SERR spectroelectrochemical study of cytochrome cd₁ nitrite reductase coimmobilized with physiological redox partner Cytochrome C₅₅₂ on biocompatible metal electrodes

Cytochrome cd₁ nitrite reductases (cd₁NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd₁NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd₁NiR from Marinobacter hydrocarbonoclasticus (Mhcd₁) depends on the presence of its physiological redox partner, cytochrome c₅₅₂ (cyt c₅₅₂), we show here a detailed surface enhanced resonance Raman characterization of Mhcd₁ and cyt c₅₅₂ attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd₁ and cyt c₅₅₂ are coimmobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag//SAM//Mhcd₁//cyt c₅₅₂ and Ag//SAM//cyt c₅₅₂//Mhcd₁ constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c₅₅₂ retains its native properties, while the redox potential of apparently intact Mhcd₁ undergoes a ∼150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd₁, reinforcing the idea that subtle and very specific interactions between Mhcd₁ and cyt c₅₅₂ govern efficient intermolecular electron transfer and catalytic activity of Mhcd₁.