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.     

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.     

Purification and characterization of an azoreductase from Escherichia coli CD-2 possessing quinone reductase activity

An oxygen-insensitive intracellular enzyme that is responsible for the decolorization of azo dyes was purified from Escherichia coli CD-2. The molecular weight of the purified enzyme was estimated as 27,000 ± 500 Da. Protein identification indicated that the enzyme had high sequence homology with E. coli K12 quinone reductase, and the enzyme was proved to have both azoreductase and quinone reductase activity. With methyl red as substrate, the optimal pH value and temperature were 6.5 and 37 °C, respectively. The enzyme was stable under different physiochemical conditions. The azoreductase activity was restrained by SDS and was almost completely inhibited by Co²⁺ and Hg²⁺. K_m and V_max values were 0.18 mM and 8.12 U mg^?1 of protein for NADH and 0.05 mM and 6.46 U mg^?1 of protein for methyl red, respectively. The purified enzyme could efficiently decolorize methyl red with both NADH and NADPH as electron donors.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.     

Selection of an endogenous 2,3-butanediol pathway in Escherichia coli by fermentative redox balance

Fermentative redox balance has long been utilized as a metabolic evolution platform to improve efficiency of NADH-dependent pathways. However, such system relies on the complete recycling of NADH and may become limited when the target pathway results in excess NADH stoichiometrically. In this study, endogenous capability of Escherichia coli for 2,3-butanediol (2,3-BD) synthesis was explored using the anaerobic selection platform based on redox balance. To address the issue of NADH excess associated with the 2,3-BD pathway, we devised a substrate-decoupled system where a pathway intermediate is externally supplied in addition to the carbon source to decouple NADH recycling ratio from the intrinsic pathway stoichiometry. In this case, feeding of the 2,3-BD precursor acetoin effectively restored anaerobic growth of the mixed-acid fermentation mutant that remained otherwise inhibited even in the presence of a functional 2,3-BD pathway. Using established 2,3-BD dehydrogenases as model enzyme, we verified that the redox-based selection system is responsive to NADPH-dependent reactions but with lower sensitivity. Based on this substrate-decoupled selection scheme, we successfully identified the glycerol/1,2-propanediol dehydrogenase (Ec-GldA) as the major enzyme responsible for the acetoin reducing activity (k_cat/K_m≈0.4 mM⁻¹ s⁻¹) observed in E. coli. Significant shift of 2,3-BD configuration upon withdrawal of the heterologous acetolactate decarboxylase revealed that the endogenous synthesis of acetoin occurs via diacetyl. Among the predicted diacetyl reductase in E. coli, Ec-UcpA displayed the most significant activity towards diacetyl reduction into acetoin (V_max≈6 U/mg). The final strain demonstrated a meso-2,3-BD production titer of 3 g/L without introduction of foreign genes. The substrate-decoupled selection system allows redox balance regardless of the pathway stoichiometry thus enables segmented optimization of different reductive pathways through enzyme bioprospecting and metabolic evolution.     

Purification and characterization of a novel carbonyl reductase involved in oxidoreduction of aromatic β-amino ketones/alcohols

Aromatic β-amino ketones/alcohols such as adrenalone play an important role in some stereoselective synthesis of pharmaceuticals. Unfortunately, the transformation of aromatic β-amino ketones to their chiral alcohols has been carried out chemically as no corresponding biocatalyst has been available. Here, a novel carbonyl reductase responsible for the reduction of adrenalone to (R)-(−)-epinephrine was identified and characterized from Kocuria rhizophila. This enzyme was purified to homogeneity by ammonium sulfate precipitation followed by ion-exchange column chromatography, hydrophobic chromatography and gel chromatography. The purified enzyme yielded pure (R)-enantiomer product with high activity and utilized NADH as the cofactor. The enzyme had special significance by showing selectivity for many aromatic β-amino ketones/alcohols such as 2-amino-acetophenone, 2-amino-4′-hydroxyacetophenone, isoproterenol and ephedrine. The maximum reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) for adrenalone and NADH were 14.62 μmol/(min mg) protein and 0.189 mM, 11.66 μmol/(min mg) protein and 0.204 mM respectively. These properties ensure the enzyme a promising future for industrial application as a replacement of chemical synthesis of aromatic β-amino chiral alcohols.     

