Cofactor assembly in the Cu-dependent nitrite reductase, AniA, from pathogenic Neisseria

Abstract

Neisseria gonorrhoeae infects the human genitourinary tract. In its O2-limited host niche, N. gonorrhoeae relies on nitrite as the terminal electron acceptor in anaerobic respiration. In this process, nitrite is reduced to nitric oxide by the copper (Cu)-dependent nitrite reductase, AniA.
AniA is essential for biofilm formation and therefore, N. gonorrhoeae infection. AniA exists on either side of the outer membrane and is a target for vaccines and novel antimicrobials. Cu insertion into the AniA active site, and thus enzyme activity, requires a periplasmic Cu-binding accessory protein AccA. AccA is thought to be specific to AniA, however AccA is part of a larger family of periplasmic Cu binding proteins. Therefore, another protein may be able to replace AccA. To test this, AccA was replaced with PCuAC from Thermus thermophilus, creating an ∆accA/pCuAC+ mutant strain. Microaerobic cultures of this mutant did not grow or consume nitrite, however, both could be rescued upon the addition of Cu salt. This suggests that in the presence of PCuAC but without AccA, AniA does not acquire Cu and is inactive unless Cu is supplied. This suggests PCuAC cannot insert Cu into AniA and AccA is required for this process in cells. This was tested using purified proteins, Cu transfer to apo-AniA via Cu(I)-PCuAC was found to be 15X slower than from Cu(I)Cu(II)-AccA. Therefore, showing PCuAC cannot insert Cu into AniA at a rate to support bacterial growth.
This study indicates that the interaction between AccA and AniA is specific and favourable for the transfer of Cu. This specificity suggests the feasibility of targeting the AccA-AniA interactions for novel antimicrobials against multi-drug-resistant N. gonorrhoeae.