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The role of the periplasmic Cu metallochaperone AccA in metalating the Cu-dependent nitrite reductase AniA in Neisseria gonorrhoea

FIRTH, SAMANTHA,JADE (2023) The role of the periplasmic Cu metallochaperone AccA in metalating the Cu-dependent nitrite reductase AniA in Neisseria gonorrhoea. Doctoral thesis, Durham University.

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Neisseria gonorrhoeae (the gonococcus) respires nitrite (NO2-). This process requires the nitrite reductase, AniA, which contains T1- and T2Cu in its active sites. We have characterised AccA, a PCuAC homologue with an extended His- and Met-rich C-terminal domain, as a likely periplasmic Cu- binding metallochaperone that metalates AniA.

In this study, biochemical examination of purified AccA and site-directed variants confirms that it binds one Cu(I) atom with femtomolar affinity in the conserved 2 His, 2 Met binding site. The C-terminal domain binds Cu(II) with picomolar affinity, although precise ligands remain unknown. Gonococcal strains lacking AccA or any conserved His and Met residue in AccA fail to grow and reduce NO2-. This phenotype is reversed when Cu(II) salts are supplemented in the growth medium. These results suggest that, in the absence of AccA, AniA is expressed as an apo-enzyme, but is re-metalated if the periplasmic buffered Cu pool increases. Interestingly, gonococcal strains lacking the C-terminal domain of AccA show reduced growth and NO2- consumption only in the presence of the Cu(I) chelator BCS, suggesting a role during Cu starvation.

Cu-transfer experiments using purified proteins confirmed AccA metalates both Cu sites in AniA with metal coordinated by both the Cu primary and C-terminal Cu binding sites. However, the C-terminal tail was required for metalation of the T2-site. AniA is expressed as a monomer and Cu binding induces protein trimerisation. This may offer an alternative role of why AccA is required to metalate AniA in vitro.

This work raises questions regarding the thermodynamics and kinetics of how metalloproteins acquire Cu from the buffered cellular pool via metallochaperones.

Item Type:Thesis (Doctoral)
Award:Doctor of Philosophy
Keywords:Cu, Metallochaperone, Nitrite Reductase, N. gonorrhoeae
Faculty and Department:Faculty of Science > Biological and Biomedical Sciences, School of
Thesis Date:2023
Copyright:Copyright of this thesis is held by the author
Deposited On:06 Jun 2023 09:10

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