochrome $\text{b}{}_5$ of the enzyme were found between young and old human erythrocytes, and also between human and rabbit red cells.     

Magnetic circular dichroism studies of hemoglobin: The reduction of ferrihemoglobin by ferrocytochrome b5 and characterization of the high-spin hydroxy species of mixed-valence hemoglobin

The final step in the erythrocyte methemoglobin reduction pathway, the transfer of an electron from cytochrome b₅, to methemoglobin, has been studied using magnetic circular dichroism spectroscopy. Spectral analysis allowed us to determine accurately the concentration of each redox species in mixtures of the two heme-proteins and to follow simultaneously the kinetics of the appearance or disappearance of each of these species during reduction reactions. Our analysis detected a substantial increase in the high-spin hydroxymethemoglobin species in the partially reduced bovine hemoglobin tetramer. This species was sensitive to the degree of reduction and pH, and was spectrally similar to fluoride methemoglobin. At pH 7.8. 100% of the hydroxide component of methemoglobin was in the high-spin form when two or more subunits were in the ferrous form. Kinetic analysis of bovine methemoglobin reduction yielded values for the apparent first-order rates for the tetrameric species possessing four, three, two, and one ferric subunit. Further analysis showed that the reduction kinetics can also be described by an equilibrium state, pure competitive inhibition model for enzyme catalysis in which ferrous and ferric subunits of hemoglobin compete for cytochrome b₅ This analysis generated a K_D that depends on ionic strength and hemoglobin tetramer conformation, a V_max that was independent of these factors, and an inhibition constant that was equal to K_d. This model is consistent with the hypothesis that the reduction of methemoglobin can be separated into two steps, the ionic interaction between cytochrome b₅ and hemoglobin and the electron transfer.     

Immunological similarity between NADH-cytochrome b5 reductase of erythrocytes and liver microsomes

In a number of animal species soluble NADH-cytochrome b₅ reductase of erythrocytes was compared with membrane-bound NADH-cytochrome b₅ reductase of liver microsomes by using an antibody to purified NADH-cytochrome b₅ reductase from rat liver microsomes. The results obtained indicated clearly that they are immunologically very similar to each other. The data with erythrocyte ghosts suggested that cytochrome b₅ and NADH-cytochrome b₅ reductase are also present in the ghost.     

Immunochemical evidence for the participation of cytochrome b5 in microsomal stearyl-CoA desaturation reaction

A rabbit antiserum was prepared against rat liver microsomal cytochrome b₅, and utilized in demonstrating the participation of this cytochrome in the microsomal stearyl-CoA desaturation reaction. The antiserum inhibited the NADH-cytochrome c reductase activity of rat liver microsorncs, but it did not inhibit either NADH-ferricyanide or NADPH-cytochrome c reductase activity of the microsomes. Thus, the inhibitory effect of the antiserum on the microsomal electron-transferring reactions seemed to be specific to those which require the participation of cytochrome b₅.The NADH-dependent and NADPH-dependent desaturations of stearyl CoA by rat liver microsomes were strongly inhibited by the antiserum. The reduction of cytochrome b₅ by NADH-cytochrome b₅ reductase as well as the reoxidation of the reduced cytochrome b₃ by the desaturase, the terminal cyanide-sensitive factor of the desaturation system, was also strongly inhibited by the antiserum. When about 90%, of cytochrome b₅ was removed from rat liver microsomes by protease treatment, the desaturation activity of the microsomes became much more sensitive to inhibition by the antiserum. These results confirmed our previous conclusion that the reducing equivalent for the desaturation reaction is transferred from NAD(P)H to the cyanidesensitive factor mainly via cytochrome b₅ in the microsomal membranes.     

