Henk Nauta

A tunnelling model to explain the reduction of ferricytochrome c by H and OH radicals

The kinetics of the reaction of OH radicals with ferricytochrome c was studied in the time range 1 microsecond to 1 s by means of pulse radiolysis. The OH radicals reduce ferricytochrome c by 40% +/- 10%. The time course of the reduction is explained by a mechanism whereby a radical formed after hydrogen has been abstracted from the outer surface of the protein reduces the iron by electron tunnelling. We have calculated that the reducing electron in the radical is bound with an energy of at least 1.75 eV and that the frequency factor of the tunnelling process is v=10(11.5)s-1. This model accounts for the observed absorbance change in time range 5 . 10(-6)--10(-1)s. The time course of the reduction of ferricytochrome c by H radicals (Lichtin, N.N., Shafferman A. and Stein, G. (1974) Biochim. Biophys. Acta 357, 386--398) is explained by the same model.

Studies on (K+ + H+)-ATPase. VI. Determination on the molecular size by radiation inactivation analysis.

(1) A (K+ + H+)-ATPase containing membrane fraction, isolated from pig gastric mucosa, has been further purified by means of zonal electrophoresis, leading to a 20% increase in specific activity and an increase in ratio of (K+ + H+)-ATPase to basal Mg2+-ATPase activity from 9 to 20. (2) The target size of (Na+ + K+)-ATPase, determined by radiation inactivation analysis, is 332 kDa, in excellent agreement with the earlier value of 327 kDa obtained from the subunit composition and subunit molecular weights. This shows that the Kepner-Macey factor of 6.4 X 10(11) is valid for membrane-bound ATPases. (3) The target size of (K+ + H+)-ATPase is 444 kDa, which, in connection with a subunit molecular weight of 110000, suggests a tetrameric assembly of the native enzyme. The ouabain-insensitive K+-stimulated p-nitrophenylphosphatase activity has a target size of 295 kDa. (4) In the presence of added Mg2+ the target sizes of the (K+ + H+)-ATPase and its phosphatase activity are decreased by about 15%, while that for the (Na+ + K+)-ATPase is not significantly changed. This observation is discussed in terms of a Mg2+-induced tightening of the subunits composing the (K+ + H+)-ATPase molecule.

The reduction mechanism of ferricytochrome c

The second-order rate constant of the reaction between the hydrated electron and ferrinitrocytochrome c exhibits a marked pH dependence that could not be fully ascribed to changes in geometrical parameters and in net charge of the protein molecule. The correlation between the pH dependence of the rate constant, the 695-nm absorbance and the ionization state of the nitrated tyrosyl-67 residue indicates that tyrosine-67 is of importance in maintaining the specific structure for the electron transfer mechanism in ferricytochrome c upon reduction.

A pulse radiolysis study of electron tunneling in an 8 M NaOH glass between 4 and 100 K

Pulse radiolysis experiments have been performed in an 8 M NaOH glass between 4 and 100 K and in the time range 10−6 to 10 s. The spur radius was estimated from the decay of the trapped electrons on the red side of the absorption maximum. The spur radius was about 4 nm at 80 K and increased to about 6 nm at 10 K. We studied the decay of trapped electrons in the presence of the following electron scavengers: CrO2−4, Fe(CN)3−6, and NO−2. No temperature dependence was found between 10 and 100 K. To explain the results distance‐dependent and/or time‐dependent Franck–Condon factors have been introduced. We show that in the time range studied it is not possible to distinguish between direct tunneling and trap‐to‐trap tunneling. Dry electron scavenging efficiencies and encounter pair formation are expressed in S37. For CrO2−4, Fe(CN)3−6, and NO−2 we found that S37 was 45, 130, and 300 M, respectively.

REDUCTION OF FERRICYTOCHROME c, METHEMOGLOBIN AND METMYOGLOBIN BY HYDROXYL AND ALCOHOL RADICALS

We have studied the reaction of ferricytochrome c, methemoglobin and metmyoglobin with OH and alcohol radicals (methanol, ethanol, ethylene glycol and glycerol). These radicals can be divided into three groups: 1. The OH radicals which reduce the ferricytochrome c with a yield of (30 +/- 10)% and methemoglobin with a yield of (40 +/- 10)%. They do not reduce metmyoglobin. The reduction is not a normal bimolecular reaction but is most probably an intramolecular electron transfer of a protein radical. 2. Methanol and ethanol radicals which reduce all three hemoproteins with a yield of (100 +/- 5)%. This reduction is a normal bimolecular reaction. 3. Glycerol radicals which do not reduce the ferrihemoproteins under our experimental conditions. Ethylene glycol radicals do not reduce ferricytochrome c and metmyoglobin but they do reduce methemoglobin with a yield of (30 +/- 10)%.

