Elucidating copper ion interactions with carbonic anhydrase: insights from fluorescence quenching and thermodynamic analysis.

Abstract

Plant carbonic anhydrases (CAs) are essential metalloenzymes catalyzing reversible hydration of CO₂ to HCO₃⁻, thereby optimizing photosynthetic efficiency and carbon fixation in plants. They facilitate CO₂ delivery to Rubisco, enhance carbon assimilation, and play a role in plant responses to stresses (such as drought, high salinity) by modulating stomatal conductance and internal CO₂ concentrations. Despite the well-established physiological importance of plant CAs, the influence of metal ions, particularly copper (Cu²⁺), on their structure and activity remains inadequately understood. Here, bCA II is used as a well-characterized model enzyme to investigate enzyme-metal interactions. We employed intrinsic tryptophan and tyrosine fluorescence quenching to elucidate the binding mechanism of Cu²⁺ with bCA II. Our results demonstrate static quenching, indicative of ground-state complex formation, with binding parameters assessed at 288 K and 298 K [Kb = (2.64 ± 0.15 and 1.68 ± 0.54) × 10³, M^-1, n ≈ 1] and negative ΔG°, ΔH°, ΔS°.