Studies on three microsomal electron transfer enzyme systems: Specificity of electron flow pathways

The routes of microsomal electron flow to the three terminal oxidative enzymes, the mixed function oxidase, the fatty acyl CoA desaturase, and the lipid peroxidase have been examined by the use of specific antibodies, by alteration of electron transfer enzyme levels, and with the inhibitor NADP⁺. From these studies a number of conclusions are drawn: (1) NADH-supported lipid peroxidation utilizes NADH-cytochrome b₅ reductase, but electron flow does not go via cytochrome b₅. (2) The positive modifier effect of type I substrates on NADPH-driven cytochrome P-450 reduction is seen also with NADH-supported cytochrome P-450 reductase activity. The latter reaction proceeds via cytochrome b₅ while the former does not. (3) Cross-reactivity can occur between NADH-cytochrome b₅ reductase and NADPH-cytochrome c reductase, but at a rate too slow to support most reactions. (4) Cytochrome b₅ appears to exist in two pools; one pool is readily inhibited by antibody and the other pool is either inaccessible to or incompletely inhibited by antibody. The various cytochrome b₅-dependent reactions show different abilities to use the noninhibited hemoprotein. NADH-cytochrome c reductase activity and NADH-synergism appear to utilize only the former pool and are completely inhibitable by antibody. Other NADH-supported reactions (Δ⁹-desaturation and mixedfunction oxidation) utilize the total cytochrome b₅ population. Fortification studies show that the extra bound cytochrome b₅ is distributed in the same manner as the endogenous cytochrome b₅.     

Role of NADPH and the NADPH-dependent methemoglobin reductase in the hydroxylase activity of human erythrocytes

Human erythrocytes were shown previously to catalyze the oxyhemoglobin-requiring hydroxylation of aniline, and the reaction was stimulated apparently preferentially by NADPH in the presence of methylene blue (K. S. Blisard and J. J. Mieyal,J. Biol. Chem.254, 5104, 1979). The current study provides a further characterization of the involvement of the NADPH-dependent electron transport system in this reaction. In accordance with the role of NADPH, the hydroxylase activity of erythrocytes or hemolysates from individuals with glucose-6-phosphate dehydrogenase deficiency (i.e., with diminished capacity to form NADPH) displayed decreased responses to glucose or glucose 6-phosphate, respectively, in the presence of methylene blue in comparison to samples from normal adults; maximal activity could be restored by direct addition of NADPH to the deficient hemolysates. Kinetic studies of the methylene blue-stimulated aniline hydroxylase activity of normal hemolysates revealed a biphasic dependence on NADPH concentrations: a plateau was observed at relatively low concentrations (K_m^NADPH ~ 20 μm), whereas saturation was not achieved at the higher concentrations of NADPH. The latter low efficiency phase (i.e., at the higher concentrations of NADPH) could be ascribed to a direct transfer of electrons from NADPH to methylene blue to hemoglobin. The high efficiency phase suggested involvement of the NADPH-dependent methemoglobin reductase; accordingly 2′-AMP, an analog of NADP⁺, effectively inhibited this reaction, but the pattern was noncompetitive. This behavior is suggestive of a mechanism by which both NADPH and methylene blue are substrates for the reductase and interact with it in a sequential fashion. The kinetic patterns observed for variation in NADPH concentration at several fixed concentrations of methylene blue, and vice versa, are consistent with this interpretation.     

Functional activity of the NADH-dependent reductase system in liver and Guerin’s carcinoma microsomal fraction in rats exposed to preliminary irradiation

The activity of liver microsomal and Guerin??s carcinoma NADH-cytochrome b ₅ reductase, the content and the rate of cytochrome b ₅ oxidation-reduction have been investigated in tumor-bearing rats exposed to preliminary irradiation. Preliminary irradiation of rats (before transplantation of Guerin??s carcinoma) resulted in the decrease of NADH-cytochrome b ₅ reductase activity and the content of cytochrome b ₅ in the Guerin??s carcinoma microsomal fraction in the latent and logarithmic phases of oncogenesis compared with the non-irradiated tumor-bearing rats. The effect of irradiation preceding transplantation of the tumor to rats results in the increase of enzymatic activities of liver microsomal NADH-cytochrome b ₅ reductase in the latent and logarithmic phases of tumor growth as compared with non-irradiated tumor-bearing rats. At the same time the content of cytochrome b ₅ decreased, while the rate of its oxidation-reduction rate simultaneously increased in the liver microsomal fraction of tumor-bearing rats.     

