Integration of a reconstituted de novo synthesized hemoprotein and native metalloproteins with electrode supports for bioelectronic and bioelectrocatalytic applications

A four-helix bundle de novo synthesized protein is assembled as a monolayer onto a Au electrode. Two of the helices include each two histidine units. This allows the reconstitution of the de novo protein with two Fe(III)-protoporphyrin IX units. Electrochemical characterization of the bis- heme-functionalized de novo protein, surface coverage 2.5 x 10^-11 mol·cm^-2, reveals that the heme site close to the electrode surface exhibits a redox potential, E°= 0.43 V (vs SCE), whereas the heme center in the remote position with respect to the electrode exhibits a more positive potential, E°= -0.36 V (vs SCE). This enabled the use of the de novo protein as a rectifier element in which rapid vectorial electron transfer occurs. The bis-heme-functionalized de novo protein assembled onto the electrode forms an affinity complex with the cytochrome b₁-dependent nitrate reductase (NR, E.C. 1.9.6.1). The affinity complex was cross-linked with glutaric dialdehyde to yield an integrated, electrically contacted, enzyme electrode for the effective bioelectrocatalyzed reduction of NO₃^-, current yield 80%. Similarly, the bis-heme-reconstituted de novo protein assembly forms an affinity complex With Co(II)-protoporphyrin-reconstituted myoglobin, Co(II)- Mb. Cross-linking of the affinity complex between the de novo synthesized hemoprotein and Co(II)-Mb with glutaric dialdehyde results in an integrated bioelectrocatalytic electrode for the electrocatalyzed hydrogenation of acetylene dicarboxylic acid (3) to maleic acid (4), current yield 85%.