Near‐Atomic Resolution Neutron Crystallography on Perdeuterated Pyrococcus furiosus Rubredoxin: Implication of Hydronium Ions and Protonation State Equilibria in Redox Changes

Abstract

Rubredoxins are small monomeric non-heme, mononuclear iron-sulfur cluster proteins found in prokaryotes [1] and in some eukaryotes. [2] Although a multitude of roles have been proposed, [3] the main functions of the rubredoxins remain uncertain. Rubredoxin from Pyrococcus furiosus (Pf) is one of the most thermostable proteins known, with a melting temperature of 200 8C, and it can resist denaturation over prolonged periods at temperatures of up to 95 8C. [4] The structure of the hyperthermophilic Pf rubredoxin does not contain an unusual number of hydrogen bonds but has a richly H-bonded N-terminus region involving residue Glu14, which is absent in mesophilic rubredoxins. It has been suggested that this may contribute to the high thermostability of Pf rubredoxin. [5] As a redox protein, rubredoxin is also an important model system for an understanding of electron transfer processes associated with catalysis. Iron-sulfur proteins occur widely, covering a wide range of redox potentials, and this variation may be attributable to variations in solvation, hydrogen bonding, and electrostatic interactions.The aim of the current work was to obtain high-resolution neutron crystallographic information from both oxidized and reduced forms of perdeuterated Pf rubredoxin by fully exploiting the power of neutrons to clearly image water molecules, hydronium ions, and the protonation of carboxylic and hydroxylic moieties. Neutron diffraction is the only method that can provide this level of detail. The D19 instrument at the Institut Laue-Langevin (ILL) is a monochromatic diffractometer that allows atomic resolution stud-ies of systems for which suitably large crystals are available. It complements the quasi-Laue instrument LADI-III, which has a much broader scope for neutron protein crystallography and which is capable of providing good results for systems having larger unit cells and smaller crystal volumes. The isotopic replacement of hydrogen by deuterium atoms allows their identification in neutron scattering density maps, with a visibility comparable to that of other elements in biological macromolecules, as well as helping to reduce the high experimental background due to the large incoherent scattering cross-section of hydrogen. [6] Perdeuterated protein was produced in the Deuteration Laboratory of ILLs Life Science Group; its use allows both the elimination of hydrogen incoherent scattering and also the enhancement of the visibility of deuterium by comparison with its lighter isotope hydrogen. The study was carried out to a resolution of 1.27 in the case of the oxidized form (PDB: 4AR3) and to 1.38 resolution for the reduced form (PDB: 4AR4). This is the first high-resolution neutron study of Pf rubredoxin in both forms. It reveals information beyond that attainable in previous lower-resolution studies of hydrogenated [7] and deuterated systems. [8] The combination of monochromatic neutron crystallography and protein perdeuteration has played a key role in obtaining this high-quality structural information.Our neutron ...

The IMAGINE instrument: first neutron protein structure and new capabilities for neutron macromolecular crystallography

“…The 1.75 Å resolution limit and the quality of the data collected from this 0.7 mm 3 Pf Rd crystal are similar to those for the data previously collected using larger crystals ($3-4 mm 3 ; Kurihara et al, 2004;Li et al, 2004;Gardberg et al, 2010;Munshi et al, 2012;Myles et al, 2012;Cuypers et al, 2013; see Table 1). The R work values for the refined structures are all similar.…”

Impact of Deuteration on the Assembly Kinetics of Transthyretin Monitored by Native Mass Spectrometry and Implications for Amyloidoses

“…These results further validate the increasingly widespread use of perdeuteration in neutron crystallography . It should be noted that while the two structures look identical at the resolution obtained, atomic resolution data (≤1 Å) may help to further clarify the small structural effects associated with H/D isotopic replacement …”

Biosynthetic preparation of selectively deuterated phosphatidylcholine in genetically modified Escherichia coli

“…This organism can be adapted to grow in minimal media in high levels of D 2 O (Leiting et al 1998), and the use of D 2 O can be combined with different deuterated carbon sources such as sodium acetate, glycerol or glucose. This type of approach is now widely used in neutron protein crystallography (Cuypers et al 2013a; Howard et al 2011), SANS (Cuypers et al 2013b; Laux et al 2008; Rochel et al 2011; Vijayakrishnan et al 2010; Zhu et al 2010) and neutron reflectivity (Grage et al 2011; Hellstrand et al 2013) and has previously also been used in neutron fibre diffraction studies of natural and synthetic polymers (Gardner et al 2004; Shotton et al 1997). Deuterated phospholipids, on the other hand, are generally produced by chemical synthesis, which allows for the combination of various fatty acyl residues to obtain the desired mixtures of partially labelled or perdeuterated, saturated phospholipid species as well as selective deuteration of specific groups (Bragina and Chupin 1997).…”