Totally synthetic microperoxidase-11

Abstract

A totally synthetic microperoxidase-11 (MP-11) is reported. Accordingly, the undecapeptide (VQKCAQCHTVE) was synthesized by solid-phase peptide synthesis followed by the thiol-ene click reaction with haemin for reconstitution. High-speed atomic force microscopy measurement conducted in water confirmed the protein reconstitution by visualizing the morphological differences as animated molecular images. The synthetic MP-11 showed a considerable magnitude of catalytic activity (27%) against the natural MP-11 in the oxidation of 3,3′,5,5′-tetramethylbenzidine by hydrogen peroxide, whereas it showed very low (2.7%) activity of a synthetic variant with a point mutation (VQKCAQCMTVE, H8M). Slab waveguide spectroscopic measurements revealed that the ferrous/ferric redox reaction occurred by the direct electron transfer with specific spectral changes. Indeed, if hydrogen peroxide existed in the solution phase, the peroxidase-modified electrode showed catalytic current–voltage behaviour regardless of whether it was prepared using natural MP-11 or the synthetic MP-11. If a substrate recycling reaction was assumed, computer simulation well reproduced the experimental curves to give a global set of electrocatalytic reaction parameters. In any of the experiments, the synthetic MP-11 and natural MP-11 gave almost identical results. Our approach will be a convenient means of preparing MP-11, as well as its mutants, that does not rely on nature.

Electrocatalytic Properties of Immobilized Heme Proteins: Basic Principles and Applications

“…Thanks to their structural features and significant electron transfer rates, MPs can function also as carriers for electron transfer reactions and promoter for enzymes adsorption on electrode surfaces, and generally are able to electrocatalyze the oxidation of various hydroperoxides in aqueous and in organic solvents. [152][153][154][155][156] Several studies exploit microperoxidases as key constituents of electrodes for biofuel cells. [155,157]…”

Folding and Assembly of Vanilloid Receptor Secondary-Structure Peptide with Hexahistidine Linker at Nickel–Nitrilotriacetic Acid Monolayer for Capsaicin Recognition

“…Liquid-phase AFM imaging was carried out using a high-speed atomic force microscope (Research Institute of Biomolecule Metrology (RIBM) Co., Ltd., Tsukuba, Japan) operated in intermittent contact mode. The detailed measurement conditions including sample preparation were as previously reported . The AFM observations in the corresponding immobilized state were made using a JSPM 5410 Scanning Probe Microscope (JEOL Ltd., Tokyo, Japan) operated in a vacuum in the noncontact mode with frequency modulation detection .…”

“…The detailed measurement conditions including sample preparation were as previously reported. 23 The AFM observations in the corresponding immobilized state were made using a JSPM 5410 Scanning Probe Microscope (JEOL Ltd., Tokyo, Japan) operated in a vacuum in the noncontact mode with frequency modulation detection. 11 Gold films comprising crystals with (111) structure (111 indicates the Miller indices; Agilent Technologies Inc., Santa Clara, CA) were used as substrates and treated using the same procedure described above.…”

Electrochemical Biosensors Employing Natural and Artificial Heme Peroxidases on Semiconductors

“…These mini-enzymes with peroxidatic activity are prepared via proteolytic digestion of cytochrome c , leading to formation of heme-peptide complexes with a polypeptide chain of typically six to eleven amino acids (MP-6 to MP-11), as shown in Figure 3 b [ 55 ]. The recently reported fully synthetic approach for the synthesis of microperoxidases further opened up new possibilities for the design of customized heme-peptides incorporating even non-natural groups [ 56 ]. Microperoxidases were shown not only to possess a peroxidase-like activity, but also to catalyze dehalogenation reactions [ 57 ] and oxygen-transfer reactions similar to cytochrome P450 enzymes [ 58 ], making them attractive tools for the construction of biosensors.…”