Immobilization of engineered cytochrome P450cam (CYP101) to gold electrode via maleimide linker for electrocatalysis

Herein, we demonstrated maleimide-based conjugation chemistry to achieve the site-specific covalent immobilization of the C334A mutant of Cytochrome P450cam (C334A CYP101) onto a gold electrode. To be specific, firstly electrode surface was functionalized with maleimide groups involving cysteamine and N-(4-Carboxyphenyl) maleimide (p-CPMI) via a facile two-step process. The enzyme's accessible cysteine residues (C58 and C136) in the phosphate buffer solution (pH 7.4) containing CYP101 employed for the covalent attachment of C334A CYP101 through a chemoselective reaction with the maleimide moieties on the p-CPMI-modified electrode. The electrochemical analysis of the immobilized enzyme on a functionalized gold electrode was performed through Cyclic Voltammetry (CV) measurement. The enzyme-modified electrode exhibited a mid-point potential of -350 ± 10 mV vs Ag/AgCl in 3 M KCl aqueous electrolyte, indicating efficient electron transfer between the enzyme and the electrode interface. This strategy for site-specific immobilization holds considerable promise for diverse electrocatalytic applications, including the enzymatic detoxification of environmental pollutants and the synthesis of high-value chemical compounds, by exploiting the catalytic efficiency of C334A CYP101.