Electrode cytochrome oxidase

The stoichiometry of proton pumping was measured by Lehninger and associates using a fast-responding 02 electrode and a glass pH electrode.189 190 They observed an export of eight H+/ O for oxidation of succinate rat liver mitochondria in the presence of a permeant cation that would prevent the buildup of Em, and four H+/ O (2 H+/ e ) for the cytochrome oxidase system. These are equivalent to two H+/ e at each of sites II and III as is indicated in Fig. 18-4. 

Figure 12.3 Coupling of photoinduced electrical interaction of cytochrome c (cyt c) with the photoisomerizable monolayer modified Au electrode to the biocatalyzed reduction of 02 by cytochrome oxidase (COX) [33]...

The activity of the enzyme is also strongly affected by the presence of inhibitors. Fluoride ions inhibit urease (173) and oxalate ions inhibit lactate oxidase (174), but the major inhibitors are heavy-metal ions, such as Ag+, Hg +, Cu " ", organophosphates, and sulfhydryl reagents (/i-chloromercuribenzoate and phenylmercury(II) acetate), which block the free thiol groups of many enzyme active centers, especially oxidase (69). Inhibiting the enzyme electrodes makes it possible to quantify the inhibitors themselves (69), for example, H2S and HCN detection using a cytochrome oxidase immobilized electrode (176). 

Direct electrochemistry has also been used (72-78) to couple the electrode reactions to enzymes for which the redox proteins act as cofactors. In the studies, the chemically reduced or oxidized enzyme was turned over through the use of a protein and its electrode reaction as the source or sink of electrons. In the first report (72, 73) of such application, the electrochemical reduction of horse heart cjd,ochrome c was coupled to the reduction of dioxygen in the presence of Pseudomonas aeruginosa nitrite reductase/cytochrome oxidase via the redox proteins, azurin and cytochrome C551. The system corresponded to an oxygen electrode in which the four-electron reduction of dioxygen was achieved relatively fast at pH 7. 

Figure 3-32. (B) The assembly of an electroswitchable, electrically-contacted electrode for the bioelectrocatalyzed reduction of O2 consisting of a crosslinked affinity complex of cytochrome c/cytochrome oxidase (cyt c/COx) on a polyacrylic acid-polyethylene imine film that includes incorporated Cu -ions. The conductivity of the film and the electrical contacting of the redox-proteins is accomphshed by applying a potential of -0.5 V vs. SCE and the generation of Cu-clusters in the film. Reproduced with permission from ref. 89. Copyright 2003 American Chemical Society.

Hill et al. (1981) proposed a CO sensor based on inhibition by the gas of cytochrome oxidase (EC 1.9.3.1) coupled to a modified platinum electrode via cytochrome c. This type of sensor has been further improved by Albery et al. (1987a). 

Albery et al. (1987a) developed a carbon monoxide sensor based on the sequence of cytochrome oxidase and cytochrome c coupled to a modified gold electrode. The inhibition by CO was detected via the decrease of the oxygen reduction rate. The sensor is also applicable to the quantitation of other inhibitors of the respiratory chain. 

A modified carbon paste electrode (CPE) using asolectin, cytochrome c, and cytochrome oxidase were applied for amperometric determination of cyanide [56]. The modified CP matrix mimics a biological membrane environment. The sensor, polarized at —0.15 V versus Ag/AgCl, generates the reduced form of cytochrome c, which in turn is oxidized by the enzyme cytochrome oxidase. The resulting current is related to the enzyme activity and is depressed by inhibitors of cytochrome oxidase such as cyanide. Concentrations of cyanide as low as 0.5 pM can be measured with half-maximal response at about 12 pM. The inhibition is reversible and reproducible (RSD = 4%), allowing cyanide determination for more than 2 months using the same probe. Possible use of this biosensor in flow systems was illustrated. 

Another application for photoswitchable enzymes attached to gold surfaces is the cytochrome c-mediated biocatalysed reduction of O2 by cytochrome oxidase, using a functional pyridine-nitrospiropyran photoiso-merisable mixed monolayer electrode (see Sect. 6.1, Fig. 39) [8,16,17]. 

Composites of gold and PANl on a glassy carbon electrode were also used by Xiang et al. [52] for the development of a glucose biosensor. They used a bienzy-matic approach where glucose was oxidized thereby producing H2O2, which was reduced by cytochrome oxidase c, absorbed on the surface of the nanocomposites. This sensor enabled a direct electron transfer to the electrode material. The sensor has a detection limit of 0.01 mM. 

PANI was also used in combination with carbon nanotubes to fabricate a sensor with cytochrome oxidase as bioelement [57]. This systan could be used for different kinds of sensing events based on the enzymatic reduction of H202- The sensitivity of a biosensor based on a PANI film on a gold electrode was improved by using a composite of PANI with pol) yrrole nanoparticles [58]. The sensor was used to detect glutamate in food products with an enzymatic reaction of the covalently bound glutamate oxidase. 

Batra, B., et al. Fabrication of a cytochrome c biosensor based on cytochrome oxidase/NiO-NPs/cMWCNT/PANI modified Au electrode. J. Biomed. Nanotechnol. 9(3), 409- 16 (2013)... 

Mesoporous materials with controlled porosity and functionality have been used to immobilize biomolecules, e.g., proteins. Enzymes of small or medium size such as cytochromes, oxidases, peroxidases, lipases or proteases, can be immobilized via physical adsorption, encapsulation, or chemical binding. Such immobilization was found to maintain some activity of the biomolecule, with applications in the biosensor field. Recently, some developments have been reported in the immobilization of redox proteins in mesoporous transparent electrodes for... 

Fig. 21. Effect of nitrite on oxygen consumption catalyzed by P. cytochrome oxidase. (A) Without addition of nitrite. (B) Nitrite added. Oxygen consumption was measured with an oxygen elec trode. As the electrode is disturbed by hydro-quinone, the zero level for the oxygen concentration does not coincide with the zero line presented in the figures.

Mayer D, Ataka K, Heberle J, Offenhaeusser A. 2005. Scanning probe microscopic studies of the oriented attachment and membrane reconstitution of cytochrome c oxidase to a gold electrode. Langmuir 21 8580-8583. 

K.V. Gobi, Y. Sato, and F. Mizutani, Mediatorless superoxide dismutase sensors using cytochrome c-modified electrodes xanthine oxidase incorporated polyion complex membrane for enhanced activity and in-vivo analysis. Electroanalysis 13, 397-403 (2001). 

Because electrode measurements of O2 uptake can detect intra- and extracellular oxidase activity, this assay can be used to measure the respiratory burst elicited by soluble and particulate stimuli. What is somewhat surprising is that, during stimulation of neutrophils with agonists such as fMet-Leu-Phe, the activated O2 uptake profile is biphasic (Fig. 5.11c). A rapid burst of O2 uptake (which coincides with measurements of cytochrome c reduction) is followed by a more sustained activity of lower magnitude. 

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