Purification and characterization of a new carbonyl reductase from Leifsonia xyli HS0904 involved in stereoselective reduction of 3,5-bis(trifluoromethyl) acetophenone

Leifsonia xyli HS0904 can stereoselectively catalyze the bioreduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to its corresponding alcohol, which is a valuable chiral intermediate in the pharmaceuticals. In this study, a new carbonyl reductase derived from L. xyli HS0904 was purified and its biochemical properties were determined in detail. The carbonyl reductase was purified by 530-fold with a specific activity of 13.2 U mg⁻¹ and found to be a homodimer with a molecular mass of 49 kDa, in which the subunit molecular-weight was about 24 kDa. The purified enzyme exhibited a maximum enzyme activity at 34 °C and pH 7.2, and retained over 90% of its initial activity at 4 °C and pH 7.0 for 24 h. The addition of various additives, such as Ca²⁺, Mg²⁺, Mn²⁺, l-cysteine, l-glutathione, urea, PEG 1000 and PEG 4000, could enhance the enzyme activity. The maximal reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) of the purified carbonyl reductase for BTAP and NADH were confirmed as 33.9 U mg⁻¹, 0.383 mM and 69.9 U mg⁻¹, 0.412 mM, respectively. Furthermore, this enzyme was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters.     

Liver microsomal electron transport systems. Properties of a reconstituted, NADH-mediated benzo[a]pyrene hydroxylation system.

An enzyme system from rat liver microsomes which catalyzes the NADH-mediated hydroxylation of benzo[a]pyrene has been reconstituted. The essential microsomal components of this NADH-dependent pathway were NADH-cytochrome b₅ reductase, cytochrome b₅, cytochrome P-448 and, phosphatidyl choline. Highly purified NADPH-cytochrome c reductase containing small amounts of deoxycholate stimulated this NADH-mediated pathway supported by 0.2 mm NADH whereas boiled reductase had little effect. Part of this stimulation could be attributed to hydroxylation of benzo[a]pyrene via a second pathway; i.e., NADPH-cytochrome c reductase in combination with cytochrome P-448 and phosphatidylcholine also supported a low rate of NADH-dependent hydroxylation. The mechanism of the remaining stimulation is not known. However, the effect of NADPH-cytochrome c reductase on the reconstituted cytochrome b₅-dependent pathway was not unique; high concentrations of deoxycholate also stimulated this pathway, perhaps by facilitating the transfer of electrons from NADH-cytochrome b₅ reductase to cytochrome b₅. The addition of NADPH-cytochrome c reductase to the cytochrome b₅-dependent reconstituted system also affected the apparent K_m of NADH for benzo[a]pyrene hydroxylation. In the absence of NADPH-cytochrome c reductase, the apparent K_m of NADH was 1.3 μm while in its presence a low (1.3 μm) and a high (1700 μm) K_m were observed, consistent with the affinities of the two flavoproteins for NADH. Our results also indicate that the relative contribution of the pathway due to NADPH-cytochrome c reductase in combination with phosphatidyl choline and cytochrome P-448 to the overall rate of NADH-supported benzo[a]pyrene hydroxylation in microsomes would be greatly dependent on the concentration of NADH chosen. The rate of benzo[a]pyrene hydroxylation by these reconstituted components was almost 10-fold greater with 10 mm NADH than with 0.2 mm NADH, a result consistent with the reduction of NADPH-cytochrome c reductase by high concentrations of NADH.     

Purification and characterization of a flavin reductase from the biodesulfurizing bacterium Mycobacterium goodii X7B

Dibenzothiophene (DBT) in fossil fuels can be efficiently biodesulfurized by a thermophilic bacterium Mycobacterium goodii X7B. Flavin reductase DszD, which catalyzes the reduction of oxidated flavin by NAD(P)H, is indispensable for the biodesulfurization process. In this work, a flavin reductase DszD in M. goodii X7B was purified to homogeneity, and then its encoding gene dszD was amplified and expressed in Escherichia coli. DszD is a homodimer with each subunit binding one FMN as cofactor. The K_m values for FMN and NADH of the purified recombinant DszD were determined to be 6.6 ± 0.3 μM and 77.9 ± 5.4 μM, respectively. The optimal temperature for DszD activity was 55 °C. DszD can use FMN or FAD as substrate to generate FMNH₂ or FADH₂ as product. DszD was coexpressed with DBT monooxygenase DszC, the enzyme catalyzing the first step of the biodesulfurization process. It was indicated that the coexpressed DszD could effectively enhance the DszC catalyzed DBT desulfurization reaction.     

