Journal of Biological Chemistry
Volume 295, Issue 39, 25 September 2020, Pages 13488-13501
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Enzymology
X-ray–induced photoreduction of heme metal centers rapidly induces active-site perturbations in a protein-independent manner

https://doi.org/10.1074/jbc.RA120.014087Get rights and content
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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 is the least trustworthy part of the structure. In this work we directly compare the kinetics of photoreduction of six different heme protein crystal species by X-ray radiation. We show that a dose of ∼40 kilograys 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.

protein crystallography
radiation damage
heme proteins
heme
enzyme structure
oxidation-reduction (redox)
X-ray crystallography
protein crystallization

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Author contributions—V. P., K. D.-C., and S. H. conceptualization; V. P., J. H. B., H. M., D. S., T. G., and S. H. data curation; V. P., J. H. B., H. M., D. S., T. G., and S. H. formal analysis; V. P. and S. H. supervision; V. P., J. H. B., and S. H. validation; V. P. and S. H. visualization; V. P., J. H. B., K. D.-C., and S. H. methodology; V. P.,J. H. B., K. D.-C., and S. H. writing-original draft; V. P. and S. H. project administration; V. P., J. H. B., C. O., K. D.-C., and S. H. writing-review and editing; J. H. B. software; C. O. and K. D.-C. resources; C. O., K. D.-C., and S. H. funding acquisition.

Funding and additional information—This project was supported by the Austrian Science Fund, FWF (Doctoral Program BioToP - Molecular Technology of Proteins (W1224) and the project P29099), Diamond Light Source Proposal MX19036, ESRF Proposal MX1998, and the iNEXT program (PID: 4482). The work of K. D.-C. was supported by Austrian Science Fund (FWF) Projects I525, I1593, P22276, P19060, and W1221; Wellcome Trust Collaborative Award 201543/Z/16/Z; by COST action BM1405—Non-globular proteins–from sequence to structure, function, and application in molecular physiopathology (NGP-NET); WWTF (Vienna Science and Technology Fund) Chemical Biology Project LS17-008; the Austrian Federal Ministry of Science, Research, and Economy; and the National Foundation for Research, Technology, and Development through funding the Christian Doppler Laboratory for High-Content Structural Biology and Biotechnology, Austria.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Present address for John H. Beale: Paul Scherrer Institute, Villigen, Switzerland.

Abbreviations—The abbreviations used are:

    Gy

    gray(s)

    AvTsdA

    thiosulfate dehydrogenase from Allochromatium vinosum

    CCld

    chlorite dismutase from Cyanothece sp. PCC7425

    hhMb

    horse heart myoglobin

    KpDyP

    dye-decolorizing peroxidase from Klebsiella pneumoniae

    LmChdC

    coproheme decarboxylase from Listeria monocytogenes

    HS

    high-spin

    LS

    low-spin

    NdCld

    chlorite dismutase from Nitrospira defluvii

    PDB

    Protein Data Bank

    XFEL

    X-ray free-electron laser

    DLS

    Diamond Light Source

    wp

    water position

    BisTris

    2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

    Bicine

    N,N-bis(2-hydroxyethyl)glycine.