One-electron oxidation-reduction properties of ascorbic acid

The one-electron oxidation-reduction properties of ascorbate were investigated by EPR. The oxidations of ascorbate by 2,6-dichlorophenolindophenol (2-equivalent oxidant) and by ferricyanide (1-equivalent oxidant) both proceeded via a one-electron transfer mechanism, yielding ascorbate free radical as an intermediate. For the reduction of both 2,6-dichlorophenolindophenol and ferricyanide, the ascorbate free radical was much more reactive than ascorbate itself. The ascorbate free radical could also act as an effective one-electron oxidant for microsomal NADH-cytochrome b₅ reductase, cytochrome b₅ and mitochondrial outer membrane cytochrome b₅. The results suggest that in biological systems the reduction of ascorbate free radical is operative in the regeneration of fully reduced ascorbate.     

Purification and properties of human erythrocyte membrane NADH-cytochrome b5 reductase

An NADH:(acceptor) oxidoreductase (EC 1.6.99.3) of human erythrocyte membrane was purified by DEAE-cellulose anion exchange, hydroxyapatite adsorption, and 5′-ADP-hexane-agarose affinity chromatographies after solubilization with Triton X-100. The purified reductase preparation was homogeneous and estimated to have an apparent molecular weight of 36,000 on SDS-polyacrylamide slab gel electrophoresis and of 144,000 on Sephadex G-200 gel filtration in the presence of 0.2% Triton X-100, whereas a soluble NADH-cytochrome b₅ reductase of human erythrocyte had a molecular weight of 32,000 by both methods, indicating the existence of a distinct membrane reductase. Digestion of the membrane reductase with cathepsin D yielded a new polypeptide chain which gave the same relative mobility as the soluble reductase on SDS-polyacrylamide slab gel electrophoresis. The membrane enzyme, the cathepsin-digested enzyme, and the soluble enzyme all cross-reacted with the antibody to rat liver microsomal NADH-cytochrome b₅ reductase. The enzyme had one mole FAD per 36,000 as a prosthetic group and could reduce K₃Fe(CN)₆, 2,6-dichlorophenolindophenol, cytochrome c, methemoglobin-ferrocyanide complex, cytochrome b₅ and methemoglobin via cytochrome b₅ when NADH was used as an electron donor. NADPH was less effective as an electron donor than NADH. The specific activity of the purified enzyme was 790 μmol ferricyanide reduced min^?1 mg^?1 and the turnover number was 40,600 mol ferricyanide reduced min^?1 mol^?1 FAD at 25 °C. The apparent K_m values for NADH and cytochrome b₅ were 0.6 and 20 μm, respectively, and the apparent V value was 270 μmol cytochrome b₅ reduced min^?1 mg^?1. These kinetic properties were similar to those of the soluble NADH-cytochrome b₅ reductase. The results indicate that the NADH:(acceptor) oxidoreductase of human erythrocyte membrane could be characterized as a membrane NADH-cytochrome b₅ reductase.     

Methemoglobin reduction under near physiological conditions

Pure methemoglobin was prepared from fresh red cells and was used as substrate for methemoglobin reduction reaction. Two sources of methemoglobin reductase were used: (a) red cell hemolysate which was prepared by freezing and thawing of unwashed red cells; (b) purified methemoglobin reductase from bank blood. Methemoglobin reduction rate was measured in a mixture of pure methemoglobin (substrate) and hemolysate (enzyme). In other experiments the rate of methemoglobin reduction was measured in the above mixture with the addition of various other compounds such as NADH, cytochrome b5, and pure methemoglobin reductase. Only the addition of pure enzyme accelerated the rate of methemoglobin reduction. In other experiments, the rate of methemoglobin reduction was measured when the reduction reaction was carried out in the presence of various amounts of deoxyhemoglobin, globin, or albumin. It was shown that all proteins tested here decreased the reduction rate. It is concluded that (a) in the red cell, under normal conditions, only the activity of the methemoglobin reductase controls the speed of methemoglobin reduction, and (b) the inhibition of methemoglobin reduction by reduced hemoglobin is mostly nonspecific suggesting a noncompetitive reaction.     

