Cytochrome titration

For example, Barlow and Margoliash [33] showed that phosphate, chloride, iodide, and sulfate, in decreasing order of effect, reduced the electrophoretic mobihty of human cytochrome c at pH 6.0 by up to a factor of 2. The cations lithium, sodium, potassium, and calcium had no effect. It is possible to account for the binding equilibria of these counterions so that the titration and electrophoresis results can be compared however, in many of the early electrophoresis experiments these data were not available and relevant conditions were not recorded or controlled. For general discussions on the extensive field of ligand binding to proteins, see Cantor and Schimmel [60] and van Holde [403]. 

The kinetics of reactions of NO with ferri- and ferro-heme proteins and models under ambient conditions have been studied by time-resolved spectroscopic techniques. Representative results are summarized in Table I (22-28). Equilibrium constants determined for the formation of nitrosyl complexes of met-myoglobin (metMb), ferri-cytochrome-c (Cyt111) and catalase (Cat) are in reasonable agreement when measured both by flash photolysis techniques (K= konlkQff) and by spectroscopic titration in aqueous media (22). Table I summarizes the several orders of magnitude range of kon and kQs values obtained for ferri- and ferro-heme proteins. Many k0f[ values were too small to determine by flash photolysis methods and were determined by other means. The small values of kQ result in very large equilibrium constants K for the... 

The first electrochemical studies of Mb were reported for the horse heart protein in 1942 (94) and subsequently for sperm whale Mb (e.g., 95) through use of potentiometric titrations employing a mediator to achieve efficient equilibriation of the protein with the electrode (96). More recently, spectroelectrochemical measurements have also been employed (97, 98). The alternative methods of direct electrochemistry (99-102) that are used widely for other heme proteins (e.g., cytochrome c, cytochrome bs) have not been as readily applied to the study of myoglobin because coupling the oxidation-reduction eqiulibrium of this protein to a modified working electrode surface has been more difficult to achieve. As a result, most published electrochemical studies of wild-type and variant myoglobins have involved measurements at eqiulibrium rather than dynamic techniques. 

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).]...

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. 

Potentiometric reductive titration, using both fresh thylakoids and PS II particles previously oxidized with ferricyanide, has revealed that the LP couple exhibits a constant midpoint redox potential ( o- +° 12 v ) above pH 7.6, but becomes pH-dependent below this pH, with a slope of about -60 mV pH pH unit, whereas the HP couple is pH-independent in the pH range between 6.5 an 8.5 ( o-+0 36 V) in general, cytochrome b-559 exhibits potential values about 40 mV lower in thylakoids than in PS II particles. After mild heating of the fresh preparations or treatment with the detergent Triton X-100, the HP couple is converted into the LP couple, which preserves its characteristic pH-dependence. In contrast, in the presence of the uncoupler CCCP, the HP couple is also converted into the LP couple, but the pH-dependence proper to the latter is now lost. 

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

The EPR-nondetectable ions in laccase function as a cooperative 2-e" unit (5). With cytochrome oxidase redox titrations based on the heme absorption bands (2) indicate the presence of a high and a low potential site (380 and 220 mV, respectively). On the other hand, the quasi equilibrium established in the rapid initial transfer of electrons from reduced cytochrome c to the primary electron acceptor in the oxidase, cytochrome a, indicates a potential of 285 mV for this site (18). 

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. 

Partially purified cytochrome c oxidase purified oxidase titrated as a single component with 71 = 1 and E = 285 mV. 

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