Finding hydrogens: elucidating the redox chemistry of cholesterol oxidase through neutron diffraction studies

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Cholesterol oxidase (COx) is a bacterial flavoenzyme catalysing the oxidation and isomerisation of cholesterol to choleste-4-en-3-one. This enzyme is utilised by bacteria in the first step of the degradation of cholesterol as a carbon source or in the depletion of cholesterol from eukaryotic membranes. In the oxidative reaction (reductive half-reaction) a proton is abstracted from the substrate hydroxyl group and a hydride transferred from the substrate to the tightly bound cofactor resulting in reduction of the cofactor and oxidation of the substrate. These reducing electrons are then passed to molecular oxygen to form hydrogen peroxide. While X-ray diffraction studies have provided significant insights into the role of the protein structure in the reaction mechanism, many questions still remain unanswered. A knowledge of the positions of hydrogen atoms in the reduced and oxidised forms of the enzyme would provide insights into the redox chemistry of COx, which involves the transfer of hydrogen atoms either in the form of hydrogen, hydrides or protons. X-ray scattering by atoms is proportional to the atomic number, therefore hydrogen atoms can only be seen in X-ray crystal structures at atomic resolution. Neutron protein crystallography (NPC) is a technique which can be used for visualising hydrogen atoms in crystal structures, as hydrogen atoms have a similar scattering magnitude to the heavier atoms in proteins (C, O, N, S) and thus can be visualised at moderate resolutions (1.5 Å - 2.5 Å). NPC studies of COx were pursued to determine the positions of these hydrogen atoms in the reduced and oxidised forms to investigate the redox chemistry of COx.

NPC requires very large crystal volumes compared to those used for X-ray diffraction at synchrotron radiation beamlines. The predominant reason for requiring large crystals is due to the low flux of beamlines. One way to increase the signal to noise ratio (SNR) of the experiment and therefore decrease the required crystal volume, is to use deuterium instead of hydrogen in the sample. Hydrogen has a negative neutron scattering length, which causes density cancellation in regions where hydrogen is bonded to a positive neutron scatterer (C, N, O and S). Hydrogen also has a large incoherent scattering component which increases the noise in the experiment and therefore lowers the SNR. Deuterium, an isotope of hydrogen, has a positive scattering length, low incoherent scattering component and twice the scattering magnitude of hydrogen, eliminating density cancellation problems and increasing the SNR. Deuterium can be incorporated into protein structures using H/D exchange - where protein is soaked in D2O-containing buffers causing exchange of labile hydrogen atoms for deuterium- or by perdeuterating the protein - where only deuterium is available for protein expression resulting in a protein with all hydrogen atoms exchanged for deuterium.

To pursue the neutron diffraction structures of COx a process of macroseeding was developed to produce large crystals suitable for neutron diffraction. A 2.2 Å resolution structure of H/D-exchanged COx was obtained from one of these macroseeded crystals and revealed insights into substrate binding for efficient hydride transfer in COx. In particular, a deuteron was observed stabilised between the protein Gly120-N and FAD-N5 reactive centre. The negative dipole over the Gly120/Asn119 peptide bond which is inferred by the position of the deuteron, is induced by a positively charged lysine side chain. Furthermore, both of these interactions are conserved in other oxidoreductase enzymes indicating that this configuration is important for enzyme function. Additionally, an X-ray diffraction structure of the reduced enzyme was obtained revealing the location of the hydride transferred to FAD in the oxidation reaction. The hydride position was in a tetrahedral geometry on the N5 atom of FAD and density functional theory calculations revealed that the interaction between Gly120 and FAD-N5 serves to tetrahedralise the N5 centre by stabilising the lone pair of electrons of N5. Taken together, these results revealed an elegant pre-formed active site which acts to position the substrate hydride donating orbital and the FAD receiving orbital simultaneously for efficient hydride transfer.

Neutron diffraction structures of the perdeuterated enzymes were also pursued. Perdeuterated protein was successfully expressed, purified and crystallised and a modified macroseeding technique produced large protein crystals. Functional and structural characterisation of the perdeuterated enzyme revealed no significant effects due to perdeuteration and confirmed that perdeuterated protein crystals were suitable for neutron diffraction studies. A 2.1 Å neutron diffraction structure was obtained which showed nuclear density for many of the deuterium atoms, however, crystal twinning decreased the quality of the maps limiting the structural insights that could be obtained from this structure. However, this indicated the viability of neutron diffraction studies of perdeuterated COx and further work will be conducted to improve the size and quality of perdeuterated crystals.
Original languageEnglish
QualificationDoctor of Philosophy
Publication statusUnpublished - Jul 2015


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