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X-ray induced photoreduction of heme metal centers rapidly induces active site perturbations in a protein-independent manner

DOI: 10.1074/jbc.RA120.014087 DOI Help

Authors: Vera Pfanzagl (BOKU - University of Natural Resources and Life Sciences) , John H. Beale (Diamond Light Source) , Hanna Michlits (BOKU - University of Natural Resources and Life Sciences) , Daniel Schmidt (BOKU - University of Natural Resources and Life Sciences) , Thomas Gabler (BOKU - University of Natural Resources and Life Sciences) , Christian Obinger (BOKU - University of Natural Resources and Life Sciences) , Kristina Djinovic-carugo (University of Vienna) , Stefan Hofbauer (BOKU - University of Natural Resources and Life Sciences)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: July 2020
Diamond Proposal Number(s): 19036

Abstract: Since the advent of protein crystallography, atomic-level macromolecular structures have provided a basis to understand biological function. Enzymologists use detailed structural insights on ligand coordination, interatomic distances and positioning of catalytic amino acids to rationalize the underlying electronic reaction mechanisms. Often the proteins in question catalyze redox reactions using metal cofactors that are explicitly intertwined with their function. In these cases, the exact nature of the coordination sphere and the oxidation state of the metal is of utmost importance. Unfortunately, the redox active nature of metal cofactors makes them especially susceptible to photoreduction, meaning that information obtained by photoreducing X-ray sources about the environment of the cofactor are the least trustworthy part of the structure. In this work we directly compare the kinetics of photoreduction of six different heme protein crystal species at by X-ray radiation. We show that a dose of approximately 40 kGy already yields 50% ferrous iron in a heme protein crystal. We also demonstrate that the kinetics of photoreduction are completely independent from variables unique to the different samples tested. The photoreduction-induced structural rearrangements around the metal cofactors have to be considered when biochemical data of ferric proteins are rationalized by constraints derived from crystal structures of reduced enzymes.

Journal Keywords: protein crystallization; enzyme structure; oxidation-reduction (redox); X-ray crystallography; heme

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Other Facilities: ID23-1 at ESRF