Inhibitory effect of α-tocopherol and its derivatives on bovine heart succinate-cytochrome c reductase

α-Tocopherol and its derivatives inhibit succinate-cytochrome c reductase activity at a concentration of 0.5 μmol/mg protein in 50 mM phosphate buffer, pH 7.4, containing 0.4 % sodium cholate when α-tocopherol is predispersed in sodium cholate solution. The inhibitory site is located at the cytochrome b-c₁ region. Succinate-ubiquinone reductase activity of succinate-cytochrome c reductase was not impaired by treatment with α-tocopherol. The α-tocopherol-inhibited succinate-cytochrome c reductase activity can be reversed by the addition of ubiquinone and its analogs. When ubiquinone- and phospholipid-depleted succinate-cytochrome c reductase was treated with α-tocopherol followed by reaction with a fixed amount of 2,3-dimethoxy-6-methyl-5-(10-bromodecyl)-1,4-benzoquinone and phospholipid, the amount of α-tocopherol needed to express the maximal inhibition was only 0.3 μmol/mg protein. When ubiquinone- and phospholipid-depleted enzyme was treated with a given amount of α-tocopherol and followed by titration with 2,3-dimethoxy-6-methyl-5-(10-bromodecyl)-1,4-benzoquinone, restoration of activity was enhanced at low concentrations of ubiquinone analog, indicating that α-tocopherol can serve as an effector for ubiquinone. The maximal binding capacity of α-[¹⁴C]tocopherol, dispersed in 50 mM phosphate buffer containing 0.25% sodium cholate, pH 7.4, to succinate-cytochrome c reductase was shown to be 0.68 μmol/mg protein. A similar binding capacity, based on cytochrome b content, was observed in submitochondrial particles. Binding of α-tocopherol to succinate-cytochrome c reductase not only caused an inhibition of enzymatic activity but also caused a reduction of cytochrome c₁ in the absence of substrate, a phenomenon analogous to the removal of phospholipids from the enzyme preparation. Furthermore, binding of α-tocopherol to succinate-cytochrome c reductase decreased the rate of reduction of cytochrome b by succinate. Since electron transfer from succinate to ubiquinone was not affected by α-tocopherol treatment, the decrease in reduction rate of cytochrome b by succinate must be due to a change in environment around cytochrome b. These results as well as the fact that reactivation of α-tocopherol-inhibited enzyme requires only low concentrations of ubiquinone were used to explain the inhibitory effect as a result of a change in protein conformation and protein-phospholipid interaction rather than the direct displacement of ubiquinone by α-tocopherol. This deduction was further supported by the fact that no ubiquinone was released from succinate-cytochrome c reductase upon treatment with α-tocopherol.     

Ascaris suum cytochrome b5, an adult-specific secretory protein reducing oxygen-avid ferric hemoglobin

The anaerobic parasitic nematode Ascaris suum has an oxygen-avid hemoglobin in the perienteric fluid, the biological function of which remains elusive. Here, we report that Ascaris cytochrome b₅ is expressed specifically in the intestinal parasitic stage and is secreted into the perienteric fluid, thus co-localizing with Ascaris hemoglobin. We also found that cytochrome b₅ reduces Ascaris non-functioning ferric methemoglobin more efficiently than mammalian methemoglobin. Furthermore, a computer graphics model of the electron transfer complex between Ascaris cytochrome b₅ and Ascaris hemoglobin strongly suggested that these two proteins are physiological redox partners. Nitric oxide has been reported to react easily with oxygen captured in hemoglobin to form nitrate, but not toxic free radicals, which may result in production of methemoglobin for the cytochrome b₅ to regenerate functional ferrous hemoglobin. Therefore, our findings suggest that Ascaris cytochrome b₅ is a key redox partner of Ascaris hemoglobin, which acts as an antioxidant.