Metal-mediated reduction of cytochrome c by thioglycolic acid

The reduction of cytochrome c by thioglycolic acid was found to be extremely sensitive to metal catalysis. The rate of the uncatalyzed reaction was negligible in comparison with rates obtained from reactions supplemented with catalytic amounts of copper or iron. Both the catalyzed and uncatalyzed reactions were independent of pH (near neutrality) but when o-phenanthroline was included in the reaction mixture, a pH dependence was induced. This pH dependence is the result of an interference of oxygen with the metal complexes. A comparison of the rate constants at zero ionic strenght, which were obtained from the application of the Debye-Hückel theory for the ionic strength dependence, demonstrated that copper complexes are superior catalysts as compared with iron complexes. Our results suggest that in the copper-mediated reaction, the catalyst is a cupric thioglycolate complex with a net charge of ?2. The addition of o-phenanthroline to the reaction mixture results in a tenfold decrease in the catalytic activity and in a change in the net charge of the catalyst to ?1. At pH 8 the iron-mediated reduction is catalyzed by a ferric thioglycolate complex, whereas at pH 7 a ferrothioglycolate complex provides the catalytic activity. Both complexes have a net negative charge of ?2. At both pH's the catalytic activity is completely abolished by the addition of o-phenanthroline. The results demonstrate the effectiveness by which metal-sulfur complexes can facilitate one-electron transfer reactions and could there-fore serve as a model in the study of various biological oxidations.     

The polyphasic reduction of oxygen to water by purified cytochrome c oxidase

The time course of oxygen consumption by purified cytochrome oxidase has been studied in reactions where the fully reduced enzyme was rapidly mixed with molecular oxygen. Similar to intact mitochondria (Reynafarje & Davies, Am. J. Physiol. 258, 1990), the enzyme reduces oxygen to water in a kinetically and well defined polyphasic event. The initial rates of O2 consumption depended hyperbolically on O2 concentration, with a bimolecular rate constant of near 10(7) M-1 s-1. The Vmax of O2 uptake was, however, a complex function of the concentrations of both enzyme and cytochrome c. It is concluded that the reduction of oxygen to water takes place in a cyclic process in which the oxidase undergoes redox changes at rates depending on the relative concentration of the enzyme and its 3 substrates: O2, electrons and protons. No evidence was found for impairments in the intramolecular flow of electrons per se.     

The reduction of Pseudomonas cytochrome c551 oxidase by chromous ions.

The reduction of cytochrome c551 oxidase from Pseudomonas aeruginosa by Cr2+ ions was followed in the stopped-flow apparatus at a number of wavelengths. The c-haem reduction proceeded in a biphasic fashion with second-order rate constants of 2.6 X 10(5)M-1-S-1 and 4.8 X 10(4)M-1-S-1 at 25 degrees C, whereas the biphasic reduction of the d1-haem appeared to be independent of reductant concentration with rate constants of approx. 1.0S-1 and 0.25S-1 respectively. The kinetically determined difference spectra (reduced minus oxidized) for the c- and d1-haems are presented.     

Measurement of free radical oxygen generation by cytochrome c reduction requirement for cytochrome c oxidase blockade

Data is presented showing that one commercial preparation of cytochrome c, used to trap and measure free radical superoxide anion, can be contaminated with cytochrome c oxidase activity. This activity can vary from lot to lot, can introduce variability into the measurement of superoxide anion and can result in falsely low estimations of free radical formation. This cytochrome c oxidase activity can be inhibited by low (0.2 mM) concentrations of KCN. Blockade of the cytochrome c oxidase activity allows reproducible measurement of superoxide anion formation at low levels by red cells.     

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.     

Binding and reduction of sulfite by cytochrome c nitrite reductase

Pentaheme cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron reduction of nitrite to ammonia as the final step in the dissimilatory pathway of nitrate ammonification. It has also been shown to reduce sulfite to sulfide, thus forming the only known link between the biogeochemical cycles of nitrogen and of sulfur. We have found the sulfite reductase activity of ccNiR from Wolinella succinogenes to be significantly smaller than its nitrite reductase activity but still several times higher than the one described for dissimilatory, siroheme-containing sulfite reductases. To compare the sulfite reductase activity of ccNiR with our previous data on nitrite reduction, we determined the binding mode of sulfite to the catalytic heme center of ccNiR from W. succinogenes at a resolution of 1.7 A. Sulfite and nitrite both provide a pair of electrons to form the coordinative bond to the Fe(III) active site of the enzyme, and the oxygen atoms of sulfite are found to interact with the three active site protein residues conserved within the enzyme family. Furthermore, we have characterized the active site variant Y218F of ccNiR that exhibited an almost complete loss of nitrite reductase activity, while sulfite reduction remained unaffected. These data provide a first direct insight into the role of the first sphere of protein ligands at the active site in ccNiR catalysis.     

