Titrations, of cytochrome

Figure 3.42 Indirect coulometric titration of cytochrome c oxidase by incremental generation of MV at Sn02 optically transparent electrode. (A) Spectra recorded during titration. (B) Titration curve, MV added in increments of 0.25 mC. [Adapted from W. R. Heineman, T. Kuwana, and C.R. Hartzell, Biochem. Biophys. Res. Commun. 49 1 (1972).]...

Figure 17.1.5 Coulometric titration of cytochrome c (17.5 /xM) and cytochrome c oxidase (6.3...

Figure 7-7 The stoichiometry of binding of cytochrome csso to cytochrome c peroxidase. A solution (0.75 mM) of cytochrome C550 in 10 him Mes pH 6.0, D2O was titrated with a solution of cytochrome c peroxidase (1.4 him) in the same buffer. H-NMR spectra were recorded (2000 scans, 299 K) in a Bruker AX 400 MHz spectrometer. The molar proportions of titrant are indicated on the right side of the individual spectra. The shift of the methionine methyl resonance caused by binding to the c)rtochrome c peroxidase is plotted. Open circles are for the titration of cytochrome C550 with cytochrome c peroxidase. Closed circles are for the complementary titration of cytochrome c peroxidase (1 him) with cytochrome C550 (1.6 mm). The upper curve is theoretical for a Kd of 5 pm the lower is theoretical for a Kd of 20 pM.

Fujihira, Y. Kuwana, T. Hartzell, C. R. Reversible redox titrations of cytochrome-c and cytochrome-c oxidase using detergent solubilized electrochemically generated mediator-titrants. Biochem. Biophys. Res. Commun. 1974, 61, 538-543. 

Figure 10. Plot of log [cytochrome c (reduced)/cytochrome c(oxidized)Jversus log [cytochrome c oxidase (reduced)/cytochrome c oxidase (oxidized)] for reductive indirect coulometric titration of cytochrome c-cyto-chrome c oxidase mixture. Data points taken from Figure 9 and solid line is computer calculation for the cytochrome c formal potential of 250 mV, the cytochrome c oxidase heme a formal potential of 210 mV, and the cytochrome c oxidase heme formal potential of 350 mV. Adapted from reference (60) with permission.

RCs from Rps. viridis were isolated as described by [1], Absorption spectra were recorded with a Aminco DW-2a spectrophotometer. The redox potential was measured with Pt, Ag/AgCl pare of electrodes. The redox-titrations of cytochrome c hemes were carried out at as in [1]. Data storage, processing and curve fitting were carried out with IBM AT computer using self-made program system GIM. 

FIGURE 1. a) The experimental absorbance-wavelength>Eh surface for high-potential redox titration of cytochrome c in Rps. viridis RCs. Experimental conditions as in [1]. 

Cells, membrane preparations, or isolated RC complexes were used for the determination of cytochromes oxidized by flash excitation under aerobic conditions and anaerobic conditions in the presence of ascorbate and DAD. In some species, redox titrations of cytochromes (flash-induced changes and total contents) were also performed. 

Fig. 3. Plot of log [cytochrome Cred]/[cytochrome vs. log [cytochrome c oxidasCred]/[cytochrome c oxidasCo ] for first reduction titration of cytochrome c - cytochrome c oxidase mixture. Data points obtained from data for reduction by MV in Fig. 2. Solid line computer simulation for E° (cyt c) = 0.250 V, (cyt a) = 0.210 V, E° (cyt 3) = 0.350 V. (Reproduced from [28], courtesy of the publisher).

Electrochemical titrations of redox proteins with spectroscopic monitoring bear the advantage that the sample is not successively diluted with redox reagents, that titrations can be performed in cycles, and that the electrode potential (and thus the ox/red equilibrium of the sample) can be set at mV precision. This precision offers the possibility to analyze redox proteins with several cofactors that may be coupled and that may exhibit cooperativity. As an example, Fig. 2 shows the complete redox titration of cytochrome c oxidase, the terminal enzyme of the respiratory chain. Provided that fast equilibration (typically within seconds) at the electrode occurs, a full spectral data set can be obtained within some minutes. 

V. CYTOCHROME C AS MEDIATOR IN INDIRECT COULOMETRIC TITRATIONS OF CYTOCHROME C OXIDASE... 

Figure 7 Difference absorption spectra for an indirect coulometric titration of cytochrome c oxidase. Solution contained 150 xM cytochrome c, 11 pM C5dochrome c oxidase, and less than IpM oxygen. Charge injected in first two reductive increments 5 nmol, 10 nmol thereafter. Optical path length 2.3 mm, solution 0.21 M Tiis, 0.24 M cacodylic acid (0.20 M ionic strength), pH 7.0, 0.5% (v/v) Tween 20.

Figure 3. NMR changes of heme 8-CH3 and 3-CH3 on titration of cytochrome c with 3.

