Philippe Carpentier

Crystallographic snapshots of the reaction of aromatic C–H with O 2 catalysed by a protein-bound iron complex

Chemical reactions inside single crystals are quite rare because crystallinity is difficult to retain owing to atomic rearrangements. Protein crystals in general have a high solvent content. This allows for some molecular flexibility, which makes it possible to trap reaction intermediates of enzymatic reactions without disrupting the crystal lattice. A similar approach has not yet been fully implemented in the field of inorganic chemistry. Here, we have combined model chemistry and protein X-ray crystallography to study the intramolecular aromatic dihydroxylation by an arene-containing protein-bound iron complex. The bound complex was able to activate dioxygen in the presence of a reductant, leading to the formation of catechol as the sole product. The structure determination of four of the catalytic cycle intermediates and the end product showed that the hydroxylation reaction implicates an iron peroxo, generated by reductive O(2) activation, an intermediate already observed in iron monooxygenases. This strategy also provided unexpected mechanistic details such as the rearrangement of the iron coordination sphere on metal reduction.

In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF

The current version of the Cryobench in crystallo optical spectroscopy facility of the ESRF is presented. The diverse experiments that can be performed at the Cryobench are also reviewed.

The Upgrade Programme for the Structural Biology beamlines at the European Synchrotron Radiation Facility – High throughput sample evaluation and automation

Automation and advances in technology are the key elements in addressing the steadily increasing complexity of Macromolecular Crystallography (MX) experiments. Much of this complexity is due to the inter-and intra-crystal heterogeneity in diffraction quality often observed for crystals of multi-component macromolecular assemblies or membrane proteins. Such heterogeneity makes high-throughput sample evaluation an important and necessary tool for increasing the chances of a successful structure determination. The introduction at the ESRF of automatic sample changers in 2005 dramatically increased the number of samples that were tested for diffraction quality. This first generation of automation, coupled with advances in software aimed at optimising data collection strategies in MX, resulted in a three-fold increase in the number of crystal structures elucidated per year using data collected at the ESRF. In addition, sample evaluation can be further complemented using small angle scattering experiments on the newly constructed bioSAXS facility on BM29 and the micro-spectroscopy facility (ID29S). The construction of a second generation of automated facilities on the MASSIF (Massively Automated Sample Screening Integrated Facility) beam lines will build on these advances and should provide a paradigm shift in how MX experiments are carried out which will benefit the entire Structural Biology community.

Krypton Derivatization of an O2‐Tolerant Membrane‐Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport

[NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2 -tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2 -tolerant and O2 -sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance.

Formation of High‐Valent Iron–Oxo Species in Superoxide Reductase: Characterization by Resonance Raman Spectroscopy

Superoxide reductase (SOR), a non-heme mononuclear iron protein that is involved in superoxide detoxification in microorganisms, can be used as an unprecedented model to study the mechanisms of O2 activation and of the formation of high-valent iron-oxo species in metalloenzymes. By using resonance Raman spectroscopy, it was shown that the mutation of two residues in the second coordination sphere of the SOR iron active site, K48 and I118, led to the formation of a high-valent iron-oxo species when the mutant proteins were reacted with H2O2. These data demonstrate that these residues in the second coordination sphere tightly control the evolution and the cleavage of the O-O bond of the ferric iron hydroperoxide intermediate that is formed in the SOR active site.

SPINE-compatible `carboloops': a new microshaped vitreous carbon sample mount for X-ray and neutron crystallography

A novel vitreous carbon mount for macromolecular crystallography, suitable for neutron and X-ray crystallographic studies, has been developed. The technology described here is compatible both with X-ray and neutron cryo-crystallography. The mounts have low density and low background scattering for both neutrons and X-rays. They are prepared by laser cutting, allowing high standards of production quality, the ability to custom-design the mount to specific crystal sizes and large-scale production.

Using Raman and fluorescence spectroscopies in protein crystallography

Small heat-shock proteins (sHSPs) are a family of evolutionary conserved ATP-independent chaperones. These proteins share a common architecture defined by a signature α-crystallin domain (ACD) flanked by highly variable N and C-terminal extensions. The ACD, which has an immunoglobulin-like fold, plays an important role in sHSP assembly, mediating dimer formation of individual protomers, that then may assemble into larger oligomers. In vertebrate sHSPs the dimer interface is mediated by the symmetrical antiparallel pairing of two β-strands (β7), resulting in the formation of an extended β-sheet on one face of the ACD dimer. Recent crystallographic studies of isolated ACDs from a number of vertebrate sHSPs suggest a variability in the register of the β7/β7 interface, which may, in part, give rise to the polydispersity often seen with the full-length proteins. To further analyse the structure of ACD dimers we have employed a combination of X-ray crystallography and solution small-angle X-ray scattering (SAXS) to study the ACD-containing fragments of human HSPB1 and HSPB6. Unexpectedly, the obtained crystal structure of the HSPB1 fragment does not reveal the typical β7/β7 dimers, but rather hexamers formed by an asymmetric contact between the β4 and β7 strands from adjacent ACDs [1,2]. Nevertheless, in solution, both ACDs form stable dimers via the symmetric antiparallel interaction of β7 strands. Using SAXS, we show that it is possible to discriminate between different putative registers of the β7/β7 interface, and that under physiological conditions there is only a single register of the strands for both proteins [2]. Furthermore, we have solved the crystal structure of a fragment of HSPB6 including a portion of its N-terminal extension, the ACD and the C-terminal extension. The structure reveals a stable tetramer, as also confirmed by the SAXS data in solution. The novel tetramer formation is possible due to specific ‘patching’ of the β4/β8 side of the ACDs by a short hydrophobic motif found in the N-terminal extension.