title: Structure-function studies of a novel two-domain copper nitrite reductase (CuNiR) from Bradyrhizobium sp. ORS 375 with low catalytic efficiency
Creators: name: Rose, Samuel
Publisher: name: University of Liverpool Repository
Year: 2024

description: Copper-containing nitrite reductases (CuNiR) are enzymes that liberate fixed nitrogen back to the atmosphere in the first committed step of the denitrification pathway, catalysing the reduction of nitrite (NO2-) to gaseous product nitric oxide (NO) in some denitrifying bacteria. Nitrous oxide (N2O) is sequentially produced which is a major greenhouse gas and ozone depleting substance. Rhizobia, which form symbiotic relationships with leguminous plants at root nodules, are major contributors to denitrification processes due to their large abundance in worldwide agriculture. CuNiRs from Achromobacter xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR) have been extensively studied. Catalysis in CuNiR is governed by the provision of two protons and one electron to a catalytic type 2 copper centre (T2Cu) when nitrite is bound. A type 1 copper centre (T1Cu) donates the electron after receiving it from a partner redox protein and two invariant catalytic residues (HisCAT and AspCAT) are essential for catalysis. This thesis presents a novel CuNiR from Bradyrhizobium ORS 375 (Br2DNiR), a Rhizobia species, which has a substantially lower catalytic efficiency compared to AxNiR and AcNiR. Efforts are presented in this thesis to understand the substantial catalytic inefficiency of Br2DNiR through a comprehensive structural and functional study. Static structures representing two key stages in the catalytic reaction (as-isolated and nitrite-bound) were obtained to atomic resolution (&lt; 1.2 Å) by synchrotron radiation crystallography (SRX), and by serial femtosecond rotation crystallography (SF-ROX) using an X-ray free electron laser (XFEL) (to obtain comparable, free from radiation induced chemistry (FRIC), structures). These structures revealed an unusual pentacoordinate (H2O)2-T2Cu-His3 site in the as-isolated state, owing to a more accessible substrate channel, as well as variations of nitrite binding and AspCAT conformations. The in-situ generation of product NO was also captured during the catalytic reaction using two approaches: a time-resolved freeze trap method, following the initiation of turnover in crystallo, and a single crystal serial crystallography method called MSOX (multiple serial structures from one crystal). Both confirmed the coordination of a controversial copper-nitrosyl intermediate to T2Cu. MSOX was also coupled with single crystal spectroscopy to simultaneously monitor the T1Cu redox state and electron transfer (ET) to T2Cu during data collection with MSOX of the as-isolated Br2DNiR also performed. The two movies revealed inter-Cu ET does not occur in as-isolated state but does occur in nitrite-bound state, although severely gated. Together with a pentacoordinate T2Cu active site requiring the displacement of two waters with nitrite are reasons for catalytic inefficiency observed. Extensive mutagenesis and functional studies of Br2DNiR and AcNiR were also performed using an electrochemical NO electrode revealing bioengineering of the enzyme is difficult but interesting results can be observed. In addition, a novel three-domain CuNiR from Thermus Oshimai (ToNiR), which contains a C-terminal tethered Cupredoxin domain, was also structurally characterised for the first time providing unprecedented insights into this enzyme and revealing a self-contained enzyme that exists with efficient internal electron transfer (ET). Both provide the potential basis for the design of biomimetic synthetic systems, where useful properties of inefficient catalysis and self-contained systems observed in nature may prove effective in modified systems.