Figure 13 Spectrocoulometric titration of cytochrome c (17.5 iiM) and cytochrome c oxidase (6.3 )iM) by reduction with electrogenerated methyl viologen radical cation (MV +) at a SnOs OTE. Each spectrum was recorded after 5x10 equivalents of charge (0.5 mC) were passed. Spectra correspond to titration from totally oxidized to totally reduced forms. The final two spectra around 605 nm were recorded after excess MV + was present. Inset shows titration curves at 550 and 605 nm. Reprinted with permission from Heineman WR, Kuwana T and Hartzell CR (1973) Biochemical and Biophysical Research Communications 50 892-900.

The titration of cytochrome oxidase-dimyristoyl phosphatidylcholine complexes of different lipid/protein ratios, labelled with the 14-PCSL spin label, is given in Fig. 3.4. The ratio of... 

In order for the heme to combine with such ligands as oxygen and carbon monoxide, one electron must be introduced to form the ferrous state (Peterson et a/., 1977). The oxidation-reduction step has been measured for the purified cytochrome P450 from adrenal cortex mitochondria and from P auaomorm putida. In each case large negative values have been obtained and correlated with the reductive titration of cytochrome P450 in rat liver microsomes (Cooper et a/., 1977). 

Mediating electron transfer in micelles seems to have been reported first by Kuwana and co-workers , who used electrogenerated ferricinium ion in non-ionic micelles for redox titrations of cytochrome C and cytochrome C oxidase. Neutral micelles have been used to solubilize cytochrome C oxidase in spectroelectrochemical titrations , but examples of other applications are few. 

Addition of cytochrome c to solutions containing some of our TPP-based receptors resulted in quenching of porphyrin fluorescence emission (Ex = 420, Em = 650 mn). In contrast, titrations with tetracationic /w-tetrakis-(4-trimethylaminophenyl) porphyrin (11,TTMAPP) and cationic TPP-based receptor 10 showed no quenching even at high concentrations. This demonstrates the clear preference for anionic species by the cationic cytochrome c recognition surface, in agreement with previously reported studies. 

Figure 9.1 Titration of 1 /xM cytochrome b2 core in 6 M guanidine with aliquots of 5.5 fiM of L-Trp.

Fig. 9. A reductive titration of the crystalline bovine heart cytochrome c oxidase with dithionite. Absolute spectra for each oxidation state are shown for the Soret (A) and visible (B) regions. The difference spectra against the spectrum in the fully reduced state are given for the near-infrared region (C). The insets show titration curves against the electron equivalent per enzyme. The reaction mixture contained 7.5 jlM bovine heart cytochrome c oxidase in 0.1 M sodium phosphate buffer, pH 7.4. The enzyme preparation was stabilized with a synthetic non-ionic detergent, CH3(CH2)ii(0CH2CH2)80H. The light path was 1 cm.

The lipase-solubilized reductase is inhibited by p-mercuribenzoate, is protected from this inhibition by NADPH, and the inhibition is relieved by thiols (10). Careful titration of this enzyme with p-mercuribenzoate at pH 6.5 results in an almost 3-fold stimulation upon addition of 2 moles of mercurial per flavin the control activity is again observed when 7 equivalents have been added. At pH 7.7, a stimulation of 70% is seen with 1 equivalent and loss of activity is complete (extrapolated) with 6 equivalents (245). The protection of the enzyme by NADPH against mercurial inhibition is reminiscent of the effects with NADH cytochrome 63 reductase (360). 

Fw. 18. Anerobic titration of NADPH-cytochrome P-450 reductase with NADPH. Curve 1, oxidized enzyme curves 2-6 after the addition of 0.16, 054, 0.49, 058, and 1.4 moles of NADPH per mole of total enzyme-bound flavin. The inset, B, shows the changes at 455 and 586 nm as a function of the NADPH added (405)-... 

The redox properties of cytochrome c oxidase have been investigated both by anaerobic reductive titrations 159) and by potentiometric titrations (160). Since measurements of the latter kind are, at least in principle, able to provide absolute potential values, they have been favored in recent studies. The inconsistencies found in the early work (161-163) may have resulted from the lack of equilibrium conditions in some cases, from differences in the preparations, or simply from some incorrect interpretations of data. The importance of establishing that equilibrium conditions are attained has recently been recognized (107, 124,1 5), but identical sets of measurements on the various types of preparations have yet to be reported. 

The fluorescence studies of the interaction of cytochrome c with the anilinonaphthalene sulfonate-apoenzyme and protoporphyrin-apoenzyme complexes provide another line of evidence (S9) in support of the above-mentioned conclusion. Both fluorescence steady-state and lifetime titrations of these fluorescence-labeled apoenzymes with ferro- and ferricytochrome c indicates the formation of a 1 1 complex, the afiSnity for ferricytochrome c being less than that for ferrocytochrome c. From the phosphorescence and fluorescence quenching, the distance between the emitter (a fluorescence label) and the quencher (the heme of cytochrome... 

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