Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes.In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxidetoform the catalytic intermediate compound I(Fe IV = Oa nd ap orphyrin cation radical). Using serial femtosecond X-rayc rystallography (SFX), we have determined the pristine structures of the Fe III and Fe IV =Oredox states of aB-type DyP.These structures reveal aw ater-free distal heme site that, together with the presence of an asparagine,imply the use of the distal arginine as ac atalytic base.Acombination of mutagenesis and kinetic studies corroborate such ar ole.O ur SFX approach thus provides unique insight into howt he distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxideh eterolysis.

A subtle positional shift of the distal haem pocket aspartate in two dye decolourising peroxidase homologs has a remarkable effect on their reactivity with H2O2.

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. Graphic abstract

Both O 2 and H 2 O 2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/ Vis, EPR, and Mçssbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe 2+ , was exposed to O 2 or H 2 O 2. We show that O 2 binds di-Fe 2+ FC reversibly, two Fe 2+ ions are oxidized in concert and a H 2 O 2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe 2+ FC, at a rate circa 1000 faster than O 2 , ensuring an overall 1:4 stoichiometry of iron oxidation by O 2. Initially formed Fe 3+ can further react with H 2 O 2 (producing protein bound radicals) but relaxes within seconds to an H 2 O 2-unreactive di-Fe 3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H 2 O 2 rather than sequester iron.

Posttranslational modifications of immunoglobulins have been a topic of great interest and have been repeatedly reported as a major factor in disease pathology. Cost-effective, reproducible, and high-throughput (HTP) isolation of immunoglobulins from human serum is vital for studying the changes in protein structure and the following understanding of disease development. Although there are many methods for the isolation of specific immunoglobulin classes, only a few of them are applicable for isolation of all subtypes and variants. Here, we present the development of a scheme for fast and simultaneous affinity purification of α (A), γ (G), and μ (M) immunoglobulins from human serum through affinity monolith chromatography. Affinity-based monolithic columns with immobilized protein A, G, or L were used for antibody isolation. Monolithic stationary phases have a high surface accessibility of binding sites, large flow-through channels, and can be operated at high flow rates, making them the ideal supports for HTP isolation of biopolymers. The presented method can be used for HTP screening of human serum in order to simultaneously isolate all three above-mentioned immunoglobulins and determine their concentration and changes in their glycosylation pattern as potential prognostic and diagnostic disease biomarkers.