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.     

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 superoxide radical with iron complexes of EDTA studied by pulse radiolysis.

The reactions of Fe3+-EDTA and Fe2+-EDTA with O2- and CO2- were investigated in the pH range 3.8--11.8. Around neutral pH O2- reduces Fe3+-EDTA with a rate constant which is pH dependent kpH 5.8--8.1 = 2 - 10(6)--5 - 10(5) M-1 - s-1. At higher pH values this reaction becomes much slower. The CO2- radical reduces Fe3+-EDTA with kpH 3.8--1- = 5 +/- 1 - 10(7) M-1 - s-1 independent of pH. At pH 9--11.8, Fe2+-EDTA forms a complex with O2- with kFe2+-EDTA + O2 = 2 - 10(6)--4 - 10(6) M-1 - s-1 which is pH dependent. We measured the spectrum of Fe2+-EDTA-O2- and calculated epsilon 290 over max = 6400 +/- 800 M-1 - cm-1 in air-saturated solutions. In O2-saturated solutions another species is formed with a rate constant of 7 +/- 2 s-1. This intermediate absorbs around 300 nm but we were not able to identify it.     

Interaction of hydrated electron with dietary flavonoids and phenolic acids: rate constants and transient spectra studied by pulse radiolysis.

The reaction rate constants and transient spectra of 11 flavonoids and 4 phenolic acids reacting with e(aq)- at neutral pH were measured. Absorption bands of the transients of e(aq)- reacting with the above compounds all located at a wavelength shorter than 400 nm. The e(aq)- scavenging abilities were divided into three groups: (+)catechin ((1.2 +/-0.1) x 10(8) M(-1)s(-1)) < 4-chromanol ((4.4 +/- 0.4) x 10(8) M(-1)s(-1)) < genistein ((6.2+/-0.4) x 10(9) M (-1) s(-1) approximately genistin ((8 +/- 1) x 10(9) M(-1)s(-1)) approximately rutin ((7.6 +/- 0.4) x M(-1)s(-1) approximately caffeic acid ((8.3 +/- 0.5) x 10(9)M(-1)s(-1)) < transcinnamic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately p-coumaric acid ((1.1 +/- 0.1) x 10(10) M(-1)s(-1) approximately 2,4,6-trihydroxylbenzoic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately baicalein ((1.1 +/- 0.5) x 10(10) M(-1)s(-1)) approximately baicalin((1.3 + 0.1) X 10(10) M(-1)s(-1)) approximately naringenin ((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately naringin ((1.0 +/- 0.1) x 10(10) M(-1)s(-1)) approximately gossypin((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately quercetin((1.3 +/- 0.5) x 10(10) M(-1)s(-1)). These results suggested that C4 keto group is the active site for e(aq)- to attack on flavonoids and phenolic acids, whereas the o-dihydroxy structure in B ring, the C2,3 double bond, the C3-OH group, and glucosylation, which are key structures that influence the antioxidant activities of flavonoids and phenolic acids, have little effects on the e(aq)- scavenging activities.     

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 x 10(7) M-1 . s-1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20 degrees C). (2) The ionic strength dependence of the cytochrome c-cytochrome aa3 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 (epsilon) of the reaction medium on the reaction of cytochrome C with cytochrome aa3 was investigated. With increasing epsilon the second-order rate constant decreased.     

Rapid calmodulin-dependent interdomain electron transfer in neuronal nitric-oxide synthase measured by pulse radiolysis

Electron transfer within rat neuronal nitric-oxide synthase (nNOS) was investigated by pulse radiolysis. Radiolytically generated 1-methyl-3-carbamoyl pyridinium (MCP) radical was found to react predominantly with the heme of the enzyme with a second-order rate constant for heme reduction of 3 x 10(8) m(-1) s(-1). In the calmodulin (CaM)-bound enzyme a subsequent first-order phase was observed which had a rate constant of 1.2 x 10(3) s(-1). In the absence of CaM, this phase was absent. Kinetic difference spectra for nNOS reduction indicated that the second phase consisted of heme reoxidation accompanied by formation of a neutral flavin semiquinone, suggesting that it is heme to flavin electron transfer. Experiments with the heme proximal surface mutant, K423E, had no second phase, confirming that the mutation blocks interdomain electron transfer. With the autoinhibitory loop deletion mutant, Delta40, the slow phase was observed even in the absence of CaM consistent with the role of the loop in impeding interdomain electron transfer. The rate of heme to FMN electron transfer observed in the wild-type enzyme is approximately 1000 times faster than the FMN to heme electron transfer rate predicted during catalysis from kinetic modeling, suggesting that the catalytic process is slowed by kinetic gating.     