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.     

Purification and properties of reductase of the three-component p-cymene methyl hydroxylase from Pseudomonas chlororaphis subsp. aureofaciens

A novel three-component p-cymene methyl hydroxylase from Pseudomonas chlororaphis subsp. aureofaciens was reported earlier on the basis of genetic characterization and their expression catalyzing methyl group hydroxylation. This enzyme system was inductively synthesized when grown on p-cymene and had an important role in initiating p-cymene metabolism in vivo. In the present study, a NADH-dependent cytochrome c reductase protein has been purified to an electrophoretically homogeneous state and found to be involved in the hydroxylation of methyl group of p-cymene. Molecular mass of the reductase appears to be 38 kDa by SDS/PAGE and 39 kDa by gel filtration apart from one molecule of tightly bound FAD and two atoms each of iron and acid-labile sulfur per molecule of the enzyme. An apparent Km value of the enzyme for NADH is 32 ± 1.2 μM. To the best of our knowledge, this is the first report on the purification of reductase component of p-cymene methyl hydroxylase.     

Identification of a marine NADPH-dependent aldehyde reductase for chemoselective reduction of aldehydes

A putative aldehyde reductase gene from Oceanospirillum sp. MED92 was overexpressed in Escherichia coli. The recombinant protein (OsAR) was characterized as a monomeric NADPH-dependent aldehyde reductase. The kinetic parameters K_m and k_cat of OsAR were 0.89 ± 0.08 mM and 11.07 ± 0.99 s⁻¹ for benzaldehyde, 0.04 ± 0.01 mM and 6.05 ± 1.56 s⁻¹ for NADPH, respectively. This enzyme exhibited high activity toward a variety of aromatic and aliphatic aldehydes, but no activity toward ketones. As such, it catalyzed the chemoselective reduction of aldehydes in the presence of ketones, as demonstrated by the reduction of 4-acetylbenzaldehyde or the mixture of hexanal and 2-nonanone, showing the application potential of this marine enzyme in such selective reduction of synthetic importance.     

Microsomal Electron Transfer in Higher Plants: Cloning and Heterologous Expression of NADH-Cytochrome b5 Reductase from Arabidopsis

AtCBR, a cDNA encoding NADH-cytochrome (Cyt) b₅ reductase, and AtB5-A and AtB5-B, two cDNAs encoding Cyt b₅, were isolated from Arabidopsis. The primary structure deduced from the AtCBR cDNA was 40% identical to those of the NADH-Cyt b₅ reductases of yeast and mammals. A recombinant AtCBR protein prepared using a baculovirus system exhibited typical spectral properties of NADH-Cyt b₅ reductase and was used to study its electron-transfer activity. The recombinant NADH-Cyt b₅ reductase was functionally active and displayed strict specificity to NADH for the reduction of a recombinant Cyt b₅ (AtB5-A), whereas no Cyt b₅ reduction was observed when NADPH was used as the electron donor. Conversely, a recombinant NADPH-Cyt P450 reductase of Arabidopsis was able to reduce Cyt b₅ with NADPH but not with NADH. To our knowledge, this is the first evidence in higher plants that both NADH-Cyt b₅ reductase and NADPH-Cyt P450 reductase can reduce Cyt b₅ and have clear specificities in terms of the electron donor, NADH or NADPH, respectively. This substrate specificity of the two reductases is discussed in relation to the NADH- and NADPH-dependent activities of microsomal fatty acid desaturases.     

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.     

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.     

