Semiquinone State Research Articles

Spectroscopic studies on the photoreaction of choline oxidase, a flavoprotein, with covalently bound flavin.

Formation of the anionic flavosemiquinone was observed spectrophotometrically during the anaerobic photo-irradiation of Alcaligenes sp. choline oxidase in the presence of EDTA. Further irradiation slowly converted the semiquinone form into the fully reduced state. The presence of a catalytic amount of riboflavin greatly enhances the photoreduction rate not only to the semiquinone state but also to the fully reduced state. This semiquinone species has low reactivity toward the substrate, choline or betaine aldehyde, as well as toward oxygen. This low reactivity toward oxygen is unique to the semiquinone form of a flavoprotein oxidase. The oxidized enzyme forms a complex with betaine, the product of the enzymatic reaction of choline oxidase. The dissociation constant for this complex was found to be 17 mM by spectroscopic titration. Anaerobic photo-irradiation of the enzyme with a saturating amount of betaine in the absence of EDTA produces, with no detectable semiquinone formation, an absorption spectrum which resembles (but significantly differs from) that of the fully reduced form. This species was found to comprise two flavin species. One of them is rapidly oxidized to the oxidized form by oxygen and is thus assigned as the fully reduced state. The other is converted slowly to the oxidized form upon aerobic standing in the dark. We tentatively assigned this latter species as a C(4a)-adduct. Formaldehyde was detected as a product of this photoreaction. The amount of formaldehyde formed coincided with that of the fully reduced enzyme. On the basis of the results obtained we propose a mechanism of the photoreaction of the enzyme in the presence of betaine where a C(4a)-adduct and the fully reduced enzyme via an N(5)-adduct are formed. Betaine also affects the dithionite reduction. In the dithionite reduction of the oxidized enzyme, the semiquinone species is an intermediate in the conversion of the oxidized to the fully reduced form, while the reduction of the oxidized enzyme-betaine complex with dithionite produces the fully reduced form without any significant formation of the semiquinone species.

Water Relaxation Measurements on Semiquinones of Various Flavoproteins

AbstractThe water relaxation rates of several flavoproteins in the semiquinone state have been investigated by the spin echo technique. The results indicate a rather unspecific interaction between water and the protein‐bound flavosemiquinones. An average interaction distance of 0.3‐0.5 nm has been estimated. From the temperature dependence of the rate constants the free energy of activation for proton exchange is calculated to be about 17 kJ/mol. The rate of proton exchange is around 1011 s−1 for the flavosemiquinones investigated are accessible to water regardless of their ionic state.The large difference in relaxation rates of water protons between D‐ and L‐ amino‐acid oxidases is noticeable. Oxynitrilase exhibits the highest whereas Azotobacter vinelandii flavodoxin shows the lowest water relaxation rate of the flavoproteins studied. The results are discussed in relation to the visible‐light absorption properties of the flavoproteins.

Structural and dynamic information on the complex of Megasphaera elsdenii apoflavodoxin and riboflavin 5'-phosphate. A phosphorus-31 nuclear magnetic resonance study.

It is shown that commercial FMN contains a considerable amount of the 4', 3', and 2' isomers and other phosphorus-containing compounds. These impurities can easily be analyzed and quantified by the 31P NMR technique. The phosphate group of FMN bound to Megasphaera elsdenii apoflavodoxin is probably in the dianionic form. Its chemical shift is almost independent of pH in the range 5.5-9.2 and of the redox state of the protein. The phosphate group of bound FMN is buried in the protein. Protons of the apoprotein and of bound water located in the vicinity of the phosphate group in native flavodoxin are not exchangeable with deuteron of the bulk solvent. These protons are, however, easily exchangeable in apoflavodoxin, as shown by reconstitution experiments. The distance between the phosphorus atom of bound FMN and the N(10) atom of the isoalloxazine moiety of FMN is calculated to be about 7.8 A. This result is in good agreement with X-ray data published for the related flavodoxin from Clostridium MP. The electron exchange between the oxidized and semiquinone state of M. elsdenii flavodoxin is rather slow (kexc' much less than 2 s-1) whereas that between the semiquinone and hydroquinone form is much more favored (kexc' much greater than 100 s-1). This indicates that the activation energy for the transition between the semiquinone and hydroquinone states must be smaller than that for the transition between the oxidized and semiquinone states. These results offer a reasonable explanation for the one-electron transfer reaction of flavodoxins in biological reactions.

