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.
Highlights
Microperoxidases (MPs) have been establishing themselves as an attractive class of alternatives to haemperoxidases [1]
We concluded that our approach is a convenient means of MP-11 synthesis that does not rely on nature
A previous report included the solution CD spectral data, which indicated that the undecapeptide became a somewhat developed secondary structure upon reconstitution: the apparent helical content improved from 13% to 14% [21]
Summary
Microperoxidases (MPs) have been establishing themselves as an attractive class of alternatives to haemperoxidases [1]. A naturally occurring undecamer (VQKCAQCHTVE, MP-11) obtained proteolytically from cytochrome c contains a haem group that covalently bonds to the polypeptide chain via. Even though the structure is minimal, MP-11 and its homologues show a substantial degree of peroxidase activity They have been widely used as models for haem active sites and for investigations of the oxidoreductase reaction mechanism [2,3,4,5,6]. Bren et al extended to a biosynthesis method that does not rely on cytochromes c expression [18]. Working constantly on those naturally originating polypeptides, we can expect any MP with an unusual function to be available. We concluded that our approach is a convenient means of MP-11 synthesis that does not rely on nature
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