The reduction of porphyrin cytochrome c by hydrated electrons and the subsequent electron transfer reaction from reduced porphyrin cytochrome c to ferricytochrome c.

1. Hydrated electrons, produced by pulse radiolysis react with porphyrin cytochrome c with a bimolecular rate constant of 3-10(10) M-1 S-1 at 21 degrees C and pH 7.4. 2. After the reduction step an absorbance change with a half-life of 5 microns is observed with the spectral range of 430-470 nm. A relatively stable intermediate then decays with a half-life of 15 s. 3. The spectrum of the intermediate observed 50 microns after the generation of hydrated electrons shows a broad absorption band between 600 and 700 nm and a peak at 408 nm. The spectrum is attributed to the protonated form of an initially produced porphyrin anion radical. 4. Reduced porphyrin cytochrome c reacts with ferricytochrome c with a bimolecular constant of 2-10(5) M-1- S-1 in 2 mM phosphate pH 7.4, at 21 degrees C and of 2 - 10(6) M-1-S-1 under the same conditions but at 1 M ionic strength. It is proposed that electron transfer in an analogous exchange reaction between ferrocytochrome c and ferricytochrome c occurs via the exposed part of the haem.     

The effect of complex formation upon the reduction rates of cytochrome c and cytochrome c peroxidase compound II

The effect of complex formation between ferricytochrome c and cytochrome c peroxidase (Ferrocytochrome-c:hydrogen peroxide oxidoreductase, EC 1.11.1.5) on the reduction of cytochrome c by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), reduced N-methylphenazonium methosulfate (PMSH), and ascorbate has been determined at low ionic strength (pH 7) and 25 degrees C. Complex formation with the peroxidase enhances the rate of ferricytochrome c reduction by the neutral reductants TMPD and PMSH. Under all experimental conditions investigated, complex formation with cytochrome c peroxidase inhibits the ascorbate reduction of ferricytochrome c. This inhibition is due to the unfavorable electrostatic interactions between the ascorbate dianion and the negatively charged cytochrome c-cytochrome c peroxidase complex. Corrections for the electrostatic term by extrapolating the data to infinite ionic strength suggest that ascorbate can reduce cytochrome c peroxidase-bound cytochrome c faster than free cytochrome c. Reduction of cytochrome c peroxidase Compound II by dicyanobis(1,10-phenanthroline)iron(II) (Fe(phen)2(CN)2) is essentially unaffected by complex formation between the enzyme and ferricytochrome c at low ionic strength (pH 6) and 25 degrees C. However, reduction of Compound II by the negatively changed tetracyano-(1,10-phenanthroline)iron(II) (Fe(phen)(CN)4) is enhanced in the presence of ferricytochrome c. This enhancement is due to the more favorable electrostatic interactions between the reductant and cytochrome c-cytochrome c peroxidase Compound II complex then for Compound II itself. These studies indicate that complex formation between cytochrome c and cytochrome c peroxidase does not sterically block the electron-transfer pathways from these small nonphysiological reductants to the hemes in these two proteins.     

The kinetics of the reduction of cytochrome c by the superoxide anion radical.

1. At neutral pH ferricytochrome c is reduced by the superoxide anion radical (O2-), without loss of enzymatic activity, by a second order process in which no intermediates are observed. The yield of ferrocytochrome c (82-104%), as related to the amount of O2- produced, is slightly dependent on the concentration of sodium formate in the matrix solution. 2. The reaction (k1 equals (1.1+/-0.1) - 10(6) M-1 - s-1 at pH 7.2, I equals 4 mM and 21 degrees C) can be inhibited by superoxide dismutase and trace amounts of copper ions. The inhibition by copper ions is removed by EDTA without interference in the O2- reduction reaction. 3. The second-order rate constant for the reaction of O2- with ferricytochrome c depends on the pH of the matrix solution, decreasing rapidly at pH greater than 8. The dependence of the rate constant on the pH can be explained by assuming that only the neutral form of ferricytochrome c reacts with O2- and that the alkaline form of the hemoprotein is unreactive. From studies at pH 8.9, the rate for the transition from the alkaline to the neutral form of ferricytochrome c can be estimated to be 0.3 s-1 (at 21 degrees C and I equals 4 mM). 4. The second-order rate constant for the reaction of O2- with ferricytochrome c is also dependent on the ionic strength of the medium. From a plot of log k1 versus I1/2-(I + alphaI1/2)-1 we determined the effective charge on the ferricytochrome c molecule as +6.3 and the rate constant at I equals 0 as (3.1+/-0.1) - 10(6) M-1 - s-1 (pH 7.1, 21 degrees C). 5. The possibility that singlet oxygen is formed as a product of the reaction of O2- with ferricytochrome c can be ruled out on thermodynamic grounds.