Purification and characterization of NADH oxidase from membranes of Acholeplasma laidlawii, a copper-containing iron-sulfur flavoprotein.

1. NADH oxidase was extracted from the membranes of Acholeplasma laidlawii with buffer containing 3% Triton X-100 and subsequently purified by several chromatographic steps. The final preparation was essentially homogeneous as judged by gel electrophoresis under nondenaturing conditions. 2. The enzyme appears to be a copper-containing iron-sulfur flavoprotein (FMN:CU:Fe:labile S = 1:1:6:6). The enzyme, containing a high fraction of hydrophobic amino acids, is composed of three subunits of molecular weight 65 000, 40 000 and 19 000. 3. When oxygen is used as electron acceptor the purified enzyme demonstrates a specific activity of 58.0 IU/mg of protein and catalyzes the formation of H2O2 in nearly stoichiometric amount. The apparent Km value for NADH is estimated to be 0.4 mM (pH 7.4). NADPH cannot serve as a substrate for the enzyme. In addition to the NADH oxidase activity, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (ferricyanide, dichloroindophenol, cytochrome c). Metal-chelating agents and mercurials are shown to inhibit the activity of the enzyme. 4. From electron paramagnetic resonance and optical absorption measurements evidence was obtained that the flavin semiquinone radical in the NADH oxidase has a high air-stability, and that the flavin shuttles between the fully reduced and the semiquinone state upon electron transport from NADH to the electron acceptors. Inhibition of the NADH oxidoreductase activities by superoxide dismutase indicates that O-2 serves as an intermediate in the electron transfer from NADH to all electron acceptors used in this work. In addition to electron transfer via the superoxide radical O-2, an alternative pathway probably involving Fe-S centers is operative. From these results and literature data we present a reaction scheme for electron transport from NADH to the various electron acceptors.

Role of tryptophanyl and tyrosyl residues of flavoproteins in binding with flavin coenzymes. X-ray structural studies using model complexes.

The crystal structures of 7,8-dimethylisoalloxazine-10-acetic acid-tryptamine (1:1) tetrahydrate and 7,8-dimethylisoalloxazine-10-acetic acid-tyramine (1:1) tetrahydrate complexes were determined by the X-ray diffraction method, as models for flavin-tryptophan and flavin-tyrosine interactions in flavoproteins. The observed parallel stackings and the intermolecular spacing distances, which were less than the normal van der Waals separation between the isoalloxazine and indole rings and between the isoalloxazine and phenol rings, suggest the existence of charge-transfer interactions in their ground states. The indole and phenol rings interact with the pyrimindinoid and pyrazinoid portions of the isoalloxazine ring and have short contacts, less than 3.4 A, with the reduction site (N1 and N5 atoms) of this ring. This suggests that the reduction of oxidized flavin to the semiquinone state may be facilitated by charge transfer from the former rings to the N1 and N5 atoms. Absorption difference spectra showed that both complexes associate with equimolar ratios in solution as well as in the crystalline state and that they have the same charge-transfer bands and association constants as flavin mononucleotide (FMN)-Trp and FMN-Tyr complexes, respectively. On the other hand, proton magnetic resonance spectra suggested that in solution, the stacking modes of the indole and phenol rings to the isoalloxazine ring are different from those observed in the crystal structures and both aromatic rings are stacked over the whole of the isoalloxazine ring.

Studies on the stabilized ubisemiquinone species in the succinate-cytochrome c reductase segment of the intact mitochondrial membrane system.

1. Evidence is presented for the presence of a stable ubisemiquinone pair in the vicinity of iron-sulphur centre S-3, based on its thermodynamic and spin relaxation properties. 2. These semiquinones are coupled by dipolar interaction; quantitative analysis of the signals of the spin-coupled semiquinones (at pH 7.4) gives midpoint redox potentials E1 (oxidized to semiquinone state) and E2 (semiquinone to fully reduced state) of 140 and 80mV, respectively, for individual ubiquinones. 3. Values of pKS (pK of the semiquinone form) below 6.5 and pKR (pK of the fully reduced ubiquinone) of about 8.0 or above were estimated from the pH-dependence of the midpoint potentials of the spin coupled signals. Thus the ubisemiquinone associated with succinate dehydrogenase (designated as SQS) functions mostly in the anionic form of the physiological pH range. 4. Theonyltrifluoroacetone, a specific inhibitor of the succinate-ubiquinone reductase segment of the respiratory chain, destabilized the intermediate redox state; thus it quenches both the g = 2.00 signal and ubisemiquinone (SQS) and split signals from the spin coupled pair. This inhibitor has no significant effect on another bound ubisemiquinone species present in the cytochrome bc1 region (designated as SQC). 5. The possible function and location of these stabilized ubisemiquinone species were discussed in connection with Site-II energy transduction.

