CuA-based chimeric T1 copper sites allow for independent modulation of reorganization energy and reduction potential.

Jonathan Szuster,Alejandro J. Vila,Alcides J. Leguto,María A. Castro,Marcos N. Morgada,Daniel H. Murgida,Ulises A. Zitare

doi:10.1039/d0sc01620a

Jonathan Szuster, Alejandro J. Vila + Show 5 more

Open Access

https://doi.org/10.1039/d0sc01620a

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Abstract

Attaining rational modulation of thermodynamic and kinetic redox parameters of metalloproteins is a key milestone towards the (re)design of proteins with new or improved redox functions. Here we report that implantation of ligand loops from natural T1 proteins into the scaffold of a CuA protein leads to a series of distorted T1-like sites that allow for independent modulation of reduction potentials (E°') and electron transfer reorganization energies (λ). On the one hand E°' values could be fine-tuned over 120 mV without affecting λ. On the other, λ values could be modulated by more than a factor of two while affecting E°' only by a few millivolts. These results are in sharp contrast to previous studies that used T1 cupredoxin folds, thus highlighting the importance of the protein scaffold in determining such parameters.

Highlights

Redox metalloproteins are ubiquitous in nature and are implicated in a broad range of catalytic and electron transfer (ET) functions that impose quite diverse thermodynamic and kinetic requirements to the redox sites.[1]
We report the functional characterization of a series of distorted T1 chimeric proteins that were obtained by engineering of the ligand loop of the CuA site from Thermus thermophilus ba[3] cytochrome c oxidase. We show that this strategy allows for the independent modulation of l and E0 through the sequence and length of the ligand loop, while preserving the native T1 ligand set
In contrast, present an intense feature around 450 nm (3max z 1500–3500 MÀ1 cmÀ1) responsible for the greenish hue, along with a much weaker 600 nm band compared to axial sites.[41,42]. Both bands have been assigned to Scys / Cu2+ ligand to metal charge transfer (LMCT) transitions, and their relative intensities were rationalized by the so-called coupled distortion model in terms of differential overlap between the cysteine-3p and copper-3dx2Ày2 orbitals

Summary

Introduction

Redox metalloproteins are ubiquitous in nature and are implicated in a broad range of catalytic and electron transfer (ET) functions that impose quite diverse thermodynamic and kinetic requirements to the redox sites.[1]. The rst two types of centers share the cupredoxin fold and the interesting feature that all but one of the coordinating amino acids are located in a single loop that connects two b-strands.[1,4] along with point mutations, replacement of the entire ligand loop by sequences from other proteins or unnatural sequences,[5] has become one of the preferred strategies for modulating the electronic properties of T1 4,6–23 and CuA sites.[24,25,26,27,28,29,30] This methodology has allowed for the successful insertion of CuA sites into the scaffold of T1 31–35 proteins and vice versa.[36]

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Conclusion