Reduction kinetics, cytochrome

Bhattacharyya, A.K., Lipka, J.J., Waskell, L. and Tollin, G. (1991) Laser flash photolysis studies of the reduction kinetics of NADPH cytochrome P-450 reductase. Biochemistry, 30 (3), 759-765. 

Care should be exercised that excess of one reactant does in fact promote irreversible reaction if this is the desired object, otherwise invalid kinetics and mechanistic conclusions will result. Consideration of the reduction potentials for cytochrome-c Fe(III) and Fe(CN)g (0.273 V and 0.420V respectively) indicates that even by using a 10 -10 fold excess of Fe(CN)j , reduction of cytochrome-c Fe(III) will still not be complete. An equilibrium kinetic treatment is therefore necessary. ... 

Examination of one real-life case may benefit the reader s understanding. Strittmatter studied the primary kinetic isotope effects arising in the NADH-dependent cytochrome bs reductase (EC 1.6.2.2). The oxidation of NADH and subsequent reduction of cytochrome bs is facilitated by the enzyme-bound FAD group, and the kinetics of the direct transfer of a hydrogen from the A-face (or pro-R) of NADH to the flavin can be monitored by the loss of the 340 run absorbance of the NADH s dihydropyridine ring. Using deuterated isotopic isomers of NADH and several related compounds, Strittmatter obtained the primary kinetic isotope effect data compiled in the table below. 

Cytochrome c552 from Euglena gracilis (also known as cytochrome / or c6) contains 87 amino acid residues, two hemes and one flavin per molecule.693 NMR studies706 indicate that the chirality of the axial methionine is similar to that of cytochrome c but different from cytochrome c5Sl. Rapid intramolecular transport has been demonstrated by the use of pulsed laser excitation, and the measurement of reduction kinetics. Both flavin and heme groups are reduced simultaneously on a multisecond time scale, with the transient formation of a protein-bound flavin anion radical.707... 

Figure 13.3 is another measurement, with the concentrations measured on the vertical axis, the time on the horizontal it displays the outcome of an experiment on the kinetics of ascorbate reduction by cytochrome c, which supports the claim that the antibody mimic does impair the function of the protein, in this case its ability to react with ascorbate. 

Arciero DM, Balny C, Hooper AB (1991) Spectroscopic and rapid kinetic studies of reduction of cytochrome c-554 by hydroxylamine oxidoreductase from Nitrosomonas europaea. Biochemistry 30 11466-11472... 

Rich, P. R. Bendall, D. S. "Ifre Kinetics and lliamodynamics of the Reduction of Cytochrome c by Substituted p-Benzoquinols in Solution,"Biochim. Biophys. Acta 1980,592,506-518. 

Nishio IN and Whitmarsh J (1993) Dissipation of the proton electrochemical potential in intact chloroplasts. II. The pH gradient monitored by cytochrome/reduction kinetics. Plant Physiol 101 89-96... 

Recently we carried out kinetic studies with Hildenborough and Miyazaki cytochrome c3 using deazariboflavin semiquinone (dRf ), MV +, and propylene diquat (PDQ +), produced by laser flash photolysis, as reductants (37). Initially, all three reactions were accurately second order, consistent with all hemes being reduced with the same rate constant or with a single site reduced, followed by fast intramolecular electron transfer to reduce the remaining three hemes. However, by measuring reduction kinetics with cytochrome c3 poised at different extents of reduction, the kinetics of reduction of individual hemes could be resolved. Thus, reduction of cytochrome c3 in approximately 5% steps and application of the known macroscopic redox potentials (see previous section) enabled calculation of the concentration of each heme (c.) at each stage of reduction. The plot of kohs versus percent reduction can thereby be fitted to solve for the rate constant for each heme (kt) ... 

Hintz, M.J. and J.A. Peterson (1981). The kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin.Biol. Chem. 256, 6721-6728. 

Electron transfer between electron donor and acceptor located in two different proteins or within the same protein induces a spectral modification in the absorption of both redox centers and thus kinetics parameters and electron transfer data can be studied with absorption. Figure 1.23 displays the absorption spectra of oxidized (a) and reduced (b) forms of cytochrome b2 core extracted from the yeast Hansenula anomala. Reduction of cytochrome c with cytochrome b2 core is followed at the isobestic point of cytochrome b2 equal to 416.5 nm. At this wavelength, only absorption of cytochrome c increases upon reduction. 

It was mentioned before that the presence of substrates accelerate the reduction of cytochrome P450. The kinetics of this process can be conveniently followed under anaerobic conditions in the presence of carbon monoxide, since this ligand forms a rather stable complex which can be measured spectrophotometrically by its ab-... 

This chemically modified electrode exhibits quasi-reversible rates of heterogeneous electron transfer for the reduction of cytochrome c as determined by single potential step chronoabsorptometry. However, reductive and oxidative experiments did not yield kinetic results in agreement with Butler-Volmer theory. 

Fig. 4A shows the time-course of P reduction and cytochrome oxidation after a saturating short flash. In less than 1 ms, 50% of P is reduced while the reduction of the second half occurs in a much longer time-range. Conversely, after a weak flash (Fig. 4B), the reduction of P approximately follows a first order kinetics. We determined that the concentration of cyt c2 was equal or larger than 0.8 times the concentration of the reaction centers therefore, the break observed when half of the P is reduced is not determined by the amount of available cyt c2. The following structural hypothesis accounts for our data ... 

We have previously reported a break in similar Arrhenius plots at a lower temperature (1). Our previous analysis did not properly take into account the behavior of the iron-sulfur center as reductant to the c cytochromes. Furthermore, the rate constants calculated from the overall fitting were underestimates of the true values, especially below 20 C, since the initial rising phase of fitting curve had to be reduced to compensate for the lag period in the bjj reduction kinetics. Therefore the rate constants obtained from the overall fitting are hardly valid. 

The result showed that cytochrome c itself is kinetically incompetent with regard to reduction of cytochrome cdi, but it is able to transfer electrons to... 

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