Modeling and mutagenesis of amino acid residues critical for CO2 hydration by specialized NDH-1 complexes in cyanobacteria

Abstract

The uptake of inorganic carbon in cyanobacteria is facilitated by an energetically intensive CO₂-concentrating mechanism (CCM). This includes specialized Type-1 NDH complexes that function to couple photosynthetic redox energy to CO₂ hydration forming the bicarbonate that accumulates to high cytoplasmic concentrations during the operation of the CCM, required for effective carbon fixation. Here we used a Synechococcus PCC7942 expression system to investigate the role of conserved histidine and cysteine residues in the CupB (also designated, ChpX) protein, which has been hypothesized to participate in a vectoral CO₂ hydration reaction near the interface between CupB protein and the proton-pumping subunits of the NDH-1 complex. A homology model has been constructed and most of the targeted conserved residues are in the vicinity of a Zn ion modeled to form the catalytic site of deprotonation and CO₂ hydration. Growth and CO₂ uptake assays show that the most severe defects in activity among the targeted residues are due to a substitution of the predicted Zn ligand, CupB-His86. Mutations at other sites produced intermediate effects. Proteomic analysis revealed that some amino acid substitution mutations of CupB caused the induction of bicarbonate uptake proteins to a greater extent than complete deletion of CupB, despite growth under CO₂-enriched conditions. The results are discussed in terms of hypotheses on the catalytic function of this unusual enzyme.

Keywords: CCM; CO2 concentrating mechanism; Inorganic carbon; NDH-1; Photosynthesis; Rubisco.