Yigal Ilan

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.

Reactions of the ferri-ferrocytochrome-c system with superoxide/oxygen and CO2−/CO2 studied by fast pulse radiolysis

The reduction of ferricytochrome c by O2- and CO2- was studied in the pH range 6.6-9.2 and Arrhenius as well as Eyring parameters were derived from the rate constants and their temperature dependence. Ionic effects on the rate indicate that the redox process proceeds through a multiply-positively charged interaction site on cytochrome c. It is shown that the reaction with O2- (and correspondingly with O2 of ferrocytochrome c) is by a factor of approx. 10(3) slower than warranted by factors such as redox potential. Evidence is adduced to support the view that this slowness is connected with the role of water in the interaction between O2-/O2 and ferri-ferrocytochrome c in the positively charged interaction site on cytochrome c in which water molecules are specifically involved in maintaining the local structure of cytochrome c and participate in the process of electron equivalent transfer.

Effects of alcohol/water mixtures on the structure and reactivity of cytochrome c.

Abstract The oxidation reaction of ferrocytochrome c (produced in situ by pulse radiolysis) by Fe(CN) 3− 6 , was used to probe the effect of alcohol/water mixtures on the reactivity of the protein. Reduced cytochrome c is oxidized in a biphasic process. The relative contribution of each phase depended on: pH, alcohol concentration and temperature. p K a values were derived from the kinetic data. These p K a values were identical with the spectroscopic p K a values determined under similar conditions by monitoring the 695 nm absorption band of the oxidized protein. The two phases of oxidation were therefore related to the oxidation of a relaxed and a nonrelaxed conformer of reduced cytochrome c produced in situ. A shift in the p K a of ferricytochrome c and a retardation of the redox reactions of both the reduced and the oxidized protein were observed at low alcohol concentrations (up to 5 mol %). These low alcohol concentrations are known to affect the structure of water (Yaacobi, M. and Ben-Naim, A. (1973) J. Sol. Chem. 5, 425−443; Ben-Naim, A. (1967) J. Phys. Chem. 71, 4002−4007 and Ben-Naim, A. and Baer, S. (1964) Trans. Faraday Soc. 60, 1736−1741) but have only minor effects on the protein. Accordingly, the kinetic results are interpreted on the basis of involvement of water molecules in the reaction complex of cytochrome c with its redox substrates.

One electron reduction of metmyoglobin and methemoglobin and the reaction of the reduced molecule with oxygen.

We have used the pulse radiolysis technique to reduce with solvated electrons (see article) a single Fe(III) site in methemoglobin and metmyoglobin. The reduction process was followed spectrophotometrically and the reactions rate constants were measured: (see article) =6.5 +/- 1-10(10) M-1-S-1. (see article)=2.5 +/- 0.3-10(10) M-1-S-1. Approx. 60% of the (see article) have reacted with the hemin group, and the rest of the (see article) have probably reacted with the globin moiety. We followed the reaction of the reduced proteins to yield the oxyderivatives and measured the rate constants of the oxygenation process k reduced methemoglobin + O2 = 2.6 +/- 0.6-10(7) M-1-S-1 and k myoglobin + O2 = 1.8 +/- 0.2-10(7) M-1-S-1, all the rate constants were measured at pH = 6.8, I = 0.004, T = 22 +/- 2 degrees C. The high rate constant for reduced methemoglobin indicates that one-site-reduced methemoglobin is probably in the R state, as predicted for methemoglobin from X-ray analysis. The spectra of the reduced and oxygenated species were measured under similar conditions at gamma = 450-650 nm. We were able to follow slight changes in the micro-second time scale, these changes were attributed to conformational changes. We were not able to detect any reaction between the radical (see article) and the hemin group (which would result in a complex such as heme O-2). This may be due to kinetic reasons.

H/2H isotope effect in redox reactions of cytochrome c.

The rate of reaction of ferro- and ferricytochrome c (C(II) and C(III) with ferri- and ferrocyanide and of C(III) with 02- and CO2- was determined in H2O and in 2H2O in the temperature range 5-35 degrees C. No isotope effect was evident in any of the reductions of C(III); the apparent energy of activation was identical in H2O and 2H2O. An isotope effect with kH2O/k2H2O = 1.25 to 1.85, depending on pH for instance was observed in the oxidation of C(II), in the slow phase of oxidation which involves conformational changes. An interpretation (supported by evidence from previous work) involving water molecules in the close vicinity of the reaction site on the protein is discussed.

Intramolecular electron transfer and binding constants in iron hexacyanide-cytochrome c complexes as studied by pulse radiolysis

Internal oxidation and reduction rates of horse cytochrome c in the complexes CII . Fe(III)(CN)6(3)- and CIII . Fe(II)(CN)6(4)-, are 4.6 . 10(4)s-1 and 3.3 . 10(2)s-1, respectively. The binding site of the iron hexacyanide ions on either CII or CIII are kinetically almost indistinguishable; binding constants range from 0.87 . 10(3) to 2 . 10(3)M-1. The present pulse radiolytic kinetic data is compared with that from NMR, T-jump and equilibrium dialysis studies.

Do copper ions influence the reduction of ferricytochrome C by O−2?

Recently, it was suggested that the measured rate of reduction of ferricytochrome C by O-2 below pH 8, was too high in the presence of high concentrations of formate (Koppenol, W.H., Van Buuren, K.J.H., Butler J. and Braams, R. (1976) Biochim. Biophys. Acta 449, 157-168). The high values were attributed to the presence of impurities of copper, which compete for O-2. This assumption is consistent with either a decrease in the reduction yield of ferricytochrome C in the presence of copper, or with a very fast reaction of Cu(I) with ferricytochrome C. It was previously shown by us and by others that the reduction yield of ferricytochrome C by O-2 IS 100%. We measured the rate of reduction of ferricytochrome C by Cu(I), and found that this reaction is slow: k = (1.5 +/- 0.5) . 10(3) M-1 . s-1. Therefore, our results rule out the possibility that below pH 8 copper impurities affect the measured rate constant of the reduction of ferricytochrome C by O-2.

Fluorescence and photochemistry of benzene in solution: Heavy atom effect of Xe

Abstract Solutions of benzene in O 2 free cyclohexane gave on illumination at 254 nm fluorescence yields which were lower significantly in the presence of Xe, than in its absence. The photochemical yield of benzvalene was unaffected. The results support the assumption that this photochemistry originates in non-relaxed states before the fluorescent level is reached.

Internal electron transfer within mitochondrial succinate-cytochrome C reductase

Abstract Internal electron transfer within succinate-cytochrome C reductase from pigeon breast muscle mitochondria was followed by the pulse radiolytic technique. The electron equivalent is transferred from an unknown donor to b type cytochrome(s) in a first order process with a rate constant of: 660±150 s −1 . This process might be the rate determining step of electron transfer in mitochondria, since it is similar in rate to the turn over number of the mitochondrial respiratory chain.