posted on 2021-08-03, 17:36authored bySuchitra Mitra, Divyansh Prakash, Khashayar Rajabimoghadam, Zdzislaw Wawrzak, Pallavi Prasad, Tong Wu, Sandeep K. Misra, Joshua S. Sharp, Isaac Garcia-Bosch, Saumen Chakraborty
Copper-containing
metalloenzymes constitute a major class of proteins
that catalyze a myriad of reactions in nature. Inspired by the structural
and functional characteristics of this unique class of metalloenzymes,
we report the conception, design, characterization, and functional
studies of a de novo artificial copper peptide (ArCuP) within a trimeric
self-assembled polypeptide scaffold that activates and reduces peroxide.
Using a first-principles approach, the ArCuP was designed to coordinate
one Cu via three His residues introduced at an a site of the peptide scaffold. X-ray crystallography, UV–vis,
and electron paramagnetic resonance data demonstrate that Cu binds
via the Nε atoms of His forming a T2Cu environment.
When reacted with hydrogen peroxide, the putative copper-hydroperoxo
species is formed where a reductive priming step accelerates the rate
of its formation and reduction. Mass spectrometry was used to identify
specific residues undergoing oxidative modification, which showed
His oxidation only in the reduced state. The redox behavior of the
ArCuP was elucidated by protein film voltammetry. Detailed characterization
of the electrocatalytic behavior of the ArCuP led us to determine
the catalytic parameters (KM and kcat), which established the peroxidase activity
of the ArCuP. Combined spectroscopic and electrochemical data showed
a pH dependence on the reactivity, which was optimum at pH 7.5.