The importance of a single amino acid substitution in reduced red blood cell carbonic anhydrase function of early-diverging fish

In most vertebrates, red blood cell carbonic anhydrase (RBC CA) plays a critical role in carbon dioxide (CO₂) transport and excretion across epithelial tissues. Many early-diverging fishes (e.g., hagfish and chondrichthyans) are unique in possessing plasma-accessible membrane-bound CA-IV in the gills, allowing some CO₂ excretion to occur without involvement from the RBCs. However, implications of this on RBC CA function are unclear. Through homology cloning techniques, we identified the putative protein sequences for RBC CA from nine early-diverging species. In all cases, these sequences contained a modification of the proton shuttle residue His-64, and activity measurements from three early-diverging fish demonstrated significantly reduced CA activity. Site-directed mutagenesis was used to restore the His-64 proton shuttle, which significantly increased RBC CA activity, clearly illustrating the functional significance of His-64 in fish red blood cell CA activity. Bayesian analyses of 55 vertebrate cytoplasmic CA isozymes suggested that independent evolutionary events led to the modification of His-64 and thus reduced CA activity in hagfish and chondrichthyans. Additionally, in early-diverging fish that possess branchial CA-IV, there is an absence of His-64 in RBC CAs and the absence of the Root effect [where a reduction in pH reduces hemoglobin’s capacity to bind with oxygen (O₂)]. Taken together, these data indicate that low-activity RBC CA may be present in all fish with branchial CA-IV, and that the high-activity RBC CA seen in most teleosts may have evolved in conjunction with enhanced hemoglobin pH sensitivity.