Carbonic anhydrase II does not exhibit Nitrite reductase or Nitrous Anhydrase Activity

Carbonic anhydrase II (CA II) is a zinc metalloenzyme that catalyzes the reversible interconversion of water and CO₂ to bicarbonate and a proton. CA II is abundant in most cells, and plays a role in numerous processes including gas exchange, epithelial ion transport, respiration, extra- and intracellular pH control, and vascular regulation. Beyond these CO₂ and pH-linked roles, it has been postulated that CA II might also reduce nitrite (NO₂^-) to nitric oxide (NO), as bicarbonate and NO₂^- both exhibit sp² molecular geometry and NO also plays an important role in vasodilation and regulation of blood pressure. Indeed, previous studies by Aamand et al. have shown that bovine CA II (BCA II) possesses nitrite dehydration activity and paradoxically demonstrated that CA inhibitors (CAIs) such as dorzolamide and acetazolamide significantly increased NO production (Aamand et al., 2009; Nielsen and Fago, 2015) [1,2]. Hence, the goal of this work was to revisit these studies using the same experimental conditions as Aamand et al. measuring NO generation by two methods, and to examine the structure of CA II in complex with NO₂^- in the presence and absence of dorzolamide. Our results contradict the previous findings and indicate that CA II does not exhibit nitrite reductase or dehydration activity, and that this is not enhanced in the presence of CA inhibitors. In addition, a structural examination of BCA II in complex with NO₂^- and superimposed with dorzolamide demonstrates that CA inhibitor binding at the active site to the zinc moiety blocks potential NO₂^- binding.