Proteins redox, reconstitution

The replacement of native haem with metal-substituted porphyrin can be performed in two ways. In the first, iron ions are removed from the protein by treating native protein with anhydrous HF, followed by insertion of the appropriate metal to metal-free protein [57-59]. In the second, haem is removed either chemically or by recombination (preparing a proper recombinant protein), and then protein is reconstituted with metal-substituted porphyrins [60-64]. The Zn-substituted metal-loproteins such as cytochrome c [65-67], myoglobin [59,61, 62, 64, 68, 69], and haemoglobin [64, 68,70] have been extensively used to study photoinduced ET (PET) between modified proteins and their physiological redox partners. Interestingly, in haemoglobin with a and /3 subunits it was possible to determine ET parameters for... 

By one method a relay-cofactor dyad is assembled on the electrode, and the respective apo-protein is reconstituted on the surface to yield an aligned protein that is linked to the conductive surface by the relay component. The second method involves the synthesis of the relay-cofactor unit and the reconstitution of the apo-protein in solution. The specific immobilization of the enzyme on the electrode by the relay unit provides the structurally organized enzyme electrodes. While the first method is technically easier, the second methodology that involves tedious synthetic and separation steps, permits the fundamental structural characterization of the reconstituted protein. In the two configurations, the redox enzymes are anticipated to be electrically contacted with the electrode by means of the relay, a conductive... 

Methods to electrically wire redox proteins with electrodes by the reconstitution of apo-proteins on relay-cofactor units were discussed. Similarly, the application of conductive nanoelements, such as metallic nanoparticles or carbon nanotubes, provided an effective means to communicate the redox centers of proteins with electrodes, and to electrically activate their biocatalytic functions. These fundamental paradigms for the electrical contact of redox enzymes with electrodes were used to develop amperometric sensors and biofuel cells as bioelectronic devices. 

A further approach to controlling electrical communication between redox proteins and their electrode support through a photo-command interface includes photo stimulated electrostatic control over the electrical contact between the redox enzyme and the electrode in the presence of a diffusional electron mediator (Scheme 12).[58] A mixed monolayer, consisting of the photoisomerizable thiolated nitrospiropyran units 30 and the semi-synthetic FAD cofactor 25, was assembled on an Au electrode. Apo-glucose oxidase was reconstituted onto the surface FAD sites to yield an aligned enzyme-layered electrode. The surface-reconstituted enzyme (2 x 10-12 mole cm-2) by itself lacked electrical communication with the electrode. In the presence of the positively charged, protonated diffusional electron mediator l-[l-(dimethylamino)ethyl]ferrocene 29, however, the bioelectrocatalytic functions of the enzyme-layered electrode could be activated and controlled by the photoisomerizable component co-immobilized in the monolayer assembly (Figure 12). In the... 

The clusters thus generated have been found to have properties closely matching those of native proteins. Further studies have shown that N-H- -S hydrogen bonding to sulfur atoms within the cluster creates a small positive shift in redox potentials. The sequence Cys-(X)3-Cys-(X)2-Cys-(X)2-Cys, which includes sequence (6) and is often found in ferredoxins, has been shown to support efficient cluster reconstitution. ... 

Reconstitution of membranes from a small number of molecular components provides simplified structures to study. Thus, cytochrome oxidase or photosynthetic reaction centers, both electron transfer proteins, may be extracted from their native membranes, purified, and reincorporated at relatively high concentration into a simple well defined lipid bilayer. Diffraction investigation then provides information about the distribution and structure of the protein in the membrane. Understanding the mechanism for electron transport in these proteins will require considerable additional information. One key element of structural informations is the location of the redox centres in the membrane profile. 

The site-specific modification of enzymes with a single electron-relay group located near to the redox cofactor and providing efficient electrical contact with the conductive support has been achieved by the reconstitution of enzymes with cofactors covalently linked to redox groups. Affinity interactions between enzymes and their cofactors at the electrode interface can allow the efficient electrical contacting of aligned proteins. 

Semi-synthetic enzymes are produced by the reconstitution of apo-proteins with artificial active sites that yield novel catalytic functions [237]. For example, reconstitution of apo-myoglobin with Co(II)-protoporphyrin IX results in a novel biocatalyst that is capable of hydrogenating acetylene derivatives or evolving hydrogen [209, 238]. By the modification of the reconstitution of apo-proteins with artificial redox-active cofactors and the covalent attachment of photosensitizer units, photo-... 

Willner, I., Katz, E., Willneg B., Blondei R., Heleg-Shabtai, V., and Buckmann, A. F. Assembly of functionalized monolayers of redox proteins on electrode surfaces novel bioelectronic and optobioelectronic systems. Biosens. Bioelectron. 1997,12, 337-356. Blonder R., Katz, E., Willneg I., Wray, V., and Buckmann A. F. A lication of a nitro-spiropyran-FAD reconstituted glucose oxidase and cfuurged electron inediators as optobioelectronic assemblies for the amperometric transduction of recorded cortical signals control of the ON - OFF direction of the photoswitch. J. Am. Chem. Soc. 1997, 119, 11747-11757. 

In contrast to ferritin, very little work has been done on the reconstitution of BFR cores, other than the experiments mentioned above that showed that, in the absence of phosphate, crystalline ferrihydrite formed inside the protein shell. The intermediate stages in this process are unknown, but the sigmoid iron uptake behavior (25) suggests there could be a similar succession of events oxidation and nucleation on the protein shell followed by direct oxidation on the core. The influence of the heme, if any, on BFR iron core formation also awaits investigation. As mentioned above, the presence of the iron core influences the heme redox potential, but it is not known whether the presence of heme influences the redox potential of the nonheme iron. 

In all the biomimetic membranes previously described and allowing the incorporation of proteins, the protein orientation in the membrane is purely casual. At most, if one of the two extremembrane domains of the protein is much bulkier than the other, incorporation in a tBLM occurs preferentially with the bulkier domain turned toward the aqueous phase, in view of the hmited spaciousness of the hydrophihc moiety of the tBLM. Moreover, the packing density of the reconstituted proteins in the hpid bilayer is not well controlled. The need for a well-defined protein orientation with respect to the electrode surface is particularly felt with redox membrane proteins, in which the electrons involved in a chain of redox couples are conveyed across the membrane in a weh-defined direction. 

The need to improve the electrical communication between redox proteins and electrodes, and the understanding that the structural orientation at the molecular level of redox proteins and electroactive relay units on the conductive surfaces is a key element to facilitate ET, introduced tremendous research efforts to nano-engineer enzyme electrodes with improved ET functionalities. The present chapter addresses recent advances in the assembly of structurally aligned enzyme layers on electrodes by means of surface reconstitution and surface crosslinking of structurally oriented enzyme/cofactor complexes on electrodes. The ET properties of the nano-structured interfaces is discussed, as well as the possible application of the systems in bioelectronic devices such as biosensors, biofuel cell elements or optical and electrical switches. 

ELECTRODES EUNCTIONALIZED WITH RECONSTITUTED REDOX PROTEINS... 

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