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The use of CO2 as a massive and polarizable drift gas is shown to greatly improve peak-to-peak resolution (Rp-p), as compared with N2, for the separation of disaccharides in a Synapt G2 traveling wave ion mobility cell. Near or baseline Rp-p was achieved for three pairs of sodiated molecules of disaccharide isomers, that is, cellobiose and sucrose (Rp-p?=?0.76), maltose and sucrose (Rp-p?=?1.04), and maltose and lactose (Rp-p?=?0.74). Ion mobility mass spectrometry using CO2 as the drift gas offers therefore an attractive alternative for fast and efficient separation of isomeric disaccharides.
Hepcidin-25 was identified as themain iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II) binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandemmass spectrometry (MS/MS), high-resolutionmass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D)model of hepcidin-25with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or referencematerial comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.
Hepcidin-25 was identified as themain iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II) binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandemmass spectrometry (MS/MS), high-resolutionmass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D)model of hepcidin-25with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or referencematerial comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.
L-edge spectroscopy of 3d transition metals provides important electronic structure information and has been used in many fields. However, the use of this method for studying dilute aqueous systems, such as metalloenzymes, has not been prevalent because of severe radiation damage and the lack of suitable detection systems. Here we present spectra from a dilute Mn aqueous solution using a high-transmission zone-plate spectrometer at the Linac Coherent Light Source (LCLS). The spectrometer has been optimized for discriminating the Mn L-edge signal from the overwhelming 0 K-edge background that arises from water and protein itself, and the ultrashort LCLS X-ray pulses can outrun X-ray induced damage. We show that the deviations of the partial-fluorescence yield-detected spectra from the true absorption can be well modeled using the state-dependence of the fluorescence yield, and discuss implications for the application of our concept to biological samples.
Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5-10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo (VI) ion with each one MPT, Mo=O, Mo-O-, and Mo=S ligand, and a weak Mo-O(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (pK similar to 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (pK similar to 8.8) is an inhibitor at alkaline pH. A similar acidic pK for the WT and Q179A. variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation.
YedY from Escherichia coil is a new member of the sulfite oxidase family of molybdenum cofactor (Moco)-containing oxidoreductases. We investigated the atomic structure of the molybdenum site in YedY by X-ray absorption spectroscopy, in comparison to human sulfite oxidase (hSO) and to a Mo(IV) model complex. The K-edge energy was indicative of Mo(V) in YedY, in agreement with X- and Q-band electron paramagnetic resonance results, whereas the hSO protein contained Mo(VI). In YedY and hSO, molybdenum is coordinated by two sulfur ligands from the molybdopterin ligand of the Moco, one thiolate sulfur of a cysteine (average Mo-S bond length of similar to 2.4 angstrom), and one (axial) oxo ligand (Mo=O, similar to 1.7 angstrom). hSO contained a second oxo group at Mo as expected, but in YedY, two species in about a 1:1 ratio were found at the active site, corresponding to an equatorial Mo-OH bond (similar to 2.1 angstrom) or possibly to a shorter M-O(-) bond. Yet another oxygen (or nitrogen) at a similar to 2.6 angstrom distance to Mo in YedY was identified, which could originate from a water molecule in the substrate binding cavity or from an amino acid residue close to the molybdenum site, i.e., Glu104, that is replaced by a glycine in hSO, or Asn45. The addition of the poor substrate dimethyl sulfoxide to YedY left the molybdenum coordination unchanged at high pH. In contrast, we found indications that the better substrate trimethylamine N-oxide and the substrate analogue acetone were bound at a similar to 2.6 angstrom distance to the molybdenum, presumably replacing the equatorial oxygen ligand. These findings were used to interpret the recent crystal structure of YedY and bear implications for its catalytic mechanism.