Limited functional compensation among subunits of the carbonic anhydrase module of mitochondrial complex I

Highly similar carbonic anhydrase subunits of the integrated CA module of complex I are not functionally interchangeable, giving rise to complex I variants adapted to different developmental and metabolic contexts. Plant mitochondrial NADH dehydrogenase complex (complex I) includes a carbonic anhydrase (CA) module composed of trimers of gamma CAs, which protrudes into the matrix and is thought to participate in complex I assembly. In Arabidopsis, the CA module is typically formed by heterotrimers containing CA1, CA2 and CAL proteins, where CA2 is the most abundant subunit. Null ca2 mutants contain lower complex I levels and higher glycine levels in leaves. Here, we tested whether the requirement for CA2 reflects only its higher expression level or a specific functional specialisation. To address this question, the CA1 coding sequence was expressed under the control of the CA2 promoter in the ca2 mutant background. Elevated CA1 expression partially restored the abundance of complex I and its NADH dehydrogenase activity, indicating that CA1 can structurally replace CA2 in the assembly of the CA module. However, oxygen consumption was reduced and photorespiratory markers such as glycine and serine were elevated, suggesting that complex I function was not fully recovered. Furthermore, metabolite profiling revealed alterations in central carbon metabolites, including changes in sugar levels and decreased fatty acid content. Structural modelling and molecular dynamics simulations suggest that replacing CA2 with CA1 destabilizes the catalytic site. Taken together, these results suggest that although CA1 can support complex I assembly, CA2 retains a specialized role required for optimal respiratory and metabolic functions.