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Stopped-flow cytochrome

Takigami, T., Takeuchi, F., Nakagawa, M., Hase, T. and Tsubaki, M. Stopped-flow analyses on the reaction of ascorbate with cytochrome b561 purified from bovine chromaffin vesicle membranes. Biochemistry 42 8110-8118,2003. [Pg.332]

The systems that we investigated in collaboration with others involved intermolecular and intramolecular electron-transfer reactions between ruthenium complexes and cytochrome c. We also studied a series of intermolecular reactions between chelated cobalt complexes and cytochrome c. A variety of high-pressure experimental techniques, including stopped-flow, flash-photolysis, pulse-radiolysis, and voltammetry, were employed in these investigations. As the following presentation shows, a remarkably good agreement was found between the volume data obtained with the aid of these different techniques, which clearly demonstrates the complementarity of these methods for the study of electron-transfer processes. [Pg.41]

M. Fabian and co-workers have studied the protein s role in internal electron transfer to the catalytic center of cytochrome c oxidase using stopped-flow kinetics. Mitochondrial cytochrome c oxidase, CcO, an enzyme that catalyzes the oxidation of ferrocytochrome c by dioxygen, is discussed more fully in Section 7.8. In the overall process, O2 is reduced to water, requiring the addition of four electrons and four protons to the enzyme s catalytic center. Electrons enter CcO from the cytosolic side, while protons enter from the matrix side of the inner mitochondrial membrane. This redox reaction. [Pg.143]

In a stopped-flow study on cytochrome cdi from P. aeruginosa, the ferrous d heme-NO species was formed despite electron transfer from the c to the d heme being relatively slow, rate constant approximately 1 s , for the relatively short distance between the two hemes (32). Such a distance would normally predict much faster electron transfer. The relatively slow interheme electron transfer rate has been observed on a number of occasions, and before the structure of the protein was known was thought to reflect the relatively large interheme separation distance and/or relative orientation of the two hemes (30). The crystal structures provide no evidence for either of these proposals there is nothing unusual about the relative orientation of the c and di heme groups. [Pg.181]

Several studies have dealt with the influence of lipids on conformational equilibria in cytochrome c via hydrophobic and electrostatic interactions. The binding of sodium dodecyl sulfate monomers and micelles was reported to cause a transition of cytochrome c to a state B2 which is of potential physiological relevance. The interplay between heme only state changes and secondary structure changes was analyzed by freeze-quench and stopped-flow experiments.276 The response of the heme spin state to lipid acyl chains in cytochrome c was... [Pg.154]

Steady state, and stopped flow, kinetics using cytochrome c l as electron acceptor subsequently confirmed that the reaction cycle is essentially the same as with a dye electron acceptor (Figure 2) (Dijkstra et al., 1989) and... [Pg.77]

A combination of stopped-flow mixing and laser-flash photolysis of the newly generated species is finding increasing use in studies of biological reactions. In a recent example [16], a ferrocytochrome a3-CO adduct was produced by the stopped-flow mixing of the fully reduced cytochrome caaj and CO. The follow-up photolysis caused rapid dissociation of CO. The recombination of the reduced ferrocytochrome and CO, and the reaction with O2 were then observed as kinetic steps. [Pg.479]

The physiological pathway of electron transfer in flavocytochrome is from bound lactate to FMN, then FMN to 52-heme, and finally 52-heme to cytochrome c (Fig. 9) (2,11, 80,102). The first step, oxidation of L-lactate to pyruvate with concomitant electron transfer to FMN, is the slowest step in the enzyme turnover (103). With the enzyme from S. cerevisiae, a steady-state kinetic isotope effect (with ferricyanide as electron acceptor) of around 5 was obtained for the oxidation of dl-lactate deuterated at the C position, consistent with the major ratedetermining step being cleavage of the C -H bond (103). Flavocytochrome 52 reduction by [2- H]lactate measured by stopped-flow spectrophotometry resulted in isotope effects of 8 and 6 for flavin and heme reduction, respectively, indicating that C -H bond cleavage is not totally rate limiting (104). [Pg.275]

Measurements made during the first 50 msec after passing the maximum and in the presence of either erythrocuprein or cytochrome c show an accelerated decay of the Oi- From Fig. 24 it can further be concluded that the superoxide anion is present in much higher concentrations at pH. 10.6 which is probably due to the higher stability of Oi- at elevated pH values (193). On the other hand Oi- is involved in autocatalytic reoxidation of reduced flavins, as demonstrated in stopped-flow studies. For example, in the presence of 0.1 erythrocuprein the reoxidation of tetra-acetyl riboflavin by O2 was diminished by a factor of 4 (151). [Pg.38]

In 1993 Weiss, Riley, and co-workers reported a study on purported SOD mimics by stopped-flow UV-vis spectroscopy (428) in which they assessed reactivity by following the decay of the superoxide absorption at 245 nm. Two of the earlier techniques that had been used to assess SOD activity included observation by UV-vis spectroscopy of the oxidation of nitroblue tetrazolium (NBT) (68) or the oxidation of a cytochrome c by superoxide (52). Both systems used superoxide from an in situ generator, frequently xanthine oxidase, wherein the complex being analyzed was compared to a calibrated oxidation of the chromophore alone and in the presence of MnSOD. The direct observation of the decrease in the superoxide signal with time by UV-vis is also possible, and superoxide may be introduced as a solution (428) or generated, in some cases, by pulsed radiolysis (79, 80). In these direct observation experiments, the rate of decay of superoxide in the presence of the complex is compared to the rates of decay of superoxide alone and in the presence of one unit of activity of MnSOD. In all cases, the systems are usually referenced, or calibrated, against the same set of conditions with MnSOD. Due to interactions with cytochrome c with components of assay mixtures other than superoxide, false readings of activity were often observed for some early SOD mimics. The NBT, stopped-flow, or pulsed radiolysis techniques have tended to provide the more accurate answers on the ability of reputed MnSOD mimics. To be considered active in any manner with respect to the decay of superoxide in the stopped-flow analyses, Weiss et al. stated that compounds based on their analyses needed to exhibit kcat values in excess of 10B 5 M 1 s 1 (428). [Pg.408]

The complex of manganese with desferrioxamine B (429-432), reported to be a MnSOD mimic in 1987, illustrates this point. In the initial studies, the cytochrome c assay was applied, and this compound was reported to be a catalytic MnSOD mimic. Follow-up studies by Riley and co-workers by stopped-flow techniques showed that this complex interacts at best on a stoichiometric level with superoxide, but it could not be considered to be a catalyst and was termed inactive (428). This occurred due to interactions with cytochrome c wherein the manganese complex interfered with the reduction of cytochrome c, leading in effect to a false positive. Thus, although some... [Pg.408]

The available results demonstrate readily the complementarity of the kinetic and thermodynamic data obtained from stopped-flow, UV-Vis, electrochemical and density measurements, and yield a mutually consistent set of trends allowing further interpretation of the data. The overall reaction volumes determined in four different ways are surprisingly similar and underline the validity of the different methods employed. The volume profile in Fig. 1.20 illustrates the symmetric nature of the intrinsic and solvational reorganization in order to reach the transition state of the electron-transfer process. In these systems the volume profile is controlled by effects on the redox parmer of cytochrome c, but this does not necessarily always have to be the case. The location of the transition state on a volume basis is informative regarding the early or late nature of the transition state, and therefore details of the actual electron-transfer route followed. [Pg.25]


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See also in sourсe #XX -- [ Pg.431 ]




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