Repurposing human Carbonic Anhydrase 2 for artificial photosynthesis: structural and functional role of selected transition metals

Natural photosynthesis has inspired the development of artificial systems designed to convert solar energy into chemical energy. In this context, artificial photosynthesis (AP) presents a promising approach for sustainable energy production, particularly through light-driven water splitting, which generates non-polluting by-products such as oxygen (O¢) and hydrogen (H¢). This study aims to develop and optimize novel biological catalysts for AP applications, focusing on human carbonic anhydrase II (hCA2), a ubiquitous enzyme that catalyses the reversible hydration and dehydration of carbon dioxide and bicarbonate. The catalytic activity of hCA2 relies on a Zn2z ion, which is tetrahedrally coordinated by three histidine residues in the enzyme's active site. Previous studies have demonstrated that substituting Zn2z with other metal ions (e.g., Co2z, Ni2z, Cu2z) can significantly alter the enzyme's catalytic properties, opening new possibilities for different applications. In this work, we explored metal-substituted hCA2, focusing on Ir3z-substituted enzyme, as previous research suggested that the combination with this ion may drive unique water-splitting properties. To investigate this, hCA2 was expressed as a recombinant enzyme in Escherichia coli, and the active protein was purified through multiple steps, including affinity and size- exclusion chromatography. Following zinc removal by chelation, the apo-protein was used to generate various metal-substituted variants. These engineered enzymes were then characterized through isothermal titration calorimetry and X-ray crystallographic analysis to determine their metal-binding properties and structural integrity. Additionally, we evaluated the water-splitting capabilities of the metal-substituted hCA2 variants for AP applications. Structural analysis of the metal-substituted enzymes highlighted the peculiar properties of each histidine residue involved in metal binding in relationship with the catalytic function. Notably, the discovery of a second binding site, located between residues 4 and 64, was explored with five alanine variants: H94A, H96A, and H119A (targeting the catalytic site) and H4A (targeting the second accessory site). Structural characterization and esterase kinetic assays of these variants provide deeper insights into the role of these residues in site stabilization and enzymatic activity. This work also advances our understanding of enzyme-metal interactions and coordination geometry in catalytic processes through the characterization of hCA2, its alanine variants, and the substitution of different metals in the catalytic centre. These findings pave the way for future studies on substrate binding and metal coordination mechanisms, ultimately contributingto the design of engineered enzymes with improved catalytic efficiency, increased oxygen production turnover rates, and enhanced long-term stability. Such advances could facilitate the large-scale implementation of biocatalysts in AP and sustainable energy technologies.