One-electron reduction of hepatic NADH-cytochrome b5 reductase as studied by pulse radiolysis

The reduction of flavin in hepatic NADH-cytochrome b5 reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced the flavin of NADH-cytochrome b5 reductase to form the red semiquinone between pH 5 and 9. The spectrum of the red semiquinone differs from that of enzyme reduced by dithionite in the presence of NAD+. After the first phase of the reduction, conversion of the red to blue semiquinone was observed at acidic pH. Resulting products are the blue (neutral) or red (anionic) semiquinone or a mixture of the two forms. The pK value for this flavin radical was approximately 6.3. Subsequently, the semiquinone form reacted by dismutation to form the oxidized and the fully reduced forms of the enzyme with a rate constant of 1 x 10(3) M-1 s-1 at pH 7.1. In the presence of NAD+, eaq- reacted with NAD+ to yield NAD(.). Subsequently, NAD. transferred an electron to NAD+-bound oxidized enzyme to form the blue and red semiquinone or mixture of the two forms of the enzyme, where pK value of this flavin radical was approximately 6.3. The blue semiquinone obtained at acidic pH was found to convert to the red semiquinone with a first order rate constant of 90 s-1, where the rates were not affected by pH or the concentration of NAD+. The final product is NAD+-bound red semiquinone of the enzyme.     

The mechanism of reduction of cytochrome c as studied by pulse radiolysis.

1. The reaction of hydrated electrons with ferricytochrome c was studied using the pulse-radiolysis technique. 2. In 3.3 mM phosphate-buffer (pH 7.2), 100 mM methanol and at a concentration of cytochrome c of less than 20 muM the reduction kinetics of ferricytochrome c by hydrated electrons is a bimolecular process with a rate constant of 4.5-10-10 M-1-S-1 (21 degrees C). 3. At a concentration of cytochrome c of more than 20 muM the apparent order of the reaction of hydrated electrons with ferricytochrome c measured at 650 nm decreases due to the occurrence of a rate-determining first-order process with an estimated rate constant of 5-10-6s-1 (pH 7.2, 21 degrees C). 4. At high concentration of cytochrome c the reaction-time courses measured at 580 and 695 nm appear to be biphasic. A rapid initial phase (75% and 30% of total absorbance change at 580 and 695 nm, respectively), corresponding to the reduction reaction, is followed by a first-order change in absorbance with a rate constant of 1.3-10-5 S-1 (pH 7.2, 21 degrees C). 5. The results are interpreted in a scheme in which first a transient complex between cytochrome c and the hydrated electron is formed, after which the heme iron is reduced and followed by relaxation of the protein from its oxidized to its reduced conformation. 6. It is calculated that one of each three encounters of the hydrated electron and ferricytochrome c results in a reduction of the heme iron. This high reaction probability is discussed in terms of charge and solvent interactions. 7. A reduction mechanism for cytochrome c is favored in which the reduction equivalent from the hydrated electron is transmitted through a specific pathway from the surface of the molecule to the heme iron.     

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa₃ were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa₃. The bimolecular rate constant for this reaction is 2·10⁷ M^?1·s^?1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20°C). (2) The ionic strength dependence of the cytochrome c-cytochromeaa₃ interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa₃ complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (?) of the reaction medium on the reaction of cytochrome c with cytochrome aa₃ was investigated. With increasing ? the second-order rate constant decreased.     

Time-resolved resonance Raman spectroscopy of cytochrome c reduced by pulse radiolysis

The first investigation of the dynamics of a redox transition of an electron-transfer enzyme by time-resolved resonance Raman spectroscopy in combination with pulse-radiolytical reduction is described by an application to cytochrome c. A long-lived transient state is observed upon reduction of the alkaline form of cytochrome c as a distinct frequency shift of one resonance Raman band. From the frequency in the stable oxidized state, 1567 cm^?1, this particular resonance Raman band shifts within less than 1 μs to 1533 cm^?1 in the transient reduced state, which has a lifetime longer than 20 ms but shorter than a few seconds. Finally, in the stable reduced state, this band is located at 1547 cm^?1. According to a previous normal coordinate analysis, this resonance Raman band can be assigned predominantly to a stretching mode of the outermost C-C bonds in the four pyrrole rings of porphyrin. This vibrational mode is influenced by the protein most directly through the covalent thioether linkages of two cysteines to porphyrin. We interpret the long lifetime of the transient state as due to the slow return of Met-80 as sixth ligand to the heme iron upon reduction of the alkaline form of cytochrome c.     

