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Rational De Novo Design of a Cu Metalloenzyme for Superoxide Dismutation.

ABSTRACT: Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. These metalloproteins contain a redox-active metal ion in their active site (Mn, Cu, Fe, Ni) with a tightly controlled reduction potential found in a close range around the optimal value of 0.36?V versus the normal hydrogen electrode (NHE). Rationally designed proteins with well-defined three-dimensional structures offer new opportunities for obtaining functional SOD mimics. Here, we explore four different copper-binding scaffolds: H₃ (His₃ ), H₄ (His₄ ), H₂ DH (His₃ Asp with two His and one Asp in the same plane) and H₃ D (His₃ Asp with three His in the same plane) by using the scaffold of the de novo protein GR?₃ D. EPR and XAS analysis of the resulting copper complexes demonstrates that they are good Cu^II -bound structural mimics of Cu-only SODs. Furthermore, all the complexes exhibit SOD activity, though three orders of magnitude slower than the native enzyme, making them the first de novo copper SOD mimics.

SUBMITTER: Mathieu E

PROVIDER: S-EPMC6944188 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Rational De Novo Design of a Cu Metalloenzyme for Superoxide Dismutation.

Mathieu Emilie E Tolbert Audrey E AE Koebke Karl J KJ Tard Cédric C Iranzo Olga O Penner-Hahn James E JE Policar Clotilde C Pecoraro Vincent V

Chemistry (Weinheim an der Bergstrasse, Germany) 20191203 1

Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. These metalloproteins contain a redox-active metal ion in their active site (Mn, Cu, Fe, Ni) with a tightly controlled reduction potential found in a close range around the optimal value of 0.36 V versus the normal hydrogen electrode (NHE). Rationally designed proteins with well-defined three-dimensional structures offer new opportunities for obtaining func ...[more]

PMID: 31710732

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Project description:BackgroundSuperoxide dismutases (SODs) are categorized as antioxidant enzymes that are involved in many processes such as stress signalling responses and cell protection against free radical species. The primary function of SOD is the removal of produced radical species like superoxide ions in different physiological processes. There are various isozymes of SODs which are classified according to the metal cofactor in their active sites into four general types of Fe-SOD, Mn-SOD, Cu/Zn-SOD and Ni-SOD. Among metal nanoparticles, gold nanoparticles (AuNPs) are useful for biological purposes as sensing probe for determining critical analysis based on surface plasmon resonance and colorimetric method. In this study, the human Cu-Zn SOD expressed, purified, and its interaction with AuNPs based on a new colorimetric method was investigated.ObjectivesIn this approach, a colorimetric detection method for SOD activity was developed based on the carboxylic stabilized AuNPs.Material and methodsThe Ni-NTA Sepharose affinity column was performed for the purification process of enzyme. Following SOD purification, the enzyme activity in presence of AuNPs due to the possible etching in the presence of free radicals which are produced by riboflavin, methionine, Na2CO3 and potassium phosphate buffer, have been performed. In addition, Fluorescence spectroscopy analysis toward SOD and gold nanoparticle were performed.ResultsSuperoxide radicals generated from the enzymatic reaction would preferentially etch AuNPs and resulted in remarkable changes of localized surface plasmon resonance of AuNPs, which is reduced in the presence of SOD. Under the optimized experimental conditions assay (pH~7.8 and 25 ˚C), better selectivity and sensitivity toward SOD activity was shown.ConclusionsIn this context, an indirect new colorimetric method for determining of SOD activity based on gold nanoparticles (AuNPs) was evaluated. According to the presented result, it may be concluded that by scavenging of free superoxide radicals in the presence of SOD, the amount of AuNP absorbance can be replenished.

Dataset Information

Rational De Novo Design of a Cu Metalloenzyme for Superoxide Dismutation.

Publications

Rational De Novo Design of a Cu Metalloenzyme for Superoxide Dismutation.

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