Copper maturation of nitrous oxide reductase in Paracoccus denitrificans

Gates, Andrew (2018) Copper maturation of nitrous oxide reductase in Paracoccus denitrificans. Doctoral thesis, University of East Anglia.

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    Abstract

    Nitrous oxide (N2O) is an important greenhouse gas that is also responsible for stratospheric ozone depletion [1, 2]. Human activity is the main source of N2O due to the use of fertilisers in agriculture. Nitrous oxide reductase (N2OR) is the only enzyme to destroy N2O as part of a biological process termed denitrification. This enzyme has a unique catalytic CuZ centre, an electron transfer CuA centre and a high demand for Cu with 12 atoms required per functional dimer. A previous transcriptomic study revealed that two putative Cu chaperones, ScoB and PCuC were upregulated under Cu limiting conditions [3]. Here we demonstrate that ScoB/PCuC is a high-affinity Cu system essential for N2O respiration.

    Deletion of scoB causes N2O accumulation under anoxic and Cu-limited growth. N2O respiration could be restored complementation in trans with recombinant full-length, or soluble, periplasmic ScoB proteins (ScoBFL and ScoBsol, respectively). ScoBsol was biochemically characterised and found to be a monomeric protein of _25 kDa that can bind Cu1+ or Cu2+ with an apparent KD value within the subfemtomolar range. In contrast, PCuC is a multidomain protein with a Ycn-like N-terminal domain [4], and a PCuAC-like C-terminal domain [5]. Recombinant periplasmic proteins for each individual domain and full-length protein were generated (i.e., PCuCNt, PCuCCt and PCuCFL). The pcuC deletion strain has an N2O-genic phenotype. Only complementation in trans with PCuCFL restored N2O reduction under anaerobic and Cu-limited conditions.

    In addition, the crystallographic structure of Cu-bound PCuCNt was solved to a resolution of 1.5 Å revealing a trimeric protein of _56 kDa with a novel histidine brace metal binding site. PCuCNt can bind 1+ or 2+ and competition assays with 1+ chelators revealed that metallation occurs with femtomolar affinity. Analysis of YcnI-type proteins revealed the presence of two defined families. Family A contains a HX22HX101W consensus Cu-binding motif and was principally found among alphaproteobacteria, while Family B contain a HX22DX90WX13H motif and are distributed in actinobacteria and firmicutes. The Cu-bound structure of PCuCCt was also solved to a resolution of 1.6 Å and reveals a _18 kDa monomer that contains a defined H(M)X10MX21HXM Cu-binding site that can bind Cu1+ with subfemtomolar affinity. Further biochemical studies of native PCuC confirmed that the full-length protein forms a _100 kDa homotrimer in solution regardless of metallation state, with the N-terminal domain driving oligomerization exposing individual C-terminal domains to bulk solution through a flexible linker region. Each trimer can bind up to 6 Cu atoms with binding affinities within the subfemtomolar range.

    Finally, the maturation of the Cu centres of N2OR was studied in P. denitrificans WT, scoB and pcuC deletion strains. A periplasmic and readily isolatable affinity-tagged N2OR protein was expressed in cis under two different Cu regimes in P. denitrificans. N2OR purified from WT cells grown under anaerobic and Cu-limited conditions only contained a recognisable CuA centre. However, N2OR from scoB and pcuC mutants lacked both Cu-centres, had significantly lower Cu content and impaired enzymatic activity. A model for the metallation process of the CuA centre of N2OR by the high affinity Cu-maturation system ScoB/PCuC has been proposed.

    Item Type: Thesis (Doctoral)
    Faculty \ School: Faculty of Science > School of Biological Sciences
    Depositing User: Gillian Aldus
    Date Deposited: 07 Sep 2018 13:07
    Last Modified: 07 Sep 2018 13:07
    URI: https://ueaeprints.uea.ac.uk/id/eprint/68203
    DOI:

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