Electron transfer in milk xanthine oxidase as studied by pulse radiolysis

Electron transfer within milk xanthine oxidase has been examined by the technique of pulse radiolysis. Radiolytically generated N-methylnicotinamide radical or 5-deazalumiflavin radical has been used to rapidly and selectively introduce reducing equivalents into the enzyme so that subsequent equilibration among the four redox-active centers of the enzyme (a molybdenum center, two iron-sulfur centers, and FAD) could be monitored spectrophotometrically. Experiments have been performed at pH 6 and 8.5, and a comprehensive scheme describing electron equilibration within the enzyme at both pH values has been developed. All rate constants ascribed to equilibration between specific pairs of centers in the enzyme are found to be rapid relative to enzyme turnover under the same conditions. Electron equilibration between the molybdenum center and one of the iron-sulfur centers of the enzyme (tentatively assigned Fe/S I) is particularly rapid, with a pH-independent first-order rate constant of approximately 8.5 x 10(3) s-1. The results unambiguously demonstrate the role of the iron-sulfur centers of xanthine oxidase in mediating electron transfer between the molybdenum and flavin centers of the enzyme.     

Determination of the equilibrium constant for the binding of ferricytochrome c to phospholipid vesicles and the effect of binding on the reduction rate of cytochrome c

The rate of reduction of cytochrome c by 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine was examined as a function of binding to liposomes prepared from mixed soybean phospholipids, asolectin, and from various purified phospholipids. Binding of cytochrome c to asolectin liposomes caused an increase in the rate of reduction by the pteridine derivative from 2900 to 16 000 M^?1 · s^?1 at pH 7. At low ionic strength (0.003 M) the binding stoichiometry between cytochrome c and asolectin vesicles is 15 ± 2 phosphospolipid/cytochrome c (mole ratio), determined by monitoring the change in reduction rate of cytochrome c by pteridine as cytochrome c is bound to the vesicles. A stoichiometry of 14 phospholipid/cytochrome c was obtained from gel filtration studies. Equilibrium association constants for the binding of cytochrome c to sites on the asolectin vesicles varied from 2.2 · 10⁶ to 1.8 · 10³ M^?1 between 0.02 and 0.10 M ionic strength, respectively. In general, liposomes prepared from purified phospholipids resulted in less binding of cytochrome c per mole of phospholipid and lower reduction rates than those prepared from asolectin.     

Kinetic behavior of the monodehydroascorbate radical studied by pulse radiolysis

The reactions of the monodehydroascorbate radical (As.-) with various biological molecules were investigated by pulse radiolysis. As.- reacted with both fully reduced and semiquinone forms of hepatic NADH-cytochrome b5 reductase with second-order rate constants of 4.3 x 10(6) and 3.7 x 10(5) M-1 s-1, respectively, at pH 7.0. In contrast, no reaction of As.- with ferrous cytochrome b5 could be detected by pulse radiolysis, whereas the oxidation of cytochrome b5 by As.- was observed by ascorbate-ascorbate oxidase method. This suggests that the rate constant of As.- with the ferrous cytochrome b5 must be several orders in magnitude smaller than that of the disproportionation of As.-. On the other hand, As.- reduced Fe3+EDTA with a second-order rate constant of 4.0 x 10(6) M-1 s-1 but did not reduce ferric hemoproteins such as metmyoglobin, methemoglobin, and cytochrome b5 by either the pulse radiolysis or the ascorbate-ascorbate oxidase method.     

Reactivity of semiquinones with ascorbate and the ascorbate radical as studied by pulse radiolysis

Free-radical interactions between hydroquinones (QH2) and ascorbate (AscH-) have a profound impact in many biological situations. Despite the obvious biological significance, not much is known about the kinetics of reactions of QH2 and AscH- with their corresponding free radicals, i.e., semiquinones, Q1.-, and the ascorbate radical, Asc.-. Furthermore, a general approach to reliably measure rate constants for the above reactions is fraught with complications. In this work, the kinetic behavior of Q.- and Asc.-, after pulse radiolytic oxidation of mixtures of a series of alkyl- and methoxysubstituted hydroquinones and ascorbate by azide radicals in aqueous buffer, pH 7.40, was monitored in submillisecond range by time-resolved UV spectroscopy. Rate constants for reactions of Q.- with AscH-(reaction [1]) and Asc.- (reaction [2]) were directly determined by using new kinetic procedures which distinguished between reactions [1] and [2]. The results show that the rate constants for reaction [2] vary only within a narrow range from 1.2 x 10(8) to 2.5 x 10(8) M(-1) s(-1) and do not display any pronounced correlation with Q.- structures. In contrast, the value of k1 for nonsubstituted Q.- was found to be (1.8 +/- 0.2) x 10(5) M(-1) s(-1) and decreases with the number of alkyl and methoxy substituents as well as with the decrease of the one-electron reduction potential E(Q.-/QH2).