Cytochrome c–poly(acrylic acid) conjugates with improved peroxidase turnover number

Abstract

Cytochrome c–poly(acrylic acid) (cyt c–PAA) conjugates with 34-fold enchancement in peroxidase turnover number (k_cat) are reported. Cyt c–PAA conjugates were prepared by carbodiimide coupling. PAA with molecular weight (M_w) ranging from 1.8k to 250k g mol⁻¹ were employed, and the effect of PAA M_w on peroxiodase kinetics was assessed. The k_cat value increased with increased M_w of PAA, ranging from 0.077(±0.002) s⁻¹ in the absence of PAA to 2.66(±0.08) s⁻¹ for the conjugate of cyt c with 250k PAA. Enzymatic activity studies over pH 6–8 indicated improved activity for cyt c–PAA conjugates at neutral or slightly alkaline pH. Examination of the cyt c heme spectroscopy in the presence of H₂O₂ revealed that formation of compound III, a reactive intermediate that leads to enzyme inactivation, was supressed in cyt c–PAA conjugates. Thus, we suggest the k_cat enhancement can be attributed to acidification of the pH microenvironment and inhibition of the formation of a reactive intermediate that deactivates cyt c during the catalytic cycle.