Characterization of d-amino acid aminotransferase from Lactobacillus salivarius

We searched a UniProt database of lactic acid bacteria in an effort to identify d-amino acid metabolizing enzymes other than alanine racemase. We found a d-amino acid aminotransferase (d-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between d-alanine and α-ketoglutarate. This is the first characterization of a d-AAT from a lactic acid bacterium. L. salivarius d-AAT is a homodimer that uses pyridoxal-5′-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60 °C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 °C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and d-alanine concentrations in the presence of several fixed concentrations of α-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The K_m values for d-alanine and α-ketoglutarate were 1.05 and 3.78 mM, respectively. With this enzyme, d-allo-isoleucine exhibited greater relative activity than d-alanine as the amino donor, while α-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than α-ketoglutarate. The substrate specificity of L. salivarius d-AAT thus differs greatly from those of the other d-AATs so far reported.     

Characterization of the N-oxygenase AurF from Streptomyces thioletus

AurF catalyzes the N-oxidation of p-aminobenzoic acid to p-nitrobenzoic acid in the biosynthesis of the antibiotic aureothin. Here we report the characterization of AurF under optimized conditions to explore its potential use in biocatalysis. The pH optimum of the enzyme was established to be 5.5 using phenazine methosulfate (PMS)/NADH as the enzyme mediator system, showing ∼10-fold higher activity than previous reports in literature. Kinetic characterization at optimized conditions give a K_m of 14.7 ± 1.1 μM, a k_cat of 47.5 ± 5.4 min⁻¹ and a k_cat/K_m of 3.2 ± 0.4 μM⁻¹ min⁻¹. PMS/NADH and the native electron transfer proteins showed significant formation of the p-hydroxylaminobenzoic acid intermediate, however H₂O₂ produced mostly p-nitrobenzoic acid. Alanine scanning identified the role of important active site residues. The substrate specificity of AurF was examined and rationalized based on the protein crystal structure. Kinetic studies indicate that the K_m is the main determinant of AurF activity toward alternative substrates.     

The benzimidazole based drugs show good activity against T. gondii but poor activity against its proposed enoyl reductase enzyme target

The enoyl acyl-carrier protein reductase (ENR) enzyme of the apicomplexan parasite family has been intensely studied for antiparasitic drug design for over a decade, with the most potent inhibitors targeting the NAD⁺ bound form of the enzyme. However, the higher affinity for the NADH co-factor over NAD⁺ and its availability in the natural environment makes the NADH complex form of ENR an attractive target. Herein, we have examined a benzimidazole family of inhibitors which target the NADH form of Francisella ENR, but despite good efficacy against Toxoplasma gondii, the IC₅₀ for T. gondii ENR is poor, with no inhibitory activity at 1 μM. Moreover similar benzimidazole scaffolds are potent against fungi which lack the ENR enzyme and as such we believe that there may be significant off target effects for this family of inhibitors.     

Respiratory Chain of a Pathogenic Fungus, Microsporum gypseum: Effect of the Antifungal Agent Pyrrolnitrin

Pyrrolnitrin has been reported to inhibit Bacillus megaterium primarily by forming complexes with phospholipids and to block electron transfer of Saccharomyces cerevisiae between succinate or reduced nicotinamide adenine dinucleotide (NADH) and coenzyme Q. We found that pyrrolnitrin inhibited respiration of conidia of Microsporum gypseum. In mitochondrial preparations, pyrrolnitrin strongly inhibited respiration and the rotenone-sensitive NADH-cytochrome c reductase. The rotenone-insensitive NADH-cytochrome c reductase, the succinate-cytochrome c reductase, and the reduction of dichlorophenolindophenol by either NADH or succinate were inhibited to a lesser extent. However, the activity of cytochrome oxidase was not affected by pyrrolnitrin. The extent of reduction of flavoproteins by NADH and succinate, measured at 465 - 510 nm, was unaltered; however, the reduction of cytochrome b, measured at 560 - 575 nm, was partially inhibited by pyrrolnitrin. The level of totally reduced cytochrome b was restored with antimycin A. We, therefore, concluded that the primary site of action of this antifungal antibiotic is to block electron transfer between the flavoprotein of the NADH-dehydrogenase and cytochrome b segment of the respiratory chain of M. gypseum.     

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.