Detailed Research Interests Dr

The study of the catalytic mechanism of a model member of the cytochrome P450 superfamily, cytochrome P450cam, using a combined high resolution X-ray crystallography and neutron crystallography analysis is our immediate focus. The aim of this work is to identify the mechanism of cytochrome P450cam to provide critical insight in the understanding of the catalytic mechanism of this important family of enzymes.

Cytochromes P450 (P450s) are ubiquitous enzymes playing diverse functional roles in a broad range of biological system. In mammals they are involved in a variety of biochemical processes including carcinogenesis, drug metabolism, biosynthesis of lipids and steroids or degradation of xenobiotics. They catalyze a two-electron activation of O₂ resulting in the formation of a water molecule and the insertion of a single oxygen atom in the substrate. Cytochrome P450cam from the soil bacteria Pseudomonas putida is the model system of this superfamily. Numerous studies of P450cam that aim to elucidate the critical details of the proton delivery pathway involved in the O₂ activation have been performed over the last decades. The X-ray crystallographic structures of individual intermediates of the reaction pathway have now been determined. However, the resolution of the currently available X-ray structures does not allow direct visualization of the hydrogen atom positions in the protein and the organization of the hydrogen-bonding network at the active site remains ambiguous. Therefore the hydrogen shuttle pathway is still a matter of debate. The enhanced visibility of hydrogen and of its deuterium isotope in neutron protein structures suggests that neutron protein crystallography could provide critical information that could help further characterize the proton shuttle pathway in the P450cam enzymatic mechanism.

P450cam at 47 kDa is a relatively large system for neutron analysis, and the determination of the P450can neutron structure raises many challenges. Many of them have been overcome. Critically we purify large amount of fully (per)deuterated P450cam using the protocol developed in our laboratory. We are currently working on crystal growth optimization.

Superposition of the active site showing the hydrogen bound between Tyr96 and the camphor carbonyl O atom, the Asp251 and Thr252 key residues and the internal water channel (W523, W566 and W687). Light blue, hydrogenated P450cam; blue, perdeuterated P450cam

We have recently determined the neutron structure of D-xylose isomerase, the largest system studied at atomic resolution. D-xylose isomerase is a 43 kDa enzyme that catalyses the first reaction in the catabolism of xylose. This enzyme is industrially used since it can catalyze the isomerization glucose to fructose for the production of high fructose corn syrup, a product of high sweetening capacity widely used in food industry. Isomerization of xylose or glucose at the active site of D-xylose isomerase relies upon a complex hydrogen transfer. In order to identify which and how active site amino acids are involved, a neutron crystallographic analysis was undertaken with the objective to provide critical insight into the enzymatic mechanism. Neutron quasi-Laue data at 2.2 Å resolution were collected at room temperature. The neutron structure shows unambiguously that residue His 53 is doubly protonated at the active site of the enzyme. This suggests that the reaction proceeds through an acid catalyzed opening of the sugar ring. This was the first direct observation of double protonation of His 53 and the first validation of the ring opening mechanism at the active site of D-xylose isomerase. We are currently working on the neutron structures of substrate and inhibitor bound enzyme.

a. Histidine 53. b.D₂O water molecules. Blue 2F _o–F _c neutron map contoured at 1.5σ level

Hydrogen has a large incoherent neutron cross-section. Whilst this incoherent signal can be exploited to provide dynamic information in inelastic neutron scattering experiments, in diffraction experiments the large hydrogen incoherent scattering gives rise to a large and significant background. Moreover, due to their negative scattering length, hydrogen atoms appear as negative density in neutron Fourier maps, in contrast to the other elements, including deuterium, which appear as positive density peaks. Whilst this difference in sign between hydrogen and deuterium neutron density can be exploited to give information on solvent accessibility of the target groups in a protein (H/D exchange), it may also unfortunately lead to neutron density cancellation around neighboring atoms (at the ~2.0 Å resolutions typical of these experiments) hampering accurate interpretation of the resulting neutron maps. In contrast, the incoherent scattering arising from deuterium is 40 times lower, whilst the neutron scattering length is positive and twice that of hydrogen. Therefore replacing hydrogen by deuterium in protein crystals both increases the coherent scattering signal and decreases the incoherent background, typically providing a near order of magnitude improvement of the signal to noise ratio of the data.

Deuterium labeling can be achieved either partially, by soaking crystals in deuterated mother liquor, or more fully, by preparing completely (per)deuterated protein samples. Fully deuterated systems offer as much as a 40-fold reduction in the background and thus enables radically smaller crystals to be used for neutron data collection. We have optimized protocols for the routine high-yield production of perdeuterated protein in vivo in Escherichia coli. These protocols were applied to cytochrome P450cam. X-ray studies have shown that the structural features of fully deuterated P450cam are not significantly altered at the resolution of the analysis, validating the use of perdeuterated P450cam for neutron protein crystallography.

Isotope	Abundance (%)	Atomic Number	Neutron incoherent cross section (Barns)	Neutron coherent scattering length (10^-12 cm)	X-ray scattering factors (10^-12 cm) sin q = 0 (sin q) / l =0.5 Å^-1
¹H	99.985	1	80.27	-0.374	0.28	0.02
²H (D)	0.015		2.05	0.667	0.28	0.02
¹²C	98.9	6	0.00	0.665	1.69	0.48
¹⁴N	99.63	7	0.49	0.937	1.97	0.53
¹⁶O	99.762	8	0.00	0.580	2.25	0.62
²⁴Mg	78.99	12	0.00	0.549	3.38	1.35
³²S	95.02	16	0.00	0.280	4.50	1.90
³⁹K	93.258	19	0.25	0.379	5.30	2.20
⁵⁵Mn	100	25	0.40	-0.375	7.00	3.10
⁵⁶Fe	91.7	26	0.00	1.012	7.30	3.30

Neutron coherent scattering lengths and incoherent cross-section and X-ray scattering factors in biological materials.

The Spallation Neutron Source is an accelerator-based neutron source in Oak Ridge, TN. It was completed in May 2006. At full power, SNS will provide the most intense pulsed neutron beams in the world for scientific research and industrial development.

Aerial view of the SNS site, part of Oak Ridge National Laboratory in Tennessee

The Spallation Neutron Source and the High Flux Isotope Reactor at Oak Ridge National Lab develop world leading instrumentation for neutron sciences. The suite of instruments includes small angle and high-resolution diffractometers, reflectometers, spectrometers optimized to study biological systems that spread across multiple time and length scales.

The development of the next generation time-of-flight protein diffractometer, MaNDI (Macromolecule Neutron diffractrometer), at the Neutron Spallation Source offers the promise of further 50-100-fold gains in performance over existing instrumentation. The major advantage of this next generation instrument is that whilst a very broad (Laue) band-pass is used, the pulsed time structure and energy resolution of the incident neutron beam reduces the number of both harmonic and spatially overlapped reflections and improves the signal-to-noise ratio of the data. Such gains in performance can be expected to significantly extend the size and complexity of systems that can be studied by neutron crystallography.