Single-crystal time-of-flight neutron Laue methods: application to manganese catalase from Thermus thermophilus HB27

Abstract

The IBARAKI biological crystal diffractometer (iBIX) was used in single‐crystal time‐of‐flight neutron diffraction experiments on manganese catalase from Thermus thermophilus. The unit‐cell dimensions were 133 × 133 × 133 Å, which is close to the designed maximum limitation of iBIX (135 × 135 × 135 Å). The optimum integration box sizes were set and the degree of integration box overlap was calculated for each Laue spot. Using the overlap ratio as the criterion, the selection of the diffraction intensity data was performed to give a minimum Rp.i.m.. Subsequently, diffraction intensity data from Laue spots with overlap ratios ≤0.1 were selected and a complete reflection data set with dmin = 2.35 Å was obtained. Joint X‐ray and neutron structure refinements were also successfully performed. It was difficult to determine the structures and protonation states of all the oxygen atoms in the manganese cluster owing to the disordered structure. No hydrogen atom was observed on the ordered μ‐bridging oxygen atom O1003. Instead, this oxygen atom probably forms a hydrogen bond with Thr39. In addition, the refinements clearly showed the protonation states of the amino acid residues and hydrogen bonds, as observed in Tyr192, Glu167 and Glu280. This first neutron crystal structure of manganese catalase shows that iBIX can provide acceptable diffraction data for neutron single‐crystal analyses of at least 2.4 Å resolution within the original targeted unit‐cell dimensions of 135 × 135 × 135 Å.

Charge neutralization and β-elimination cleavage mechanism of family 42 L-rhamnose-α-1,4-D-glucuronate lyase revealed using neutron crystallography

“…2 A , lower panel), suggesting that H/D exchange occurred at the C–H bond. Exchange of the HE1 atom of histidine residues with DE1 has been reported in the neutron crystallography of myoglobin (23, 30) and catalase (31). H/D exchange of the C–H bond was also observed in the guanine base of Z-DNA (32).…”

“…The purified protein solution was exchanged with 20 mM Tris pD 8.0 buffer using Amicon Ultracel-10K and concentrated to 24 mg/mL. A large-scale sitting-drop vapor diffusion sitting drop method was adopted using a Falcon 60 × 15 mm center well organ culture dish (Corning Inc., Corning, New York) (31). In the outside well of the culture dish, 2 mL of reservoir solution consisting of 0.1 M Tris pD 8.5, 0.1 M Rha, and 33.0% (w/v) PEG1500 in heavy water was poured into the outside well of the culture dish.…”

Charge neutralization and β-elimination cleavage mechanism of family 42 L-rhamnose-α-1,4-D-glucuronate lyase revealed using neutron crystallography

“…S5 ). The exchange of the HE1 atom of histidine residues with the DE1 atom occurred in the neutron crystallography of myoglobin ( 22 , 23 ) and catalase ( 24 ). H/D exchange of the C–H bond was also observed in the guanine base of Z-DNA ( 25 ).…”

Metalloprotein catalysis: structural and mechanistic insights into oxidoreductases from neutron protein crystallography

“…The identity of the bridging ligands during catalysis remains to be established; however, it has been proposed that in the resting Mn(II)Mn(III) state the active site contains a -oxo and a -OH À bridge, which converts to Mn(II)Mn(II) with a -OH À and a -H 2 O bridge following H 2 O 2 binding and oxidation (Boelrijk & Dismukes, 2000). To structurally investigate the protonation states in the active site of the thermostable MnCat from Thermus thermophilus, Yamada and coworkers collected a 2.35 Å resolution neutron diffraction data set as well as a 1.37 Å resolution X-ray diffraction data set from the same crystal for joint refinement (Yamada et al, 2019). Crystallization was performed under basic conditions to ensure the Mn(III)Mn(III) oxidation state.…”

“…Figure 9The active site and protonation states of glutamate residues with backbone carbonyl O atoms (PDB entry 6kk8, H/D exchanged;Yamada et al, 2019). 2F o À F c NSLD map ( = 1.00) is displayed as a blue mesh; H and D atoms are displayed in white and turquoise, respectively.…”