Cu insertion into Cu-dependent nitrite reductase AniA from Neisseria gonorrhoeae.

Abstract

AniA is a copper dependent nitrite reductase expressed in pathogenic Neisseria gonorrhoea. It catalyses the first step of the denitrification pathway and is known to be important in gonococcal pathogenesis. The active protein is a homotrimer containing three T1Cu and three T2Cu sites. In vivo copper is inserted into these sites by metallochaperone AccA however little is known about how this process occurs. Though previous work indicates that the apo-form of AniA is a monomer, non-specific cleavage of AniA during protein purification hindered further research into the process.
This project therefore attempting to improve the protein purification process by changing the protease used during purification or eliminating its use altogether. These efforts where however unsuccessful as AniA for two reasons: AniA cannot be purified without a purification tag and AniA is highly susceptible to non-specific cleavage in its C-terminal domain. This suggests that this region is highly flexible in apo-AniA.
This project also investigated the insertion of aqueous copper into apo-AniA using fluorometry and UV-vis Spectroscopy. This showed that copper loading into the T1Cu site quenches the tryptophan fluorescence. It confirmed that copper inserts into T1Cu site before the T2Cu site and showed that the T1Cu site competes with NTA for copper providing a potential method for measuring the T1Cu sites affinity. Kinetic UV-vis spectroscopy studies suggested that copper loading occurs in a 2-stage process: Initial copper loading forming a “green” T1Cu centre which then undergoes an intramolecular transition to form the final “blue” T1Cu centre. However more research is required to understand the role of the T2Cu site and trimerisation in this process.