Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.762775
Title: Femtosecond serial crystallography of two classes of copper nitrite reductase
Author: Halsted, T.
ISNI:       0000 0004 7658 6722
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Abstract:
Copper-containing nitrite reductases (CuNiR) catalyse the reduction of nitrite to the gaseous product nitric oxide. The enzyme forms part of the denitrification pathway which, in some bacteria, forms part of their respiratory network where nitrogen reduction is coupled to the synthesis of adenosine triphosphate. Denitrifying bacteria, through the production of nitrous oxide, have a large greenhouse gas impact and are also opportunist pathogens often found infecting immunocompromised patients. The structure and biochemical properties of CuNiRs have been extensively studied revealing a common functional core of one type 1 copper (T1Cu) site and one type 2 copper (T2Cu) site, linked by a cysteine-histidine bridge. The T1Cu is reduced by a partner redox protein and that electron is transferred to the T2Cu to reduce nitrite along with two protons. The order of this reaction is unknown, however with T2Cu reduction possible before or after nitrite binding. The structural study of this reaction is complicated by radiation damage effecting the copper sites when probed by synchrotron X-ray crystallography (SRX). The copper sites are highly prone to reduction by photoelectrons generated by the radiolysis of solvent inside the crystal. As the probe alters the redox states of the copper sites during data collection, the redox state of multiple species in the crystal change over the course of data collection, preventing them from being refined individually and obscuring the active site intermediates that may establish the enzyme mechanism order. To overcome the radiation damage, data collection was carried out at the SPring-8 angstrom compact free electron laser (SACLA), an X-ray free electron laser (XFEL), which produces femtosecond scale X-ray pulses that allow the diffraction to be captured before the onset of any radiation damage. The size of the pulse width also precludes any molecular rotation or vibration producing a 'time-frozen' structure. XFEL collection operates on the principle of 'diffraction before destruction' where the high intensity of the X-ray pulse destroys the crystal material and so new crystalline material must be inserted into the beam after each shot. Using this approach, the oxidised structure of two CuNiRs were collected from Achromobacter xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR) along with the reduced and nitrite-bound structures of AcNiR. This XFEL data along with other structural data revealed a basis for the oxidase activity in AxNiR and the pH dependence of AcNiR suggesting an ordered mechanism. This thesis reveals the specific changes that SRX can cause to radiation sensitive metalloproteins and the importance of overcoming it.
Supervisor: Hasnain, Samar Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.762775  DOI:
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