Although calcium oxalates are relevant biominerals, their formation mechanisms remain largely unresolved. Here, we investigate the early stages of calcium oxalate formation in pure and citrate-bearing solutions. Citrate is used as a well-known oxalate precipitation inhibitor; moreover, it resembles the functional domains of the biomolecules that modulate biomineralization. Our data suggest that calcium oxalate forms after Ca²⁺ and C₂O₄²⁻ association into polynuclear stable complexes that aggregate into larger assemblies, from which amorphous calcium oxalate nucleates. Previous work has explained citrate inhibitory effects according to classical theories. Here we show that citrate interacts with all early stage CaC₂O₄ species (polynuclear stable complexes and amorphous precursors), inhibiting calcium oxalate nucleation by colloidal stabilization of polynuclear stable complexes and amorphous calcium oxalate. The control that citrate exerts on calcium oxalate biomineralization may thus begin earlier than previously thought. These insights provide information regarding the mechanisms governing biomineralization, including pathological processes (e.g., kidney stone formation).

Carbonic anhydrase (CA), a ubiquitous enzyme that catalyzes the reversible hydration of CO₂, is known to be involved in the formation of CaCO₃ biominerals and is currently used for biomimetic CO₂ mineral sequestration. However, its specific role(s) in calcium carbonate (bio)mineralization is poorly understood. Here we show that CA catalyzes the formation of the reactive precursors (i.e., HCO₃ ⁻ and CO₃ ²⁻ ions) required for mineralization, accelerates the precipitation of metastable amorphous calcium carbonates and their solution-mediated conversion into crystalline calcite, which grows via a non-classical nanoparticle aggregation mechanism that facilitates CA occlusion. Ca⁺ and CO₃ ²⁻ ions promote the partial unfolding and oligomerization of CA, resulting in fibril- and sheet-like supramolecular assemblies that template nanostructured calcium carbonate crystallization. By losing its catalytic activity following the observed conformational changes, CA elicits a mechanism for arresting calcium carbonate mineralization. Our results show that CA can play multiple, until now unrecognized, critical roles, namely, as an enzyme and structural protein in CaCO₃ biomineralization and also helps to explain the observed loss of enzymatic activity during ex situ CO₂ mineral sequestration.