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

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.