A pulse radiolysis study of the reactions of OH/O− with linoleic acid in oxygen-free aqueous solution

Abstract Absorption spectra of products resulting from the reaction of OH and O− with linoleate in an aqueous solution have been measured. An OH adduct, absorbing at ∼240 nm, as well as the allyl and the dienyl radical, were observed. The dienyl radical is formed by the reaction of linoleate with OH (k = (1.15 ∓ 0.10) × 10 10 M −1 s −1 ) and O− radicals (k = 2.5 ∓ 0.4) × 109M−1s−1). The greater selectivity for the formation of dienyl radicals by O- as compared to OH is due to the formation of an OH. adduct. The dependence of the absorption band of the adduct on linoleate concentration can be explained in terms of competition between reactions of OH with linoleate molecules in micelles and in solution. It is assumed that OH radicals neither form adducts nor dienyl radicals with micellized linoleate, but still react with these molecules, albeit in a different way. Since the rate constant for the reaction of OH with micellized linoleate is comparable to the rate constant for the OH addition reaction and much lower than the rate constant for dienyl production, the absorbance at 280 nm (dienyl) is less affected by the linoleate concentration increase than the absorbance at ∼240 nm (OH adduct).

A pulse radiolysis study of the dienyl radical in oxygen-free linoleate solutions: Time and linoleate concentration dependence

Abstract The time dependence of absorption signals of the dienyl radical in linoleate solutions at pH 12 was measured using a pulsed Xenon lamp. Absorption changes of 0.5% over a period of 1.5 ms were determined from difference signals. The time dependence of the dienyl radical concentration can be described in terms of rate equations for formation and decay reactions. A growth of the dienyl radical absorption with first-order kinetics takes place only in linoleate micelles. A concentration-dependent growth parameter k 4 ⩽ 6 × 10 3 ( C - cmc )/ C s −1 , has been determined. Radical recombination occurs both in bulk water, 2 k = (2.46 ± 0.09) × 10 8 M −1 s −1 , and in micelles with a multiple radical occupancy. The competition between the growth of the dienyl absorption and its decay by recombination is shown to be affected by the micellar equilibrium. The model accounts for the dependence of the maximum yield of the dienyl radical on the linoleate and added salt concentrations.

Deconjugation of glucuronides catalysed by UDPglucuronyltransferase

Evidence was found for UDPglucuronyltransferase-catalysed deconjugation of p-nitrophenol-, 4-methylumbelliferone- and phenolphthalein-glucuronides. The evidence is based on the following observations: 1, deconjugation is UDP-dependent and the reactions show Michaels-Menten kinetics with respect to UDP and glucuronide saturability; 2, UDP-glucuronic acid was identified as reaction product; 3, all studies were done in the presence of a beta-glucuronidase inhibitor; 4, induction profiles, using 3-methylcholanthrene and phenobarbital as inducing agents, were identical for conjugation and deconjugation reactions. Optimal deconjugation rates for p-nitrophenol- and 4-methylumbelliferone-glucuronides were at pH 5.1 and for phenolphthalein-glucuronide at pH 6.5. Only conjugation reactions showed latency; the corresponding deconjugation reactions were not latent. UDPglucuronyltransferase is a group of oligomeric isoenzymes with different molecular masses. The molecular masses of the isoenzyme species catalysing the forward and reverse reactions were determined by radiation-inactivation analysis. The molecular masses of the isoenzyme species mediating the catalyses of deconjugation reactions were significantly smaller than those mediating catalyses of conjugation reactions: 66 +/- 4 kDa vs. 109 +/- 7 kDa for p-nitrophenol; 82 +/- 8 kDa vs. 105 +/- 6 kDa for 4-methylumbelliferone; and 74 +/- 8 kDa vs. 159 +/- 14 kDa for phenolphthalein. This suggests that for catalyses of deconjugation reactions only part of a UDPglucuronyltransferase isoenzyme is needed, whereas for forward reactions the complete isoenzymes are required.

A study of the temperature dependence of the visible absorption band of trapped electrons in ethylene glycol-water and LiC1 glasses between 4 and 100 K

Abstract Spectra of electrons trapped in ethylene glycol-water and in 8.5 M LiC1 glasses after pulse radiolysis have been measured between 4 and 100 K. In (50/50) ethylene glycol-water and in 8.5 M LiC1 glasses the yield of deep-trapped electrons (e - vis decreases by approximately a factor of three when the temperature is lowered from 100 to 4 K. This decrease is ascribed to an increase in the formation of shallow-trapped electrons (e - IR , λ max ≧ 3 μm). Hardly any e - IR is converted to e - vis . The growth observed in the yield of e - vis after the pulse is ascribed to deep trap relaxation. In the (50/50) ethylene glycol-water glass at 4 K the presence of large amounts of salts (≧ 2 M) causes an increase in the yield of e - vis . In the (70/30) ethylene glycol-water glass hardly any e - IR will form. A model is proposed in which an electron trapped in a water dimer is considered to be responsible for the IR absorption (λ max ≧3 μm).

Inverted micelles of AOT as electron scavengers in 3-MP at 77 K in the absence and presence of water

Pulse radiolysis of bis (2-ethylhexyl)sulfosuccinate (AOT) solutions in 3 methylpentane (3 MP) at 77 K leads to the formation of a species absorbing at around 450 nm. This absorption is believed to result from the scavenging of electrons by AOT inverted micelles. The electron scavenging capacities of an AOT solution depend on the aggregation number of the micelles. The addition of water leads to less efficient electron capture which is explained by an increase of the aggregation number. This increase is of the same order of magnitude as measured by other techniques at room temperature.