Heme-reconstituted de novo protein

Figure 22. (A) The assembly of a nitrate-sensing electrode by the cross-linking of an affinity complex formed between nitrate reductase (cytochrome-dependent, EC 1.9.6.1), NR and an Fe(III)-protoporphyrin reconstituted de novo four-helix-bundle protein. (B) Cyclic voltammograms of the NR-two heme-reconstituted de novo protein-layered Au electrode at nitrate concentrations of (a) 0, (b) 12, (c) 24, (d) 46 and (e) 68 mM. Inset calibration curve for the amperometric response of the electrode at different nitrate concentrations (at E = —0.48 V vs. SCE). Potential scan rate, 5 mV s" 0.1 M phosphate buffer, pH 7.0, under argon electrode roughness factor, ca. 20.

Figure 25. (A) Vectorial electron transfer in the two-heme-reconstituted de novo protein molecules organized as a monolayer at an electrode surface. (B) Transient current recorded with the two-heme reconstituted de novo protein monolayer during the double-potential step chronoamperometric experiment. The potential steps from -0.2 to -0.5 V (vs. SCE) to reduce the hemes in the protein, and after 70 ms the potential steps back, from —0.5 to —0.2 V, to oxidize the reduced hemes. The experiment was performed in 0.1 M phosphate buffer, pH 7.0, under argon.

The reconstitution of a tailored de novo protein with Fe(III)-protoporphyrin IX units, (15), on an electrode surface generates a hemoprotcin-type assembly that mimics ET functions of cytochrome c. A four-helix bundle de novo protein, (16), consisting of 128 amino acids and a mass of 14728 was synthesized. It included two pairs of identical helices A and B. The B helices are terminated with Gly-Gly-Cys units for the assembly of the structures on surfaces, whereas the helices A include each two histidines acting as ligand. The position of the histidine ligands on the opposite helices A permits, in principle, the axial ligation of two Fe(III)-heme sites into the protein structure. The four helix-bundle de novo protein was assembled as a monolayer on an Au-electrode as depicted in Fig. 3-16. 

Interaction of the functionalized-monolaycr electrode with Fe(III)-protoporphyrin IX, (15), resulted in the reconstitution of the protein with the two heme units. The surface coverage of the reconstituted de novo hemoprotein was found to be 2.5x10 mole cm Assuming that the protein footprint area is ca. 25 A, the saturated surface coverage of the de novo protein corresponds to 3.5x 10" mole cm and thus the experimental value... 

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