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

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 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. 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. Enzyme-mediated transfer of electrons is an integral component of numerous fundamental biological reactions, such as respiration, photosynthesis, catabolic and anabolic transformations in metabolism, molecular signaling, or cellular defense. Any catalytic reaction mediated by redox-active enzymes is principally dependent on the nature and electronic state of the respective transition metals, which are modulated by inner- and outer-sphere ligands and solvent exposure of the active site. To elucidate the reaction mechanism of a given redox enzyme, X-ray crystallography has been the principal means of high-resolution structural characterization of these enzymes and their intermediate states. Here, the main focus lies on the metal coordination and ligation state, together with inter- and intramolecular distances, which depend primarily on the oxidation state of the metal center itself. However, X-rays cause rapid reduction of metal centers, thereby inducing subsequent changes in their stereochemistry (1Hough M.A. Antonyuk S.V. Strange R.W. Eady R.R. Hasnain S.S. Crystallography with online optical and X-ray absorption spectroscopies demonstrates an ordered mechanism in copper nitrite reductase.J. Mol. Biol. 2008; 378 (18353369): 353-36110.1016/j.jmb.2008.01.097Crossref PubMed Scopus (78) Google Scholar). Consequently, care must be taken when interpreting X-ray crystal structures obtained from single-crystal data sets through routine X-ray diffraction experiments, here nominally defined as a 360° rotation data set collected at 100 K, with a total mean diffraction weighted dose (calculated by RADDOSE-3D version 4.0) of more than 1 MGy. X-rays induce both site-specific and global damage in protein crystals (2Holton J.M. A beginner's guide to radiation damage.J. 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M.A. and structures of PubMed Scopus Google Scholar). proteins have been by to electrons an active The catalytic of at PubMed Scopus Google Scholar). iron by and as a in electrons K. reduction of heme by and X-ray Synchrotron Radiat. PubMed Scopus Google already by The catalytic of at PubMed Scopus Google in about the oxidation state of redox proteins in the To this protein have to the dose crystal in X-ray diffraction be by using a of the heme in and Biol. PubMed Scopus Google Scholar, and single-crystal of the PubMed Scopus Google and has already been to ferric of heme X-ray structures of the intermediate by to be derived from or with crystal a dose of or of the heme in and Biol. PubMed Scopus Google Scholar, and single-crystal of the PubMed Scopus Google Scholar). A on obtained a ferric from crystals with data collected crystal. 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