Enzyme-bound redox properties

The extent of CPO immobilized on the sol-gel was determined by the difference between the activity of the initial enzyme solution and that measured in cumulative washes. Based on the cumulative activity lost in six washes, a second preparation of the CPO-bound sol-gel contained 10, 24, and 55 mg of CPO/g of sol-gel for the 50-, 150-, and 200-A CPO sol-gels, respectively. In prior experiments, the total activity was measured and an estimated 80% of the bound CPO was active. The sol-gel immobilization is expected to limit the unfolding of the protein bound inside pores of the sol-gel. Thus, immobilization is expected to affect solvent stability and thermostability. Immobilization would probably not impact peroxide stability, since the mechanism of peroxide inactivation is associated with changes in the redox properties and oxidation state of the heme iron and the active center, which cannot be protected by immobilization. Experimental studies of immobilized CPO were therefore limited to temperature and solvent stability. 

In dehalogenating enzymes of anaerobic microorganisms, corrinoid cofactors have a newly discovered further role in the redox catalysis of the energy conserving dehalogenation of chloro(hydro)carbon compounds ( dehalorespiration ), and the specific redox properties of the protein-bound unusual corrinoids are of particular current interest. 

Another method of immobilizing enzyme and mediator is the attachmait of a mediator-containing monolayer to an electrode surface. If this monolayer is also functionalized with an enzyme cofactor meant to bind the enzyme, a catalytic electrode results. For example, gold electrode surfaces ean be eovered with a PQQ monolayer. The monolayer retains the redox properties of PQQ and ean be functionalized further. If FAD eofaetors are then covalently bound, GOx apoenzyme can bind the FAD centers. A covalent molecular chain results and electrons may hop from FAD to PQQ to the electrode surface, thus mediating from the enzyme [87]. PQQ monolayers can also link to covalently bound NAD" ", for mediation to NAD" -dependent dehydrogenase enzymes [88,89]. Functionalizing with monolayers of cytochrome c has also been demonstrated [90]. 

It is presently thought that the most obvious reason for the changed properties upon conversion from the unready to the ready state and then to the active state is the removal of the bound oxygen species (e.g. by reduction to OH, subsequent protonation to H2O and removal of this molecule from the active site). As indicated above, reduction causes a slow Niy-S Ni -S transition. It has been shown with the D. gigas enzyme (De Facey et al. 1997) that at 40°C and the appropriate redox potential, the species with the v(CO) of 1,914cm (Ni -S in our scheme) prevails at high pH, whereas a species with v(CO) at 1,934 cm (Nig-S) is the major form at low pH. It could well be that protonation of an OH bound to nickel in the Ni -S state forms a water molecule, which then leaves the active site (at 40°C, but not at 2°C see also Section 5.7), whereafter the site becomes accessible for a rapid reaction with H2. 

Cytochrome c oxidase is the terminal member of the respiratory chain in all animals and plants, aerobic yeasts, and some bacteria." " This enzyme is always found associated with a membrane the inner mitochondrial membrane in higher organisms or the cell membrane in bacteria. It is a large, complex, multisubunit enzyme whose characterization has been complicated by its size, by the fact that it is membrane-bound, and by the diversity of the four redox metal sites, i.e., two copper ions and two heme iron units, each of which is found in a different type of environment within the protein. Because of the complexity of this system and the absence of detailed structural information, spectroscopic studies of this enzyme and comparisons of spectral properties with 02-binding proteins (see Chapter 4) and with model iron-porphyrin and copper complexes have been invaluable in its characterization. 

Table II summarizes the sources and key properties of isolated HiPIPs, almost all of which have been isolated from photosynthetic organisms, and there has been extensive speculation on their involvement in respiratory electron transport chains (18, 21, 91-93, 95, 96, 102-105). Evidence in support of such a hypothesis has recently emerged from studies of a partially reconstructed reaction center (RC) complex from Rhodoferax fermentans (93, 95). The kinetics of photo-induced electron transfer from HiPIP to the reaction center suggested the formation of a HiPIP-RC complex with a dissociation constant of 2.5 fx,M. In vivo and in vitro studies by Schoepp et al. (94) similarly have demonstrated that the only high-redox-potential electron transfer component in the soluble fraction of Rhodocyclus gelatinosus TG-9 that could serve as the immediate electron transfer donor to the reaction-center-bound C3d ochrome was a HiPIP. In vitro experiments have shown HiPIP to be an electron donor to the Chromatium reaction center (106). Fukumori and Yamanaka (107) also reported that Chromatium vinosum HiPIP is an efficient electron acceptor for a thiosulfate-oxidizing enzyme isolated from that organism.

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