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Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial dye-decolorizing peroxidases

DOI: 10.1007/s00775-021-01896-2 DOI Help

Authors: Marina Lucic (University of Essex) , Michael T. Wilson (University of Essex) , Dimitri A. Svistunenko (University of Essex) , Robin L. Owen (Diamond Light Source) , Michael A. Hough (University of Essex) , Jonathan A. R. Worrall (University of Essex)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Jbic Journal Of Biological Inorganic Chemistry , VOL 65

State: Published (Approved)
Published: September 2021
Diamond Proposal Number(s): 18565

Open Access Open Access

Abstract: Structure determination of proteins and enzymes by X-ray crystallography remains the most widely used approach to complement functional and mechanistic studies. Capturing the structures of intact redox states in metalloenzymes is critical for assigning the chemistry carried out by the metal in the catalytic cycle. Unfortunately, X-rays interact with protein crystals to generate solvated photoelectrons that can reduce redox active metals and hence change the coordination geometry and the coupled protein structure. Approaches to mitigate such site-specific radiation damage continue to be developed, but nevertheless application of such approaches to metalloenzymes in combination with mechanistic studies are often overlooked. In this review, we summarize our recent structural and kinetic studies on a set of three heme peroxidases found in the bacterium Streptomyces lividans that each belong to the dye decolourizing peroxidase (DyP) superfamily. Kinetically, each of these DyPs has a distinct reactivity with hydrogen peroxide. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (FeIV = O) heme species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis with this set of redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal heme pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis.

Subject Areas: Biology and Bio-materials


Instruments: I24-Microfocus Macromolecular Crystallography

Added On: 06/09/2021 10:53

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Lučić2021_Article_AspartateOrArginineValidatedRe.pdf

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