Preferred sites for electron transfer between cytochrome c and iron and cobalt complexes

The kinetics of oxidation of eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse ferrocytochromes c, modified at lysine 7, 13, 25, 27, 60, 72, 86, or 87, and of one trinitrophenyl horse ferrocytochrome c, modified at lysine 13, by the 3- and 3+ inorganic complexes hexacyanoferrate(III) (Fe(CN)6(3-) ) and tris(1,10-phenanthroline)cobalt(III) (Co(phen)3(3+) ) have been characterized. The influence of the modified residues on the bimolecular rate constants for these reactions define the protein molecular surface involved. The site of electron exchange for both oxidants appears to be the solvent accessible edge of the heme prosthetic group or a closely related structure on the "front" surface of the molecule. The reaction with Fe(CN)6(3-) is most strongly influenced by modification of lysine 72, a residue to the left of the exposed heme edge. (CDNP lysine 72 cytochrome c yields a 3.6-fold decrease in the bimolecular rate constant, as compared to that for the native protein.) However, it is the region around lysine 27, to the right of the heme edge, that is most influential in the reaction with Co(phen)3(3+). (CDNP-lysine 27 cytochrome c exhibits a 7.3-fold increase in the rate constant, as compared to that for the native protein.) The kinetics of reaction of the CDNP-lysine 13, 60, 72, and 87 modified cytochromes c with Fe(CN)5(4-aminopyridine)2- as oxidant and Fe(CN)5(4-aminopyridine)3- and Fe(CN)5-(imidazole)3- as reductants have also been determined and further illustrate the influence of electrostatics on the kinetics of such protein-small molecule electron exchanges.     

Reactivity of nitro-thiophene derivatives with electron and oxygen radicals studied by pulse radiolysis and polarographic techniques

Summary A new series of nitrothiophene derivatives have been synthetized and some of the physico-chemical parameters which can influence both their radiosensitizing efficiency and toxicity have been investigated. These include octanol/water partition coefficient (P), one-electron reduction potential (E ₇ ¹ ) and reactivity of the drugs towards primary radical species. For these studies, pulse radiolysis techniques and conventional polarography have been extensively used. Biological responses (both sensitization and toxicity) have been tested towards Chinese Hamster cells in vitro. The results are valuable in selecting, among the tested compounds, 5-NTMA, 5-NTM, 4- and 5-NTCA as the nitrothiophenes promising for in vivo applications.     

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.     

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).     

Reaction of cythchrome c with one-electron redox reagents. I. Reduction of ferricytochrome c by the hydrated electron produced by pulse radiolysis

Pulse radiolysis-kinetic spectrometry has been used to investigate the reaction of hydrated electrons with ferricytochrome c in dilute aqueous solution at pH 6.5–7.0. Time resolutions from 2·10^?7 to 1 s were employed. Transient spectra from 320 to 580 nm were characterized with a wavelength resolution of ±0.5 nm. 1 In neutral salt-free solution, k(ferricytochrome c+e^?_aq)=(6.0±0.9)·10¹⁰ M^?1·s^?1 and k(ferricytochrome c+H)=(1.2±0.2)·10¹⁰ M^?1·s^?1. The reaction of ferricytochrome c with hydrated electrons is sensitive to ionic strength; in 0.1 M NaClO₄, k(ferricytochrome c+e^?_aq)=(2.4±0.4)·10¹⁰ M^?1·s^?1. In contrast, k(ferricytochrome c+H) is insensitive to ionic strength. Time resolution of three spectral stages has been accomplished. The primary spectrum is the first observable spectrum detectable after irradiation and is formed in a second-order process. Its rate of formation is indisting-uishable from the rate of disappearance of the electron spectrum. The secondary spectrum is generated in a true first order intramolecular process, k(p→s)=(1.2±0.1)·10⁵ s^?1. The tertiary spectrum is also generated in a true first-order process, k(s→t)=(1.3±0.2)·10² s^?1. The specific rates of both transformations are independent of the wavelength of measurement. The tertiary spectrum, observable 50 ms after initial reaction and remaining unchanged thereafter for at least 1 s, shows that relaxed ferrocytochrome c is the only detectable product. This product is not autoxidizable, as expected for native reduced enzyme. It is more probable that the intramolecular changes responsible for the p→s and s→t spectral transformations involve the influence of conformational relaxation of ferrocytochrome c upon electronic energy states then that they are intramolecular transmission of reducing equivalents from primary sites of electron attachment.     

Intramolecular electron transfer in cytochrome c oxidase: a cascade of equilibria.

Intramolecular electron redistribution in cytochrome c oxidase after photolysis of the partially reduced CO-bound enzyme was followed at a number of different wavelengths by absorption spectroscopy. Spectra were constructed for the first two phases of this process. The first phase (tau = 3 microseconds) has a spectrum essentially identical to the difference between the Fea and Fea3 reduced-minus-oxidized spectra, indicating a 1:1 stoichiometry between the amount of Fea3 oxidized and Fea reduced. It is not necessary to invoke reduction or oxidation of other redox carriers in this phase. The second phase (tau = 35 microseconds) spectrum appears to be a linear combination of the Fea3 and Fea reduced-minus-oxidized difference spectra, reflecting the oxidation of four parts of Fea3 for every part of Fea oxidized. This process can be described in terms of transfer to CuA of electrons from the Fea3<==>Fea equilibrium system established in the first phase. The relative contributions of Fea3 and Fea in the second phase allow us to calculate the equilibrium constant for Fea3<==>Fea electron exchange, which yields a delta Em of 36 mV for the two centers (Fea3 more positive). Together with the apparent rate constant for the fast phase, this equilibrium constant yields, in turn, the forward (kf) and reverse (kr) rates for electron transfer from Fea to Fea3 as follows: kf = 2.4 x 10(5) s-1 and kr = 6 x 10(4) s-1. kf is much faster than any observed step in the reaction of the reduced enzyme with O2. Thus, the catalytic mechanism of O2 reduction to water is not rate-limited by electron transfer from Fea to the binuclear Fea3/Cu(B) site.