Determining the oxidation state of elements by X-ray crystallography.

Frank Lennartz,Holger Dobbek,Stefan Ruenger,Jae-Hun Jeoung,Manfred S. Weiss

doi:10.1107/s2059798321013048

Abstract

Protein-mediated redox reactions play a critical role in many biological processes and often occur at centres that contain metal ions as cofactors. In order to understand the exact mechanisms behind these reactions it is important to not only characterize the three-dimensional structures of these proteins and their cofactors, but also to identify the oxidation states of the cofactors involved and to correlate this knowledge with structural information. The only suitable approach for this based on crystallographic measurements is spatially resolved anomalous dispersion (SpReAD) refinement, a method that has been used previously to determine the redox states of metals in iron-sulfur cluster-containing proteins. In this article, the feasibility of this approach for small, non-iron-sulfur redox centres is demonstrated by employing SpReAD analysis to characterize Sulfolobus tokodaii sulerythrin, a ruberythrin-like protein that contains a binuclear metal centre. Differences in oxidation states between the individual iron ions of the binuclear metal centre are revealed in sulerythrin crystals treated with H2O2. Furthermore, data collection at high X-ray doses leads to photoreduction of this metal centre, showing that careful control of the total absorbed dose is a prerequisite for successfully determining the oxidation state through SpReAD analysis.

Highlights

Current estimates suggest that 30–50% of all proteins bind metal ions and, more than 35% of all structures currently deposited in the Protein Data Bank are of such metalloproteins (Waldron et al, 2009; Putignano et al, 2018)
Data collection at high X-ray doses leads to photoreduction of this metal centre, showing that careful control of the total absorbed dose is a prerequisite for successfully determining the oxidation state through spatially resolved anomalous dispersion (SpReAD) analysis
While methods such as electron paramagnetic resonance (EPR), X-ray absorption near-edge structure (XANES) or extended X-ray absorption fine structure (EXAFS) can be used with crystalline samples, they require additional experiments or specialized equipment or lack the spatial resolution needed to differentiate between individual atoms

Summary

Introduction

Current estimates suggest that 30–50% of all proteins bind metal ions and, more than 35% of all structures currently deposited in the Protein Data Bank are of such metalloproteins (Waldron et al, 2009; Putignano et al, 2018). Metalloproteins can mediate complex chemical reactions through their metal cofactors, including essential biological processes such as respiration and photosynthesis. The metals coordinated by these proteins can be isolated ions or parts of more complex cofactors such as haem groups or iron–sulfur clusters (Harding et al, 2010). They often are first-row transition metals, including iron, zinc, manganese and copper, which play key roles in structure stabilization, oxygen and lipid metabolism, detoxification of reactive oxygen species, DNA replication and electron transport (Waldron et al, 2009; Bowman et al, 2016). In order to understand the exact mechanism of reactions catalysed by proteins harbouring such redox-active centres, it is crucial to know the 3D structures of the protein and the catalytic site, and to assign the redox state of its cofactor

Methods

Results

Conclusion