Bifunctional redox proteins

Electron Transfer Kinetics of Bifunctional Redox Protein Maquettes... 

We prepared three bifunctional redox protein maquettes based on 12 16-, and 20-mer three-helix bundles. In each case, the helix was capped with a Co(III) tris-bipyridyl electron acceptor and also functionalized with a C-terminal viologen (l-ethyl-V-ethyl-4,4 -bipyridinium) donor. Electron transfer (ET) was initiated by pulse radiolysis and flash photolysis and followed spectrometrically to determine average, concentration-independent, first-order rates for the 16-mer and 20-mer maquettes. For the 16-mer bundle, the a-helical content was adjusted by the addition of urea or trifluoroethanol to solutions containing the metal-loprotein. This conformational flexibility under different solvent conditions was exploited to probe the effects of helical secondary structure on ET rates. In addition to describing experimental results from these helical systems, this chapter discusses several additional metalloprotein models from the recent literature. 

A different example of complementarity is explored in this chapter. We report studies of a bifunctional redox protein maquette based on the triple-helix bundle design of Ghadiri, Sasaki, and co-workers (4-S). Attempts to monitor electron transfer from an N-terminal ruthenium tris-bipyridyl excited state were fruitless, since electron transfer could not compete with the excited state decay of the Ru(IP) tris-bpy. However, as described below, replacement of photoactive ruthenium by a radiolytically accessible Co couple has allowed initial exploration of electron transfer in this synthetic protein couple. In turn, photochemistry initiated at the viologen chromophore helped confirm and extend the radiolytic results. 

More interestingly, electron transfer reaction of cytochrome c and other redox proteins occurs with fast rates at modified electrodes. The most efficient electrode surface promoters tend to be bifunctional molecules containing surface-active functional groups (thiol, disulphide, or pyridyl) which bind the promoter to the electrode surface and weakly basic or anionic functional groups (such as pyridyl nitrogen, carboxy-late, sulphonate or phosphate) which can bind the positively charged cytochrome c [188-190,204-211]. For instance, if a gold electrode is modified with 4,4 -bipyridine, this promoter forms a monolayer at the... 

There now is no difficulty in achieving the electrochemistry of small redox proteins (see, for example, Armstrong et al., 1987, 1988, Frew and Hill, 1987, 1988) whether, e.g., cytochrome c, (Figure 1) ferredoxin, azurin, rubredoxin or flavodoxin (Figure 2), A variety of electrode surfaces exist at which the direct electrochemistry of proteins proceeds without the need for mediators. At some metal electrodes, what is required is the presence of some compound, called a promoter (Eddowes and Hill, 1977), which binds to the surface yet allows electron transfer to proceed (Figure 3). Over the years, many promoters have been reported (Taniguchi et al., 1982 Allen et al., 1984) and they are all bifunctional molecules, one part of which causes... 

The investigation on the use of molecules suitable for modifying the electrode surface so as to favour electron transfers with proteins (so-called promoters, which are non-redox active molecules and therefore unable to act as redox mediators) has determined that they must be bifunctional molecules X—V /—Y, in which X is a group able to... 

The simple coordination chemistry characteristic of the majority of protein-metal interactions is replaced in certain cases by irreversible covalent modifications of the protein mediated by the metal ion. These modifications are essential for the function and are templated by the structure of the protein, as no other proteins are required for the reaction to occur. These self-processing reactions result in the biogenesis of redox cofactors in some enzymes (amine oxidases, galactose oxidase, cytochrome c oxidase) and activation of hydrolytic sites in others (nitrile hydratase). The active sites of all of these enzymes are bifunctional, directing not only the catalytic turnover reaction of the mature enzyme but the modification steps required for maturation. 

All known ACS enzymes are bifunctional in that they possess a C cluster with COdFI activity in addition to an A cluster (the ACS active site. Scheme 9). In the enzymes, a CO tunnel is described through which GO can pass directly from the C cluster, where it is generated from CO2, to the A cluster, where acetyl GoA synthesis takes place. Again, two mechanisms were proposed that differ in the order of binding events and redox states involved. In essence, however, GO binds to an Ni-GHs species, followed by insertion and generation of an Ni-acetyl species, which upon reaction with GoA liberates the acetyl GoA product. It is interesting to note that methylation of Ni occurs by reaction with methyl cobalamin (Scheme 7). In M. thermoacetica, the cobalamin is the cofactor for a rather unique protein called the corrinoid iron sulfur protein (GFeSP). The above process, even if mechanistic details still remain in question, resembles the industrial Monsanto acetic acid synthesis process (Scheme 9, bottom). In this case, however, the reaction is catalyzed by a low-valent Rh catalyst. 

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