Investigation of the interaction of superoxide ion with adriamycin and the possible origin of cardiotoxicity of the anthracycline anticancer antibiotics

Interaction of superoxide ion with adriamycin in an aprotic medium has been studied. It was shown that superoxide ion reacts irreversibly with adriamycin, giving a diamagnetic product (the dimer or oligomer of semiquinone) which can be reoxidized to adriamycin. This product was also obtained when adriamycin reacted with benzosemiquinone, ubisemiquinone, and the semiquinones of tocopherylquinone and vitamin K 1. It is suggested that the cardiotoxicity of adriamycin and other anthracycline anticancer antibiotics is caused by the high elcctron-attracting properties of these antibiotics, while the ability of natural quinones to reduce cardiotoxicity and to induce recovery of respiration in mitochondria is due to their interaction with the semiquinone states of the antibiotics.

Purified liver microsomal NADPH-cytochrome P-450 reductase. Spectral characterization of oxidation-reduction states.

NADPH-cytochrome P-450 reductase was isolated from liver microsomes of phenobarbital-induced rats. The enzyme exhibits an apparent minimal molecular weight of 76,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 molecule each of FMN and FAD. Trypsin treatment of the reductase yields an enzyme with an apparent minimal molecular weight of 69,000 which retains the ability to reduce cytochrome c but has no activity toward cytochrome P-450. Various spectrophotometric titrations were performed to examine the electron-accepting properties of the purified NADPH-cytochrome P-450 reductase and, in particular, to determine the oxidation state of the stable semiquinone form produced by air oxidation of NADPH-reduced enzyme. Titration of the air-stable semiquinone form of the reductase with ferricyanide indicated that 1 mol/2 mol of flavin was required for complete oxidation. Furthermore, a spectrum corresponding to that of the air-stable semiquinone form was produced by the addition of approximately 0.5 mol of reductant/2 mol of flavin when the oxidized enzyme was titrate with NADPH or dithionite under anaerobic conditions. The spectral changes which accompanied the overall reduction of oxidized enzyme to the reduced form with dithionite produced four sets of isosbestic points, and the spectrophotometric titration curve consisted of four approximately equal phases. In the titration with NADPH, no significant further reduction was observed after the addition of approximately 1.5 mol/2 mol of flavin. However, the enzyme was fully reduced by NADPH when an NAPH-generating system was used to prevent the accumulation of NADP. Our results establish that the air-stable semiquinone form is a 1-electron-reduced form, rather than a half-reduced (2-electron-reduced) form as maintained by others and are in agreement with earlier studies (Iyanagi, T., Makino, N., and Mason, H.S. (1974) Biochemistry 13, 1701-1710) with the purified trypsin-solubilized reductase. Accordingly, the air-stable species represents a form of the NADPH-cytochrome P-450 reductase in which one of the two flavins exists in the semiquinone state and the other in the oxidized state.

Crystal and molecular structure of a donor–acceptor complex involving protonated riboflavin, quinol, and bromide ions

Black crystals of stoicheiometry riboflavin:quinol:hydrogen bromide:water 1:1:2:2 have been grown. The crystals are monoclinic, a= 20·55(5), b= 13·69(4), c= 10·18(4)A, β= 91·2(1)°, with four ‘molecules’ of this composition in a cell of space group P21. The structure was solved from photographic data by Patterson and Fourier methods, and refined by least squares to R 0·145 for 2358 independent reflections. There are charge-transfer interactions between riboflavin molecules (as acceptors) and quinol molecules and bromide ions (as donors). The riboflavin molecules are probably in the semiquinone state and their interaction with the donors is not confined to a specific region of the isoalloxazine nuclei.

The reduction of flavodoxin from Azotobacter vinelandii by pyruvate.

Pyruvate is shown to be a substrate for the reduction of flavodoxin from Azotobacter vinelandii, using the phosphoroclastic enzyme system from Clostridium pasteurianum. In this reaction, the reduction of this flavoprotein proceeds beyond the semiquinone state partly to the hydroquinone form. Pyruvate is possibly the substrate for the reduction of flavodoxin in the intact organisms.