Overview

Molecular oxygen is implicated in many fundamental biochemical processes acting as ligand, substrate or product. Main examples of such processes concern oxygen-transport/storage proteins (like globins) or redox enzymes where oxygen acts in the oxidation step. Obviously, there is long list of oxygen processes in proteins. Investigations of interactions between biomolecules and oxygen species, particularly the oxygen binding sites and diffusion pathways are of fundamental interest to explain protein function. To investigate protein-oxygen interactions in crystallo we have designed a cryogenic oxygen pressure cell (figure 1). Users who have crystallized proteins sensitive to oxygen, and who are interested in studying protein/oxygen interactions of any kind are welcome to use our system.

Figure 1. On the right, a picture of the oxygen pressure cell installed in the fume-hood of the HPMX lab. On the left, a schematic represenation of the main components of the system.

Experimental setup

We have developed a standalone pressure cell to expose macromolecular crystal in a pressurized oxygen environment prior to cryo-cool them in liquid oxygen (figure 2). The concept is based on the phase diagram of oxygen, which is in a gas phase at ambient temperature and in a liquid phase at cryogenic temperature. The crystal is harvested and cryo-proteted, installed in the top part of a vertical pressure tube and then pressurized with oxygen at ambient temperature (the oxygen atoms bind proteins). Once pressurised, the sample is directly cryo-cooled in liquid O2. After returning to ambient pressure, the cryo-cooled sample is kept at cryogenic temperatures. The crystal is mounted using a SPINE pin that can be used in any sample changer of the MX beamlines.With this method, samples are pressurize at 50 bar in pure O2, therefore the concentration of oxygen disolved inside the crystal increases by a factor of 200.


Figure 2. The oxygen pressure cell. Top left, the soak-and-freeze method based on the phase diagram of oxygen. Top right, the different steps during oxygen pressurisation, described at the bottom.

Examples

1)  Molecular oxygen in urate oxidase

We tested our oxygen pressure cell with a crystal of urate oxidase. The structure of this complex was already characterized by N. Colloc’h & T. Prange (Biophys J., 2008). The enzyme uses the reduction of molecular oxygen to catalyze the oxidation of urate. Crystals have been cryo-cooled at either 10 or 40 bars of O2. For both cases, we obtain equivalent results as those published in 2008, but with an improved resolution. The water molecule that is located at ambient pressure next to the substrate (here an inhibitor) is substituted by a molecule of oxygen when the crystal is pressurised in pure O2. The oxygen molecule is identified without ambiguity and the binding geometry at 1.4 Å is perfectly described (figure 3). This results provides a part of the molecular mechanism of this redox enzyme.

Figure 3.  Structure of the complex urate-oxidase/oxygen obtained with our oxygen pressure cell

 

2)  Molecular oxygen in neuroglobin

We used the method to detect directly the existence of O2 docking sites in a neuroglobin crystal. Neuroglobin (Ngb) is the member of the globin protein family expressed in the brain. It is supposed to be involved in the protection of nervous tissues against ischemia. The Ngb function is unknown, but few hypotheses put forward its capability of binding and transporting O2. Diffraction data was collected on a crystal of ferric Ngb cryo-cooled at 50 and 80 bar of pure molecular oxygen, respectively. A single O2-docking site is observed at both pressures, though the occupancy increases from 50% to 85% (figure 4). The O2 molecule binds in a cavity already identified using xenon (Xe-III site; Colloc'h, Carpentier et al., (2017). Biophys. J.). This result was published by C. Ardiccioni et al. IUCRJ (2019), and allowed the authors to hypothesise that the Xe-III site is a preferential path for ligand migration and/or a storage place for diatomic molecules.

Figure 4.  Structure of the complexes A) Neuglobin/xenon and B) Neuglobin/oxygen.

 

3)  Molecular oxygen in a hydrognase

NiFe hydrogenases catalyse the reversible conversion of molecular hydrogen into protons. Generally, NiFe hydrogenases are irreversibly inactivated by O2, but few of them appear to be tolerant to atmospheric oxygen, and are therefore interesting for applications. The hydrogenase of Ralstonia eutropha (ReMBH) belongs to this subgroup, and is an attractive targets for studies on the mechanisms of O2-tolerance in hydrogenases. Previous studies using krypton labeling revealed major differences in the hydrophobic tunnel network between O2-sensitive and O2-tolerant hydrogenases (J. Kalms et al., Angew. Chem. (2016)). The routes though for O2 through these enzymes remained elusive, since there is a technical bottleneck for visualizing small and weakly interacting molecules like O2 in crystal structures. To overcome this difficulty, we used the present cryogenic oxygen pressure cell based on the soak-and-freeze technique to track the route of O2 in in ReMBH crystals. Twelve crystals were O2-derivatised in a cryogenic oxygen pressure cell at 56 – 70 bar for between 15 – 70 minutes. Crystals were first flash-frozen using the cryogenic oxygen pressure cell, and thereafter transferred in liquid nitrogen at beamline ID29 for diffraction data collection. Seven molecular oxygen positions were observed within the ReMBH tunnel system, describing a continuous pathway between the protein exterior and the active site (figure 5). The O2-derivatisation led to structural changes at the proximal FeS-cluster in the active site. Molecular dynamics simulations confirmed that O2 and H2 molecules both diffuse specifically within the same gas tunnels, and corroborated the high occupancy of O2 positions determined by the crystallographic experiment. Simulations also demonstrated that O2 diffuses preferably away from the active site, and confirmed that the gas tunnel network of ReMBH holds a mechanism to separate H2 from O2 and thereby protects the enzyme from inhibition. This result is published in J. Kalms et al. Proc. Nat. Acad. Sci. (2018).

 

Figure 5.  Crystallographic structure of the O2-derivative of hydrogenase of Ralstonia eutropha (ReMBH).